Cancer diagnostic and cancer therapeutic Patent Application (2025)

U.S. patent application number 13/817687 was filed with the patent office on 2014-10-30 for cancer diagnostic and cancer therapeutic. This patent application is currently assigned to Thomas Jefferson University. The applicant listed for this patent is Michael P. Lisanti, Federica Sotgia. Invention is credited to Michael P. Lisanti, Federica Sotgia.

Cancer diagnostic and cancer therapeutic

Abstract

This invention relates to methods and kits for making aprognosis of disease course in a neoplastic disease patient bydetermining the level, expression and/or activity of abiomarker.

Related U.S. Patent Documents

Claims

1. A method for making a prognosis of disease course in a humanneoplastic disease patient, the method comprising the steps of: (a)obtaining a sample from the patient, said sample comprising a tumortissue, a body fluid or stromal cells adjacent to a neoplasm, orstromal cells within a neoplasm and/or a total tumor extract: (b)determining the level of a biomarker selected from the groupconsisting of ACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2,BNIP3, BNIP3L, miR-3 1, miR-34c, ACAT1, ACAT2, OXCT1, OXCT2, ADMA,3-hydroxybutyrate, and combinations thereof, in the sample; whereinsaid prognosis is made when the level of biomarker in sample ishigher than the level of biomarker in a control.

2. The method for making a prognosis of claim 1, wherein thebiomarker is selected from the group consisting of ACLY, HMGCS1,HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, and combinationsthereof, in the sample.

3. The method for making a prognosis of claim 1, wherein making theprognosis of disease course comprises determining the likelihoodthat a carcinoma is of a grade likely to become an invasivecarcinoma.

4. The method for making a prognosis of claim 3, wherein thebiomarker is selected from the group consisting of ACLY, HMGCS1,HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, and combinationsthereof, in the sample.

5. The method for making a prognosis of claim 3, further comprisingcorrelating the amount of labeling signal in the test sample with acontrol, wherein the carcinoma is of a grade likely to becomeinvasive when the level of biomarker in the sample is higher thanthe level of biomarker in a control.

6. The method of claim 3, wherein the carcinoma is a carcinoma ofthe breast.

7. The method of claim 3, wherein the carcinoma is selected fromthe group consisting of carcinoma of the breast, skin, kidney,parotid gland, lung, bladder and prostate.

8. The method of claim 3, wherein the detection reagent is labeledantibody capable of binding to human ACLY, HMGCS1, HMGCS2, HMGCL,HMGCLL1, BDH1, BDH2, BNIP3, and/or BNIP3L.

9. The method of claim 3, wherein the detection reagent is labelednucleic acid capable of binding to human ACLY, HMGCS1, HMGCS2,HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, and/or BNIP3L.

10. The method of claim 3, wherein the amount of labeling signal ismeasured by a technique selected from the group consisting ofemulsion autoradiography, phosphorimaging, light microscopy,confocal microscopy, multi-photon microscopy, and fluorescencemicroscopy.

11. The method of claim 3, wherein the amount of labeling signal ismeasured by autoradiography and a higher signal intensity in a testsample compared to a control prepared using the same steps as thetest sample is used to diagnose a high grade carcinoma possessing ahigh probability the carcinoma will progress to an invasivecarcinoma.

12. The method of claim 3, wherein the amount of labeling signal ismeasured by a technique selected from the group consisting of is anassay selected from the group consisting of RT-PCR, QRT-PCR,rolling circle amplification and nucleic acid sequenced-basedamplification assays.

13. The method for making a prognosis of claim 1, wherein the humanneoplastic disease patient has a neoplasm selected from the groupconsisting of breast, skin, kidney, lung, pancreas, rectum andcolon, prostate, bladder, epithelial, non-epithelial, lymphomas,sarcomas, melanomas, and the like.

14. The method for making a prognosis of claim 1, wherein the humanneoplastic disease patient has a breast neoplasm subtype selectedfrom the group consisting of ER(+), PR(+), HER2(+), triple-negative(ER(-)/PR(-)/HER2(-)), ER(-), PR(-), all tumor and nodal stages,and all tumor grades.

15. The method for making a prognosis of claim 1, wherein the levelof biomarker is determined by immunohistochemical staining.

16. The method for making a prognosis of claim 1, wherein the levelof biomarker is determined by an assay selected from the groupconsisting of RT-PCR, QRT-PCR, rolling circle amplification andnucleic acid sequenced-based amplification assays.

17. The method of claim 1, wherein the level of biomarker isdetermined by enzymatic assay.

18. The method for making a prognosis of claim 1, wherein theprognosis of disease course includes a risk for metastasis,recurrence and relapse of neoplastic disease.

19. The method for making a prognosis of claim 1, wherein increaseof biomarker predicts early disease recurrence, metastasis,survival, and tamoxifen-resistance at diagnosis.

20. The method for making a prognosis of claim 1, wherein INCREASEof biomarker in the sample predicts the prognosis of lymph-nodepositive (LN(+)) patients.

21. The method for making a prognosis of claim 1, wherein INCREASEof biomarker in the sample is associated with a poor prognosis.

22. The method for making a prognosis of claim 1, wherein theup-regulation or presence of biomarker in the sample is associatedwith a bad prognosis.

23. The method for making a prognosis of claim 1, wherein theneoplasm is a pre-malignant lesions selected from the groupconsisting of ductal carcinoma in situ (DCIS) of the breast andmyelodysplastic syndrome of the bone marrow.

24. The method for making a prognosis of claim 1, wherein theprognosis of disease course includes staging malignant disease in ahuman neoplastic disease patient.

25. The method for making a prognosis of claim 1, wherein INCREASEof biomarker in the sample is a surrogate marker for stromal RBtumor suppressor functional inactivation by RBhyper-phosphorylation.

26. The method for making a prognosis of claim 1, wherein the bodyfluid is selected from the group consisting of plasma, serum,blood, lymphatic fluid, cerebrospinal fluid, synovial fluid, urine,saliva, mucous, phlegm, sputum, and combinations thereof.

27. The method for making a prognosis of claim 1, wherein thesample comprises DCIS breast tissue and stromal cells adjacent to aneoplasm, stromal cells within a neoplasm, and/or a total tumorextract from a human neoplastic disease patient.

28. The method for making a prognosis of claim 27, wherein thesample comprises ductal carcinoma in situ (DCIS) pre-invasivecancerous breast tissue, wherein the level of the biomarker in thestromal cells of the sample is compared with the level of biomarkerwith the level of biomarker in a control, and wherein the course ofdisease progression is made when the level of biomarker in thestromal cells of the sample is higher than the level of biomarkerin the control.

Description

BACKGROUND OF THE INVENTION

[0001] 1. Field of Invention

[0002] This invention relates to methods and kits for making aprognosis of disease course in a neoplastic disease patient bydetermining the level, expression and/or activity of abiomarker.

[0003] 2. Description of Related Art

[0004] It is now well-recognized that the tumor micro-environmentplays a primary role in determining tumor progression andmetastasis in many different types of epithelial cancers. In thisregard, "activated or myofibroblastic" cancer-associated fibroblasthave emerged as one of the most prominent cell types in the tumorstroma that may determine clinical outcome in breast and prostatecancers. We have recently identified a loss of stromal Cav-1 in thetumor associated fibroblast compartment as a critical event in theprogression of human breast cancers (Mercier I, Casimiro M C, WangC, Rosenberg A L, Quong J, Allen K G, Danilo C, Sotgia F,Bonnucelli G, Jasmin J F, Xu H, Bosco E, Aronow B, Witkiewicz A,Pestell R G, Knudsen E S, Lisanti M P. Human BreastCancer-Associated Fibroblasts (CAFs) Show Caveolin-1Down-regulation and RB Tumor Suppressor Functional Inactivation:Implications for the Response to Hormonal Therapy. Cancer Biol Ther2008; 7:1212-25; Sotgia F, Del Galdo F, Casimiro M C, Bonuccelli G,Mercier I, Whitaker-Menezes D, Daumer K M, Zhou J, Wang C, KatiyarS, Xu H, Bosco E, Quong A A, Aronow B, Witkiewicz A K, Minetti C,Frank P G, Jimenez S A, Knudsen E S, Pestell R G, Lisanti M P.Caveolin-1-/- null mammary stromal fibroblasts sharecharacteristics with human breast cancer-associated fibroblasts. AmJ Pathol 2009; 174:746-61; Witkiewicz A K, Dasgupta A, Sotgia F,Mercier I, Pestell R G, Sabel M, Kleer C G, Brody J R, Lisanti M P.An Absence of Stromal Caveolin-1 Expression Predicts Early TumorRecurrence and Poor Clinical Outcome in Human Breast Cancers. Am JPathol 2009; 174:2023-34.). More specifically, a loss of stromalCav-1 is associated with early tumor recurrence, lymph nodemetastasis, and tamoxifen-resistance, resulting in poor clinicaloutcome (Witkiewicz A K, Dasgupta A, Sotgia F, Mercier I, Pestell RG, Sabel M, Kleer C G, Brody J R, Lisanti M P. An Absence ofStromal Caveolin-1 Expression Predicts Early Tumor Recurrence andPoor Clinical Outcome in Human Breast Cancers. Am J Pathol 2009;174:2023-34). Similar results were obtained with DCIS (10) andprostate cancer patients (Di Vizio D, Morello M, Sotgia F, PestellR G, Freeman M R, Lisanti M P. An Absence of Stromal Caveolin-1 isAssociated with Advanced Prostate Cancer, Metastatic Disease andEpithelial Akt Activation. Cell Cycle 2009; 8:2420-4.), indicatingthat a loss of stromal Cav-1 in cancer-associated fibroblasts istightly associated with tumor progression and metastasis. Thesefindings have now been replicated in several independent patientcohorts (Witkiewicz A K, Casimiro M C, Dasgupta A, Mercier I, WangC, Bonuccelli G, Jasmin J F, Frank P G, Pestell R G, Kleer C G,Sotgia F, Lisanti M P. Towards a new "stromal-based" classificationsystem for human breast cancer prognosis and therapy. Cell Cycle2009; 8:1654-8; Sloan E K, Ciocca D, Pouliot N, Natoli A, RestallC, Henderson M, Fanelli M, Cuello-Carrion F, Gago F. Anderson R.Stromal Cell Expression of Caveolin-1 Predicts Outcome in BreastCancer. Am J Pathol 2009; 174:2035-43.), and also extended to othermore lethal forms of breast cancer, such as the triple-negative andbasal-like breast cancer sub-types (Witkiewicz A K, Dasgupta A,Sammons S, Er O, Potoczek M B, Guiles F. Sotgia F, Brody J R,Mitchell E P, Lisanti M P. Loss of stromal caveolin-1 expressionpredicts poor clinical outcome in triple negative and basal-likebreast cancers. Cancer Biol Ther 2010; 10: In Press). For example,in triple-negative breast cancers, patients with high stromal Cav-1have a 75.5% survival rate at 12 years, while patients with anabsence of stromal Cav-1 have a survival rate of less than 10% at 5years post-diagnosis (Wikiewicz, 2010). Thus, the inventors havedissected the molecular basis of this phenomenon, to design bettertherapeutics targeting this high-risk patient population.

[0005] To mechanistically understand the lethality of aCav-1-deficient stromal microenvironment, Cav-1 (-/-) null mice wasused as a model system (Pavlides S, Whitaker-Menezes D,Castello-Cros R, Flomenberg N, Witkiewicz A K, Frank P G, CasimiroM C, Wang C, Fortina P, Addya S, Pestell R G, Martinez-Outschoorn UE, Sotgia F, Lisanti M P. The reverse Warburg effect: aerobicglycolysis in cancer associated fibroblasts and the tumor stroma.Cell Cycle 2009; 8:3984-4001). Using this approach, bone-morrowstromal cells were isolated (thought to be the precursors ofcancer-associated fibroblasts (Mishra P J, Humeniuk R, Medina D J,Alexe G, Mesirov J P, Ganesan S, Glod J W, Banerjee D.Carcinoma-associated fibroblast-like differentiation of humanmesenchymal stem cells. Cancer Res 2008; 68:4331-9), and subjectedthem to unbiased proteomics and genome-wide transcriptionalprofiling (Pavlides, 2009). As a result, via our proteomicsanalysis, it was observed that Cav-1 (-/-) null stromal cells showthe upregulation of i) 8 myo-fibroblast markers (includingvimentin, calponin, and collagen 1), 8 glycolytic enzymes (such aspyruvate kinase (PKM2) and lactate dehydrogenase (LDHA), and 2markers of oxidative stress (namely catalase and peroxiredoxin)(Bissell M J, Radisky D. Putting tumours in context. Nat Rev Cancer2001; 1:46-54) (Wikiewicz, 2010). These results were alsoindependently supported by our data from transcriptional profiling.An informatics analysis of these findings suggested that a loss ofstromal Cav-1 results in oxidative stress, driving aerobicglycolysis (a.k.a., the Warburg effect) in cancer-associatedfibroblasts (Pavlides S, Tsirigos A, Vera I, Flomenberg N, Frank PG, Casimiro M C, Wang C, Fortina P, Addya S, Pestell R G, RigoutsosI, Martinez-Outschoorn U E, Sotgia F, Lisanti M P. Loss of StromalCaveolin-1 Leads to Oxidative Stress, Mimics Hypoxia, and DrivesInflammation in the Tumor Microenvironment, Conferring the "ReverseWarburg Effect": A Transcriptional Informatics Analysis withValidation. Cell Cycle 2010; In Press; Pavlides S, Tsirigos A, VeraI, Flomenberg N, Frank P G, Casimiro M C, Wang C, Pestell R G,Martinez-Outschoorn U E, Howell A, Sotgia F, Lisanti M P.Transcriptional evidence for the "Reverse Warburg Effect" in humanbreast cancer tumor stroma and metastasis: similarities withoxidative stress, inflammation, Alzheimer's disease, and"Neuron-Glia Metabolic Coupling". Aging (Albany N.Y.) 2010;2:185-99.). This would then provide a feed-forward mechanism bywhich Cav-1-deficient cancer-associated fibroblasts could literally"feed" adjacent cancer cells, by providing lactate and pyruvate ina paracrine fashion (Wikiewicz, 2010). The inventors have termedthis novel idea the "Reverse Warburg Effect", as the originalWarburg effect was originally thought to occur only in epithelialcancer cells and not cancer-associated fibroblasts (Wikiewicz,2010).

[0006] The inventors have now performed an unbiased metabolomicsanalysis on the mammary fad pads derived from Cav-1 (-/-) null miceto validate the existence of the "Reverse Warburg Effect". However,what was observed was far more complex and extensive, and wascharacteristic of a profound catabolic phenotype. The results areconsistent with oxidative stress, mitochondrial dysfunction, andautophagy/mitophagy--which would also induce aerobic glycolysis inthe tumor stroma (the "Reverse Warburg Effect").

[0007] Interestingly, autophagy, mitophagy, and aerobic glycolysisare all induced by oxidative stress and are all controlled by thesame key transcription factor, namely hypoxia-induciblefactor-1-alpha (HIF1-alpha). In this regard, the inventors directlyshow that a loss of stromal Cav-1 leads to the up-regulation ofmiR-31, which is a known activator of HIF1alpha transcriptionalactivity (Liu C J, Tsai M M, Hung P S, Kao S Y, Liu T Y, Wu K J,Chiou S H, Lin S C, Chang K W. miR-31 ablates expression of the HIFregulatory factor FIH to activate the HIF pathway in head and neckcarcinoma. Cancer Res 2010; 70:1635-44.). Thus, the lethality of aCav-1-deficient tumor microenvironment could be explained by anautophagic/catabolic tumor stroma, which would then provide bothnutrients and energy to epithelial cancer cells in a paracrinefashion. This is the "autophagic tumor stroma model of cancer".This represents a unique therapeutic opportunity, as blockingautophagy in the tumor stroma should halt cancer growth, while aninduction of autophagy in the epithelial cancer cells should havethe same effect, thereby halting tumor growth. This new model ofcompartmentalized autophagy clarifies and explains thecontroversial role of autophagy in tumor pathogenesis andfacilitate the design of novel anti-cancer therapies.

[0008] Based on these and other supporting findings, this is a newmodel for understanding the Warburg effect in tumor metabolism. Inthis model, epithelial cancer cells induce aerobic glycolysis inadjacent cancer-associated fibroblasts, directing them to produceenergy-rich metabolites (such as lactate and 3-hydroxy-butryate).Then, these metabolites would be transferred to the epithelialcancer cells, where they can then enter the mitochondrial TCAcycle, undergo oxidative phosphorylation, resulting high ATPproduction. The inventors have termed this new model "The ReverseWarburg Effect" Pavlides S, Whitaker-Menezes D, Castello-Cros R,Flomenberg N, Witkiewicz A K, Frank P G, Casimiro M C, Wang C,Fortina P, Addya S, Pestell R G, Martinez-Outschoorn U E, Sotgia F,Lisanti M P. The reverse Warburg effect: aerobic glycolysis incancer associated fibroblasts and the tumor stroma. Cell Cycle2009; 8:3984-4001; Pavlides S, Tsirigos A, Vera I, Flomenberg N,Frank P G, Casimiro M C, Wang C, Pestell R G, Martinez-Outschoorn UE, Howell A, Sotgia F, Lisanti M P. Transcriptional evidence forthe "Reverse Warburg Effect" in human breast cancer tumor stromaand metastasis: similarities with oxidative stress, inflammation,Alzheimer's disease, and "Neuron-Glia Metabolic Coupling". Aging(Albany N.Y.) 2010; 2:185-99.

[0009] In direct support of these findings, it was recently shownusing a co-culture model, that MCF7 epithelial cancer cells havethe ability to down-regulate both Cav-1 expression and mitochondriain adjacent fibroblasts via the induction of autophagy/mitophagy.This then drives aerobic glycolysis in the fibroblast compartment.More specifically, MCF7 cells induce oxidative stress in adjacentfibroblasts. Oxidative stress is then sufficient to drive theinduction of autophagy/mitophagy in fibroblasts, leading to Cav-1lysosomal degradation and aerobic glycolysis. Conversely, duringco-culture, it was observed that MCF7 epithelial cancer cellsdramatically increase their mitochondrial mass and mitochondrialactivity 11. Moreover, it was possible to pheno-copy these effectsby simply adding L-lactate (an end product of glycolysis) to thetissue culture media of MCF7 cells. Under these conditions,L-lactate treatment was sufficient to dramatically increasemitochondrial mass in MCF7 cancer cells. Martinez-Outschoorn U E,Balliet R, Rivadeneira D, Chiavarina B, Pavlides S, Wang C,Whitaker-Menezes D, Daumer K M, Lin Z, Witkiewicz A K, FlomenbergN, Howell A, Pestell R G, Knudsen E, Sotgia F, Lisanti M P.Oxidative stress in cancer fibroblasts drives tumor-stromaco-evolution: A new paradigm for understanding tumor metabolism,the field effect and genomic instability in cancer cells. CellCycle 2010; 9: In Press; Martinez-Outschoom U E, Pavlides S,Whitaker-Menezes D. Daumer K M, Milliman J N, Chiavarina B, MignecoG, Witkiewicz A K, Martinez-Cantarin M P. Flomenberg N, Howell A,Pestell R G, Lisanti M P, Sotgia F. Tumor cells induce the cancerassociated fibroblast phenotype via caveolin-1 degradation:Implications for breast cancer and DCIS therapy with autophagyinhibitors. Cell Cycle 2010; 9: In Press.

[0010] Based on the above biomarker and mechanistic experiments,the Cav-1 (-/-) mammary fat pad can be used as a pre-clinical modelof a "lethal" tumor microenvironment. Pavlides S, Tsirigos A, VeraI, Flomenberg N, Frank P G, Casimiro M C, Wang C, Fortina P, AddyaS, Pestell R G, Rigoutsos I, Martinez-Outschoom U E, Sotgia F,Lisanti M P. Loss of Stromal Caveolin-1 Leads to Oxidative Stress,Mimics Hypoxia, and Drives Inflammation in the TumorMicroenvironment, Conferring the "Reverse Warburg Effect": ATranscriptional Informatics Analysis with Validation. Cell Cycle2010; In Press. With this in mind, Cav-1 (-/-) null mammary fatpads were subjected to an unbiased metabolomics analysis. Theresults obtained provided independent validation for the idea thata loss of stromal Cav-1 induces oxidative stress, which in turnactivates autophagy/mitophagy, leading to aerobic glycolysis.Importantly, 3-hydroxy-butyrate (a ketone body) is one of the keymetabolites that was most significantly elevated, over 4-fold.3-Hydroxy-butyrate is another metabolic end-product of glycolysis(which can be derived from pyruvate) that accumulates duringstarvation, and mitochondrial dysfunction, and is elevated indiabetic patients. Pavlides S, Tsirigos A, Migneco G,Whitaker-Menezes D, Chiavarina B, Flomenberg N, Frank P G, CasimiroM C, Wang C, Pestell R G, Martinez-Outschoorn U E, Howell A, SotgiaF, Lisanti M P. The Autophagic Tumor Stroma Model of Cancer: Roleof Oxidative Stress and Ketone Production in Fueling Tumor CellMetabolism. Cell Cycle 2010: Submitted.

[0011] There are several important parallels between3-hydroxy-butyrate and L-lactate. Both can be considered metabolicend-products of glycolysis, derived from pyruvate.3-hydroxy-butyrate and L-lactate are both secreted and take up bythe same monocarboxylate transporters (MCTs). After uptake by MCTs,they can both re-enter the TCA cycle as acetyl-CoA and undergooxidative metabolism, resulting the production of high levels ofATP. Thus, based on these findings, both ketones and lactate(produced via aerobic glycolysis in fibroblasts) could fuel tumorgrowth and metastasis in epithelial cancer cells.

[0012] Here, a xenograft model of human breast cancer was used toassess the possible tumor promoting properties of the end-productsof aerobic glycolysis, namely ketones and lactate. For thispurpose, MDA-MB-231 human breast cancer cells, which show a markerprofile most consistent with triple negative and basal-like breastcancers. MDA-MB-231 cells were grown in athymic nude mice as solidtumors via flank injections, or were induced to undergo lungmetastasis via tail vein injections. Then, 3-hydroxy-butyrate orL-lactate was systemically administered via intra-peritoneal (i.p.)injections. Our results clearly show that 3-hydroxy-butyrate orL-lactate "fuel" tumor growth and metastasis, without a measurableincrease in tumor angiogenesis. Thus, our results providemetabolic/functional evidence to directly support "The ReverseWarburg Effect". Via an informatics analysis of the existing rawtranscriptional profiles of epithelial cancer cells and adjacentstromal cells, the inventors also provide evidence for theupregulation of oxidative phosphorylation, the TCA cycle, andmitochondrial metabolism in human breast cancer cells in vivo.Casey T, Bond J, Tighe S, Hunter T, Lintault L, Patel O, Eneman J,Crocker A, White J, Tessitore J, Stanley M, Harlow S. Weaver D,Muss H, Plaut K. Molecular signatures suggest a major role forstromal cells in development of invasive breast cancer. BreastCancer Res Treat 2009; 114:47-62.

[0013] All references cited herein are incorporated herein byreference in their entireties.

BRIEF SUMMARY OF THE INVENTION

[0014] The invention provides a method for making a prognosis ofdisease course in a human neoplastic disease patient, the methodcomprising the steps of (a) obtaining a sample of stromal cellsadjacent to a neoplasm, stromal cells within a neoplasm, and/or atotal tumor extract; (b) determining the level of protein and/ormRNA expression of a biomarker selected from the group consistingof ACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L,and combinations thereof, in the sample; wherein said prognosis ismade when the level of biomarker in the sample is higher than thelevel of biomarker in a control. The invention further provides themethod wherein the human neoplastic disease patient has a neoplasmselected from the group consisting of breast, skin, kidney, lung,pancreas, rectum and colon, prostate, bladder, epithelial,non-epithelial, lymphomas, sarcomas, melanomas, and the like. Theinvention further provides the method wherein the human neoplasticdisease patient has a breast neoplasm subtype selected from thegroup consisting of ER(+), PR(+), HER2(+), triple-negative(ER(-)/PR(-)/HER2(-)), ER(-), PR(-), all tumor and nodal stages,and all tumor grades. The invention further provides a methodwherein the level of biomarker expression is determined byimmunohistochemical staining. The invention further provides amethod wherein the level of biomarker expression is determined byan assay selected from the group consisting of RT-PCR, QRT-PCR,rolling circle amplification and nucleic acid sequenced-basedamplification assays. The invention further provides a methodwherein the prognosis of disease course includes a risk formetastasis, recurrence and relapse of neoplastic disease. Theinvention further provides a method wherein increase of biomarkerpredicts early disease recurrence, metastasis, survival, andtamoxifen-resistance at diagnosis. The invention further provides amethod wherein increase of biomarker in the sample predicts theprognosis of lymph-node positive (LN(+)) patients. The inventionfurther provides a method wherein increase of biomarker in thesample is associated with a poor prognosis. The invention furtherprovides a method wherein the up-regulation or presence ofbiomarker in the sample is associated with a bad prognosis. Theinvention further provides a method wherein the neoplasm is apre-malignant lesions selected from the group consisting of ductalcarcinoma in situ (DCIS) of the breast and myelodysplastic syndromeof the bone marrow. The invention further provides a method whereinthe prognosis of disease course includes staging malignant diseasein a human neoplastic disease patient. The invention furtherprovides a method wherein an increase of biomarker in the sample isa surrogate marker for stromal RB tumor suppressor functionalinactivation by RB hyper-phosphorylation.

[0015] The invention provides a method for determining thelikelihood that a carcinoma is of a grade likely to become aninvasive carcinoma comprising: (a) obtaining a sample of stromalcells adjacent to a neoplasm, stromal cells within a neoplasm,and/or a total tumor extract from a neoplastic disease patient; (b)determining the labeling level of protein and/or mRNA expression ofa biomarker selected from the group consisting of ACLY, HMGCS1,HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, and combinationsthereof, in the sample; and (c) correlating the amount of labelingsignal in the test sample with a control, wherein the carcinoma isof a grade likely to become invasive when the level of biomarker inthe sample is higher than the level of biomarker in a control. Theinvention further provides a method wherein the carcinoma is acarcinoma of the breast. The invention further provides a methodwherein the carcinoma is selected from the group consisting ofcarcinoma of the breast, skin, kidney, parotid gland, lung, bladderand prostate. The invention further provides a method wherein thedetection reagent is labeled antibody capable of binding to humanACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, and/orBNIP3L. The invention further provides a method wherein thedetection reagent is labeled nucleic acid capable of binding tohuman ACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3,and/or BNIP3L. The invention further provides a method wherein theamount of labeling signal is measured by a technique selected fromthe group consisting of emulsion autoradiography, phosphorimaging,light microscopy, confocal microscopy, multi-photon microscopy, andfluorescence microscopy. The invention further provides a methodwherein the amount of labeling signal is measured byautoradiography and a higher signal intensity in a test samplecompared to a control prepared using the same steps as the testsample is used to diagnose a high grade carcinoma possessing a highprobability the carcinoma will progress to an invasive carcinoma.The invention further provides a method wherein the amount oflabeling signal is measured by a technique selected from the groupconsisting of is an assay selected from the group consisting ofRT-PCR, QRT-PCR, rolling circle amplification and nucleic acidsequenced-based amplification assays.

[0016] The invention provides a kit for making a prognosis ofdisease course in a human neoplastic disease patient, comprising:(a) at least one label that labels a biomarker selected from thegroup consisting of ACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1,BDH2, BNIP3, BNIP3L, and combinations thereof; and (b) a usageinstruction for performing a screening of a sample of said subjectwith said label such as that an amount of biomarker present in thesample is determined. The invention further provides a kit whereinthe subject is a mammal. The invention further provides a kitwherein the subject is a human. The invention further provides akit wherein the label comprises an antibody that specifically bindsto biomarker. The invention further provides a kit wherein theantibody is a monoclonal antibody. The invention further provides akit wherein the antibody is a polyclonal antibody. The inventionfurther provides a kit further comprising a multiwell plate.

[0017] The invention provides a kit for making a prognosis ofdisease course in a human neoplastic disease patient, comprising:(a) at least one label that labels the protein expression of aprotein selected from the group consisting of ACLY, HMGCS1, HMGCS2,HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, and combinationsthereof; and (b) a usage instruction for performing a screening ofa sample of said subject with said label such as that an amount ofthe protein expression of the protein. The invention furtherprovides a kit wherein the subject is a mammal. The inventionfurther provides a kit wherein the subject is a human. Theinvention further provides a kit wherein the label comprises anantibody that specifically binds to the protein. The inventionfurther provides a kit wherein the antibody is a monoclonalantibody. The invention further provides a kit wherein the antibodyis a polyclonal antibody. The invention further provides a kitfurther comprising a multiwell plate.

[0018] The invention provides a kit for making a prognosis ofdisease course in a human neoplastic disease patient, comprising:(a) a collection of isolated polynucleotides which bind selectivelyto the RNA products of biomarkers, wherein the biomarkers areselected from the group of genes consisting of ACLY, HMGCS1,HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, and combinationsthereof; (b) a usage instruction for performing a screening of asample of said patient with said label such as that an amount ofthe mRNA expression of biomarker present in the sample isdetermined. The invention further provides a kit wherein thescreening is a nucleic acid amplification assay selected from thegroup consisting of RT-PCR, QRT-PCR, rolling circle amplificationand nucleic acid sequenced-based amplification assays. Theinvention further provides a kit wherein the subject is a mammal.The invention further provides a kit wherein the subject is ahuman. The invention further provides a kit wherein the labelcomprises a nucleic acid that specifically binds to biomarker. Theinvention further provides a kit further comprising a multiwellplate.

[0019] The invention provides a method of predicting response toanti-neoplasm therapy or predicting disease progression neoplasticdisease, the method comprising: (a) obtaining a sample of stromalcells adjacent to a neoplasm, stromal cells within a neoplasm,and/or a total tumor extract from a neoplastic disease patient; (b)determining the labeling level of protein and/or mRNA expression ofa biomarker selected from the group consisting of ACLY, HMGCS1,HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, and combinationsthereof, in the sample and comparing the labeling level ofbiomarker in the sample with the labeling level of biomarker in acontrol; (c) analyzing the obtained neoplasm test sample forpresence or amount of one or more molecular markers of hormonereceptor status, one or more growth factor receptor markers, andone or more tumor suppression/apoptosis molecular markers; (d)analyzing one or more additional molecular markers both proteomicand non-proteomic that are indicative of cancer disease processesselected from the group consisting of angiogenesis, apoptosis,catenin/cadherin proliferation/differentiation, cell cycleprocesses, cell surface processes, cell-cell interaction, cellmigration, centrosomal processes, cellular adhesion, cellularproliferation, cellular metastasis, invasion, cytoskeletalprocesses, ERBB2 interactions, estrogen co-receptors, growthfactors and receptors, membrane/integrin/signal transduction,metastasis, oncogenes, proliferation, proliferation oncogenes,signal transduction, surface antigens and transcription factormolecular markers; and then correlating (b) the presence or amountof biomarker, with (d) clinicopathological data from said tissuesample other than the molecular markers of cancer diseaseprocesses, in order to ascertain a probability of response totherapy or future risk of disease progression in cancer for thesubject. The invention further provides a method wherein the humanneoplastic disease patient has a breast neoplasm subtype selectedfrom the group consisting of ER(+), PR(+), HER2(+), triple-negative(ER(-)/PR(-)/HER2(-)), ER(-), PR(-), all tumor and nodal stages,and all tumor grades. The invention further provides a methodwherein the human neoplastic disease patient has a neoplasmselected from the group consisting of breast, skin, kidney, lung,pancreas, rectum and colon, prostate, bladder, epithelial,non-epithelial, lymphomas, sarcomas, melanomas, and the like. Theinvention further provides a method wherein the neoplasm is apre-malignant lesions selected from the group consisting of ductalcarcinoma in situ (DCIS) of the breast and myelodysplastic syndromeof the bone marrow. The invention further provides a method whereinthe correlating to ascertain a probability of response to aspecific anti-neoplasm therapy drawn from the group consisting oftamoxifen, anastrozole, letrozole or exemestane. The inventionfurther provides a method wherein the one or more additionalmarkers includes, in addition to markers ER, PR, and/or HER-2. Theinvention further provides a method wherein the one or moreadditional markers includes, in addition to markers ER, PR, and/orHER-2. The invention further provides a method wherein the neoplasmis breast cancer. The invention further provides a method whereinthe analyzing is of both proteomic and clinicopathological markers;and wherein the correlating is further so as to a clinicaldetection of disease, disease diagnosis, disease prognosis, ortreatment outcome or a combination of any two, three or four ofthese actions. The invention further provides a method wherein theobtaining of the test sample from the subject is of a test sampleselected from the group consisting of fixed, paraffin-embeddedtissue, breast cancer tissue biopsy, tissue microarray, freshneoplasm tissue, fine needle aspirates, peritoneal fluid, ductallavage and pleural fluid or a derivative thereof. The inventionfurther provides a method wherein the molecular markers of estrogenreceptor status are ER and PGR, the molecular markers of growthfactor receptors are ERBB2, and the tumor suppression molecularmarkers are TP-53 and BCL-2; wherein the additional one or moremolecular marker(s) is selected from the group consisting ofessentially: ER, PR, HER-2, MKI67, KRT5/6, MSN, C-MYC, CAV1,CTNNB1, CDH1, MME, AURKA, P-27, GATA3, HER4, VEGF, CTNNA1, and/orCCNE; wherein the clinicopathological data is one or more datumvalues selected from the group consisting essentially of: tumorsize, nodal status, and grade; wherein the correlating is by usageof a trained kernel partial least squares algorithm; and theprediction is of outcome of anti-neoplasm therapy for breastcancer.

[0020] The invention provides a kit comprising: a panel ofantibodies comprising: at least one antibody or binding fragmentthereof specific for a biomarker selected from the group consistingof ACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L,and combinations thereof, whose binding with stromal cells adjacentto a neoplasm and/or total tumor extract has been correlated withbreast cancer treatment outcome or patient prognosis; at least oneadditional antibody or binding fragment thereof specific for aprotein whose expression is correlated with breast cancer treatmentoutcome or patient prognosis, reagents to perform a binding assay;a computer algorithm, residing on a computer, operating, inconsideration of all antibodies of the panel historically analyzedto bind to samples, to interpolate, from the aggregation of allspecific antibodies of the panel found bound to the stromal cellsadjacent to a neoplasm sample, a prediction of treatment outcomefor a specific treatment for breast cancer or a future risk ofbreast cancer progression for the subject. The invention furtherprovides a kit wherein the anti-biomarker antibody comprises: apoly- or monoclonal antibody specific for biomarker protein orprotein fragment thereof correlated with breast cancer treatmentoutcome or patient prognosis. The invention further provides a kitwherein the panel of antibodies further comprises: a number ofimmunohistochemistry assays equal to the number of antibodieswithin the panel of antibodies. The invention further provides akit wherein the antibodies of the panel of antibodies furthercomprise: antibodies specific to ER, PR, and/or HER-2. Theinvention further provides a kit wherein the treatment outcomepredicted comprises the response to anti-neoplastic therapy orchemotherapy. The invention further provides a kit furthercomprising a multiwell plate.

[0021] The invention provides a method for making a prognosis ofdisease course in a human patient by detecting differentialexpression of at least one marker in ductal carcinoma in situ(DCIS) pre-invasive cancerous breast tissue, said method comprisingthe steps of: (a) obtaining a sample of DCIS breast tissue andstromal cells adjacent to a neoplasm, stromal cells within aneoplasm, and/or a total tumor extract from a human neoplasticdisease patient; (b) determining the level of protein and/or mRNAexpression of a biomarker selected from the group consisting ofACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L,and combinations thereof, in the stromal cells of the sample as theat least one marker and comparing the level of biomarker in thestromal cells of the sample with the level of biomarker in acontrol; wherein said prognosis of further progression is made whenthe level of biomarker in the stromal cells of the sample is higherthan the level of biomarker in the control. The invention furtherprovides a method wherein the size of said abnormal tissue samplesubstantially conforms to an isolatable tissue structure whereinonly cells exhibiting abnormal cytological or histologicalcharacteristics are collected. The invention further provides amethod further comprising confirming said differential expressionof said marker in said normal tissue sample and in said abnormaltissue sample by using an immunological technique. The inventionfurther provides a method wherein said immunological technique isselected from the group consisting of radioimmunoassay (RIA), EIA,ELISA, and immunofluorescence assays. The invention furtherprovides a method wherein said immunological technique is selectedfrom the group consisting of is determined by an assay selectedfrom the group consisting of RT-PCR. QRT-PCR, rolling circleamplification and nucleic acid sequenced-based amplificationassays. The invention further provides a method wherein saidabnormal breast tissue cells are non-comedo ductal carcinoma insitu cells. The invention further provides a method wherein theamount of labeling signal is measured by a technique selected fromthe group consisting of is an assay selected from the groupconsisting of RT-PCR. QRT-PCR, rolling circle amplification andnucleic acid sequenced-based amplification assays.

[0022] The invention provides a method for making a prognosis ofdisease course in a human neoplastic disease patient, the methodcomprising the steps of: (a) obtaining a sample of stromal cellsadjacent to a neoplasm, stromal cells within a neoplasm, and/or atotal tumor extract; (b) determining the level of one or more RNAtranscripts expressed in the sample obtained from said patient,wherein said one or more RNA transcripts corresponds to a biomarkerselected from the group consisting of miR-31, miR-34c, andcombinations thereof; and (c) comparing the level of each of saidone or more RNA transcripts in said sample according to step (a)with the level of each of said one or more RNA transcripts in acontrol sample; (d) comparing the level of each of said one or moreRNA transcripts in said sample according to step (a) with the levelof each of said one or more RNA transcripts in a control sample;wherein said prognosis is made when the level of one or more RNAtranscripts in the stromal cells of the sample is higher than thelevel of one or more RNA transcripts in a control.

[0023] The invention provides a method for making a prognosis ofdisease course in a human neoplastic disease patient, the methodcomprising the steps of: (a) obtaining a sample of stromal cellsadjacent to a neoplasm, stromal cells within a neoplasm, and/or atotal tumor extract; (b) determining the level of a biomarkerselected from the group consisting of ADMA, 3-hydroxybutyrate, andcombinations thereof, in the sample; wherein said prognosis is madewhen the level of biomarker in the sample is higher than the levelof biomarker in a control. The invention further provides a methodwherein the human neoplastic disease patient has a neoplasmselected from the group consisting of breast, skin, kidney, lung,pancreas, rectum and colon, prostate, bladder, epithelial,non-epithelial, lymphomas, sarcomas, melanomas, and the like. Theinvention further provides a method wherein the human neoplasticdisease patient has a breast neoplasm subtype selected from thegroup consisting of ER(+), PR(+), HER2(+), triple-negative(ER(-)/PR(-)/HER2(-)), ER(-), PR(-), all tumor and nodal stages,and all tumor grades.

[0024] The invention provides a method for making a prognosis ofdisease course in a human neoplastic disease patient, the methodcomprising the steps of: (a) obtaining a sample of stromal cellsadjacent to a neoplasm, stromal cells within a neoplasm, and/or atotal tumor extract; (b) determining the level of protein and/ormRNA expression of a biomarker selected from the group consistingof ACAT1, ACAT2, OXCT1, OXCT2, and combinations thereof, in thesample; wherein said prognosis is made when the level of biomarkerin the sample is higher than the level of biomarker in a control.The invention further provides a method wherein the humanneoplastic disease patient has a neoplasm selected from the groupconsisting of breast, skin, kidney, lung, pancreas, rectum andcolon, prostate, bladder, epithelial, non-epithelial, lymphomas,sarcomas, melanomas, and the like. The invention further provides amethod wherein the human neoplastic disease patient has a breastneoplasm subtype selected from the group consisting of ER(+),PR(+), HER2(+), triple-negative (ER(-)/PR(-)/HER2(-)), ER(-),PR(-), all tumor and nodal stages, and all tumor grades. Theinvention further provides a method wherein the level of biomarkeris determined by immunohistochemical staining. The inventionfurther provides a method wherein the level of biomarker isdetermined by an assay selected from the group consisting ofRT-PCR, QRT-PCR, rolling circle amplification and nucleic acidsequenced-based amplification assays. The invention furtherprovides a method wherein the prognosis of disease course includesa risk for metastasis, recurrence and relapse of neoplasticdisease. The invention further provides a method wherein increaseof biomarker predicts early disease recurrence, metastasis,survival, and tamoxifen-resistance at diagnosis. The inventionfurther provides a method wherein increase of biomarker predictsthe prognosis of lymph-node positive (LN(+)) patients. Theinvention further provides a method wherein increase of biomarkeris associated with a poor prognosis. The invention further providesa method wherein the up-regulation or presence of biomarker isassociated with a bad prognosis. The invention further provides amethod wherein the neoplasm is a pre-malignant lesions selectedfrom the group consisting of ductal carcinoma in situ (DCIS) of thebreast and myelodysplastic syndrome of the bone marrow. Theinvention further provides a method wherein the prognosis ofdisease course includes staging malignant disease in a humanneoplastic disease patient. The invention further provides a methodwherein increase of biomarker is a surrogate marker for stromal RBtumor suppressor functional inactivation by RBhyper-phosphorylation.

[0025] The invention provides a method for making a prognosis ofdisease course in a human neoplastic disease patient, the methodcomprising the steps of: (a) obtaining a sample of a body fluidfrom the patient; (b) determining the level of a biomarker selectedfrom the group consisting of ACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1,BDH1, BDH2, BNIP3, BNIP3L, miR-31, miR-34c, ACAT1, ACAT2, OXCT1,OXCT2, ADMA, 3-hydroxybutyrate, and combinations thereof, in thesample; wherein said prognosis is made when the level of biomarkerin sample is higher than the level of biomarker in a control. Theinvention further provides a method wherein the human neoplasticdisease patient has a neoplasm selected from the group consistingof breast, skin, kidney, lung, pancreas, rectum and colon,prostate, bladder, epithelial, non-epithelial, lymphomas, sarcomas,melanomas, and the like. The invention further provides a methodwherein the human neoplastic disease patient has a breast neoplasmsubtype selected from the group consisting of ER(+), PR(+),HER2(+), triple-negative (ER(-)/PR(-)/HER2(-)), ER(-), PR(-), alltumor and nodal stages, and all tumor grades. The invention furtherprovides a method wherein the level of biomarker is determined byimmunohistochemical staining. The invention further provides amethod wherein the level of biomarker is determined by an assayselected from the group consisting of RT-PCR, QRT-PCR, rollingcircle amplification and nucleic acid sequenced-based amplificationassays. The invention further provides a method wherein the levelof biomarker is determined by enzymatic assay. The inventionfurther provides a method wherein the prognosis of disease courseincludes a risk for metastasis, recurrence and relapse ofneoplastic disease. The invention further provides a method whereinincrease of biomarker predicts early disease recurrence,metastasis, survival, and tamoxifen-resistance at diagnosis. Theinvention further provides a method wherein increase of biomarkerin the sample predicts the prognosis of lymph-node positive (LN(+))patients. The invention further provides a method wherein increaseof biomarker in the sample is associated with a poor prognosis. Theinvention further provides a method wherein the up-regulation orpresence of biomarker in the sample is associated with a badprognosis. The invention further provides a method wherein theneoplasm is a pre-malignant lesions selected from the groupconsisting of ductal carcinoma in situ (DCIS) of the breast andmyelodysplastic syndrome of the bone marrow. The invention furtherprovides a method wherein the prognosis of disease course includesstaging malignant disease in a human neoplastic disease patient.The invention further provides a method wherein increase ofbiomarker in the sample is a surrogate marker for stromal RB tumorsuppressor functional inactivation by RB hyper-phosphorylation. Theinvention further provides a method wherein the body fluid isselected from the group consisting of plasma, serum, blood,lymphatic fluid, cerebrospinal fluid, synovial fluid, urine,saliva, mucous, phlegm, sputum, and combinations thereof.

[0026] The invention provides a method for treating neoplasticdisease in a patient, comprising the steps of: (a) obtaining asample of stromal cells adjacent to a neoplasm, stromal cellswithin a neoplasm, and/or a total tumor extract from the neoplasticdisease patient; (b) determining the level of a biomarker selectedfrom the group consisting of ACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1,BDH1, BDH2, BNIP3, BNIP3L, miR-31, miR-34c, ACAT1, ACAT2, OXCT1,OXCT2, ADMA, 3-hydroxybutyrate, and combinations thereof, in thesample and comparing the level of biomarker in the sample with thelevel of biomarker in a control; (c) predicting if the neoplasmwill respond effectively to treatment with an anti-angiogenicagent, wherein said prediction is made when the level of biomarkerin the sample is higher than the level of biomarker in the control;and administering to said patient a therapeutically effectiveamount of an anti-angiogenic agent. The invention further providesa method wherein the anti-angiogenic agent comprises an agentselected from the group consisting of angiostatin, bevacizumab,arresten, canstatin, combretastatin, endostatin, NM-3,thrombospondin, tumstatin, 2-methoxyestradiol, Vitaxin, Getfitinib,ZD6474, erlotinib, CI1033, PKI1666, cetuximab, PTK787, SU6668,SU11248, trastuzumab, Marimastat, COL-3, Neovastat, 2-ME, SU6668,anti-VEGF antibody, Medi-522 (Vitaxin II), tumstatin, arrestin,recombinant EPO, troponin I, EMD121974, IFN, celecoxib, PD0332991,and thalidomide. The invention further provides a method whereinone or more additional anti-neoplastic agents are co-administeredsimultaneously or sequentially with the anti-angiogenic agent. Theinvention further provides a method wherein the at least one ormore additional anti-neoplastic agent comprises a proteasomeinhibitor. The invention further provides a method wherein theproteasome inhibitor is bortezomib. The invention further providesa method wherein the human neoplastic disease patient has a breastneoplasm subtype selected from the group consisting of ER(+),PR(+), HER2(+), triple-negative (ER(-)/PR(-)/HER2(-)), ER(-),PR(-), all neoplasm and nodal stages, and all neoplasm grades. Theinvention further provides a method wherein the human neoplasticdisease patient has a neoplasm selected from the group consistingof breast, skin, kidney, lung, pancreas, rectum and colon,prostate, bladder, epithelial, non-epithelial, lymphomas, sarcomas,melanomas, and the like. The invention further provides a methodwherein the neoplasm is a pre-malignant lesion selected from thegroup consisting of ductal carcinoma in situ (DCIS) of the breastand myelodysplastic syndrome of the bone marrow.

[0027] The invention provides a diagnostic kit for assaying theindividual sensitivity of target cells towards angiogenesisinhibitors, comprising: (a) at least one molecule that specificallybinds to a biomarker selected from the group consisting of ACLY,HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, miR-31,miR-34c, ACAT1, ACAT2, OXCT1, OXCT2, ADMA, 3-hydroxy butyrate, andcombinations thereof; and (b) a pharmaceutically acceptablecarrier.

[0028] The invention provides a method of predicting whether aneoplastic disease patient is afflicted with a neoplasm that willrespond effectively to treatment with an anti-angiogenic agent,comprising: (a) obtaining a sample of stromal cells adjacent to aneoplasm, stromal cells within a neoplasm, and/or a total tumorextract from the neoplastic disease patient; (b) determining thelevel of a biomarker selected from the group consisting of ACLY,HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, miR-31,miR-34c, ACAT1, ACAT2, OXCT1, OXCT2, ADMA, 3-hydroxybutyrate, andcombinations thereof, in the sample and comparing the level ofbiomarker in the sample with the level of biomarker in a control;(c) predicting if the neoplasm will respond effectively totreatment with an anti-angiogenic agent, wherein higher expressionlevels of biomarker in the sample relative to biomarker levels inthe control correlate with a neoplasm that will respond effectivelyto treatment with an anti-angiogenic agent. The invention furtherprovides a method wherein the anti-angiogenic agent comprises anagent selected from the group consisting of angiostatin,bevacizumab, arresten, canstatin, combretastatin, endostatin, NM-3,thrombospondin, tumstatin, 2-methoxyestradiol, Vitaxin, Getfitinib,ZD6474, erlotinib, CI1033, PKI1666, cetuximab, PTK787, SU6668,SU11248, trastuzumab, Marimastat, COL-3, Neovastat, 2-ME, SU6668,anti-VEGF antibody, Medi-522 (Vitaxin II), tumstatin, arrestin,recombinant EPO, troponin I, EMD121974, IFN, celecoxib, PD0332991,and thalidomide.

[0029] The invention provides a method of predicting thesensitivity of neoplasm cell growth to inhibition by ananti-neoplastic agent, comprising: (a) obtaining a sample ofstromal cells adjacent to a neoplasm, stromal cells within aneoplasm, and/or a total tumor extract from a neoplastic diseasepatient; (b) determining a level of a biomarker selected from thegroup consisting of ACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1,BDH2, BNIP3, BNIP3L, miR-31, miR-34c, ACAT1, ACAT2, OXCT1, OXCT2,ADMA, 3-hydroxybutyrate, and combinations thereof, in the sampleand comparing the level of biomarker in the sample with the levelof biomarker in a control; and (c) predicting the sensitivity ofneoplasm cell growth to inhibition by an anti-neoplastic agent,wherein higher levels of the biomarker compared the level ofbiomarker in a control correlates with high sensitivity toinhibition by anti-neoplastic agent. The invention further providesa method wherein the anti-angiogenic agent comprises an agentselected from the group consisting of angiostatin, bevacizumab,arresten, canstatin, combretastatin, endostatin, NM-3,thrombospondin, tumstatin, 2-methoxyestradiol, Vitaxin, Getfitinib,ZD6474, erlotinib, CI183J, 033, PKI1666, cetuximab, PTK787, SU6668,SU11248, trastuzumab, Marimastat, COL-3, Neovastat, 2-ME, SU6668,anti-VEGF antibody, Medi-522 (Vitaxin II), tumstatin, arrestin,recombinant EPO, troponin I, EMD121974, IFN, celecoxib, PD0332991,and thalidomide. The invention further provides a method whereinthe angiogenesis inhibitor is selected from the group consisting ofangiostatin, bevacizumab, arresten, canstatin, combretastatin,endostatin, NM-3, thrombospondin, tumstatin, 2-methoxyestradiol,Vitaxin, Getfitinib, ZD6474, erlotinib, CI1033, PKI1666, cetuximab,PTK787, SU6668, SU11248, trastuzumab, Marimastat, COL-3, Neovastat,2-ME, SU6668, anti-VEGF antibody, Medi-522 (Vitaxin II), tumstatin,arrestin, recombinant EPO, troponin I, EMD121974, IFN, celecoxib,PD0332991, and thalidomide. The invention further provides adiagnostic kit wherein the target cell is a cancer cell.

[0030] The invention provides a kit for determining targetneoplastic cells susceptible to anti-angiogenesis inhibitortreatment, comprising: (a) at least one antibody which specificallybinds a biomarker selected from the group consisting of ACLY,HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, miR-31,miR-34c, ACAT1, ACAT2, OXCT1, OXCT2, ADMA, 3-hydroxybutyrate, andcombinations thereof; and (b) a pharmaceutically acceptablecarrier. The invention further provides a diagnostic kit whereinthe antibody is a polyclonal antibody. The invention furtherprovides a diagnostic kit wherein the antibody is a monoclonalantibody.

[0031] The invention provides a kit for determining targetneoplastic cells susceptible to anti-angiogenesis inhibitortreatment, comprising: (a) a collection of isolated polynucleotideswhich bind selectively to the RNA products of biomarkers, whereinthe biomarkers are selected from the group of genes consisting ofACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L,miR-31, miR-34c, ACAT1, ACAT2, OXCT1, OXCT2, ADMA,3-hydroxybutyrate, and combinations thereof; (b) a usageinstruction for performing a screening of a sample of said patientwith said label such as that an amount of the mRNA expression of abiomarker present in the sample is determined. The inventionfurther provides a kit wherein the screening is a nucleic acidamplification assay selected from the group consisting of RT-PCR,QRT-PCR, rolling circle amplification and nucleic acidsequenced-based amplification assays. The invention furtherprovides a kit wherein the subject is a mammal. The inventionfurther provides a kit wherein the subject is a human. Theinvention further provides a kit wherein the label comprises annucleic acid that specifically binds to a biomarker selected fromthe group consisting of ACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1,BDH11, BDH2, BNIP3, BNIP3L, miR-31, miR-34c, ACAT1, ACAT2, OXCT1,OXCT2, ADMA, and 3-hydroxybutyrate.

[0032] The invention provides a method for treating neoplasticdisease in a patient, comprising the steps of: (a) obtaining asample of stromal cells adjacent to a neoplasm, stromal cellswithin a neoplasm, and/or a total tumor extract a neoplasm from thepatient and/or total tumor extract; (b) determining the level of abiomarker selected from the group consisting of ACLY, HMGCS1,HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, miR-31, miR-34c,ACAT1, ACAT2, OXCT1, OXCT2, ADMA, 3-hydroxybutyrate, andcombinations thereof, in the sample and comparing the level ofbiomarker in the sample with the level of biomarker in a control;(c) predicting if the neoplasm will respond effectively totreatment with a lactate transporter inhibitor, wherein higherexpression levels of the biomarker compared the level of biomarkerin a control correlates with high sensitivity to treatment with alactate transporter inhibitor; and (d) administering to saidpatient a therapeutically effective amount of a lactate transporterinhibitor. The invention further provides a method wherein thelactate transporter inhibitor comprises an agent which inhibits anenzyme selected from the group consisting of triose-phosphateisomerase, fructose 1,6 bisphosphate aldolase, glycero-3-phosphatedehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase,enolase, pyruvate kinase, lactate dehydrogenase. The inventionfurther provides a method wherein one or more additionalanti-neoplastic agents are co-administered simultaneously orsequentially with the lactate transporter inhibitor. The inventionfurther provides a method wherein the human neoplastic diseasepatient has a breast neoplasm subtype selected from the groupconsisting of ER(+), PR(+), HER2(+), triple-negative(ER(-)/PR(-)/HER2(-)), ER(-), PR(-), all neoplasm and nodal stages,and all neoplasm grades. The invention further provides a methodwherein the human neoplastic disease patient has a neoplasmselected from the group consisting of breast, skin, kidney, lung,pancreas, rectum and colon, prostate, bladder, epithelial,non-epithelial, lymphomas, sarcomas, melanomas, and the like. Theinvention further provides a method wherein the neoplasm is apre-malignant lesion selected from the group consisting of ductalcarcinoma in situ (DCIS) of the breast and myelodysplastic syndromeof the bone marrow.

[0033] The invention provides a method for treating neoplasticdisease in a patient, comprising the steps of: (a) obtaining asample of stromal cells adjacent to a neoplasm, stromal cellswithin a neoplasm, and/or a total tumor extract a neoplasm from thepatient and/or total tumor extract; (b) determining the level of abiomarker selected from the group consisting of ACLY, HMGCS1,HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, miR-31, miR-34c,ACAT1, ACAT2, OXCT1, OXCT2, ADMA, 3-hydroxybutyrate, andcombinations thereof, in the sample and comparing the level ofbiomarker in the sample with the level of biomarker in a control;(c) predicting if the neoplasm will respond effectively totreatment with a monocarboxylate transporter inhibitor, whereinhigher expression levels of the biomarker compared the level ofbiomarker in a control correlates with high sensitivity totreatment with a monocarboxylate transporter inhibitor; and (d)administering to said patient a therapeutically effective amount ofa monocarboxylate transporter inhibitor. The invention furtherprovides a method wherein the monocarboxylate transporter inhibitorcomprises an agent which inhibits an enzyme selected from the groupconsisting of triose-phosphate isomerase, fructose 1,6 bisphosphatealdolase, glycero-3-phosphate dehydrogenase, phosphoglyceratekinase, phosphoglycerate mutase, enolase, pyruvate kinase, lactatedehydrogenase. The invention further provides a method wherein oneor more additional anti-neoplastic agents are co-administeredsimultaneously or sequentially with the lactate transporterinhibitor. The invention further provides a method wherein thehuman neoplastic disease patient has a breast neoplasm subtypeselected from the group consisting of ER(+), PR(+), HER2(+),triple-negative (ER(-)/PR(-)/HER2(-)), ER(-), PR(-), all neoplasmand nodal stages, and all neoplasm grades. The invention furtherprovides a method wherein the human neoplastic disease patient hasa neoplasm selected from the group consisting of breast, skin,kidney, lung, pancreas, rectum and colon, prostate, bladder,epithelial, non-epithelial, lymphomas, sarcomas, melanomas, and thelike. The invention further provides a method wherein the neoplasmis a pre-malignant lesion selected from the group consisting ofductal carcinoma in situ (DCIS) of the breast and myelodysplasticsyndrome of the bone marrow. The invention further provides amethod wherein the monocarboxylate transporter inhibitor isAR-C117977. The invention provides a method of predicting thesensitivity of neoplasm cell growth to inhibition by a lactatetransporter inhibitor, comprising: (a) obtaining a sample ofstromal cells adjacent to a neoplasm, stromal cells within aneoplasm, and/or a total tumor extract from a neoplastic diseasepatient; (b) determining the level of a biomarker selected from thegroup consisting of ACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1,BDH2, BNIP3, BNIP3L, miR-31, miR-34c, ACAT1, ACAT2, OXCT1, OXCT2,ADMA, 3-hydroxybutyrate, and combinations thereof, in the sampleand comparing the level of biomarker in the sample with the levelof biomarker in a control; and (c) predicting the sensitivity ofneoplasm cell growth to inhibition by a lactate transporterinhibitor, wherein higher expression levels of the biomarkercompared the level of biomarker in a control correlates with highsensitivity to inhibition by a lactate transporter inhibitor. Theinvention further provides a method wherein the lactate transporterinhibitor comprises an agent which inhibits an enzyme selected fromthe group consisting of triose-phosphate isomerase, fructose 1,6bisphosphate aldolase, glycero-3-phosphate dehydrogenase,phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvatekinase, lactate dehydrogenase.

[0034] The invention provides a method of screening forantineoplastic activity of a potential therapeutic agentcomprising: (a) providing a cell expressing of a biomoleculeselected from the group consisting of ACLY, HMGCS1, HMGCS2, HMGCL,HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, miR-31, miR-34c, ACAT1, ACAT2,OXCT1, OXCT2, ADMA, 3-hydroxybutyrate, combinations thereof, andfragments thereof; (b) providing a potential therapeutic agent; (c)contacting the cell with the potential therapeutic agent; and (d)monitoring an effect of the potential therapeutic agent onbioactivity and/or expression of the biomolecule in the cell. Theinvention further provides a method of screening for antineoplasticactivity further comprising: (e) comparing the level of bioactivityin the absence of said potential therapeutic agent to the level ofexpression in the presence of the potential therapeutic agent,wherein a potential therapeutic agent is identified when thebioactivity and/or expression of the biomolecule in the absence ofsaid potential therapeutic agent is different than the level ofbioactivity and/or expression in the presence of the candidatebioactive agent. The invention further provides a method ofscreening for the potential therapeutic agent wherein the potentialtherapeutic agent affects the expression of the biomoleculeselected from the group consisting of ACLY, HMGCS1, HMGCS2, HMGCL,HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, miR-31, miR-34c, ACAT1, ACAT2,OXCT1, OXCT2, ADMA, 3-hydroxybutyrate, combinations thereof, andfragments thereof. The invention further provides a method ofscreening for the potential therapeutic agent wherein the potentialtherapeutic agent affects the bioactivity of the biomoleculeselected from the group consisting of ACLY, HMGCS1, HMGCS2, HMGCL,HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, miR-31, miR-34c, ACAT1, ACAT2,OXCT1, OXCT2, ADMA, 3-hydroxybutyrate, combinations thereof, andfragments thereof.

[0035] The invention provides a method for screening for apotential therapeutic agent capable of modulating the bioactivityand/or expression of a biomolecule selected from the groupconsisting of ACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2,BNIP3, BNIP3L, miR-31, miR-34c, ACAT1, ACAT2, OXCT1, OXCT2, ADMA,3-hydroxybutyrate, and combinations thereof, said methodcomprising: a) combining said biomolecule and a candidate bioactiveagent; b) determining the effect of the candidate bioactive agenton the bioactivity and/or expression of said biomolecule; c)comparing the level of bioactivity and/or expression of biomoleculein the absence of said potential therapeutic agent to the level ofbioactivity and/or expression in the presence of the candidatebioactive agent, wherein a potential therapeutic agent isidentified when the bioactivity and/or expression of thebiomolecule in the absence of said potential therapeutic agent isdifferent than the level of bioactivity and/or expression in thepresence of the candidate bioactive agent. The invention furtherprovides a method of screening for the bioactive agent wherein thepotential therapeutic agent affects the expression of thebiomolecule selected from the group consisting of ACLY, HMGCS1,HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, miR-31, miR-34c,ACAT1, ACAT2, OXCT1, OXCT2, ADMA, 3-hydroxybutyrate, andcombinations thereof. The invention further provides a method ofscreening for the bioactive agent wherein the potential therapeuticagent affects the bioactivity of the biomolecule selected from thegroup consisting of ACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1,BDH2, BNIP3, BNIP3L, miR-31, miR-34c, ACAT1, ACAT2, OXCT1, OXCT2,ADMA, 3-hydroxybutyrate, and combinations thereof.

[0036] The invention provides a method for treating neoplasticdisease in a patient, comprising the steps of: (a) obtaining asample of stromal cells adjacent to a neoplasm, stromal cellswithin a neoplasm, and/or a total tumor extract from a neoplasticdisease patient; (b) determining the level of a biomarker selectedfrom the group consisting of ACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1,BDH1, BDH2, BNIP3, BNIP3L, miR-31, miR-34c, ACAT1, ACAT2, OXCT1,OXCT2, ADMA, 3-hydroxybutyrate, and combinations thereof, in thesample and comparing the level of biomarker in the sample with thelevel of biomarker in a control; (c) predicting if the neoplasmwill respond effectively to treatment with a therapeutic agent,wherein higher expression levels of the biomarker compared thelevel of biomarker in a control correlates with high sensitivity toinhibition by a therapeutic agent; and administering to saidpatient a therapeutically effective amount of a therapeutic agent.The invention further provides a method wherein the therapeuticagent comprises an agent selected from the group consisting of17-AAG, AR-C117977, Abraxane, albumin-bound Paclitaxel, AlbuminNanoparticle Paclitaxel, Apatinib, Ascomycin, Axitinib, Bexarotene,Bortezomib, Bosutinib, Bryostatin 1, Bryostatin 2, Canertinib,Carboplatin, Cediranib, Cisplatin, Cyclopamine, Dasatinib, 17-DMAG,Docetaxel, Doramapimod, Dovitinib, Erlotinib, Everolimus,Gefitinib, Geldanamycin, Gemeitabine, Imatinib, Imiquimod, Ingenol3-Angelate, Ingenol 3-Angelate 20-Acetate, Irinotecan, Lapatinib,Lestaurtinib, Nedaplatin, Masitinib, Mubritinib, Nilotinib,NVP-BEZ235, OSU-03012, Oxaliplatin, Paclitaxel, Pazopanib,Picoplatin, Pimecrolimus, PKC412, Rapamycin, Satraplatin,Sorafenib, Sunitinib, Tandutinib, Tivozanib, Thalidomide,Temsirolimus, Tozasertib, Vandetanib, Vargatef, Vatalanib,Zotarolimus, ZSTK474, Bevacizumab (Avasti), Cetuximab, Herceptin,Rituximab, Trastuzumab, Apatinib, Axitinib, Bisindolylmaleimide I,Bisindolylmaleimide I, Bosutinib, Canertinib, Cediranib,Chelerythrine, CP690550, Dasatinib, Dovitinib, Erlotinib, Fasudil,Gefitinib, Genistein, Go 6976, H-89, HA-1077. Imatinib, K252a,K252c, Lapatinib, Di-p-Toluenesulfonate, Lestaurtinib, LY 294002,Masitinib, Mubritinib, Nilotinib, OSU-03012, Pazopanib, PD 98059,PKC412, Roscovitine, SB 202190, SB 203580, Sorafenib, SP600125,Staurosporine, Sunitinib, Tandutinib, Tivozanib, Tozasertib,Tyrphostin AG 490, Tyrphostin AG 1478, U0126, Vandetanib, Vargatef,Vatalanib, Wortmannin, ZSTK474, Cyclopamine, Carboplatin,Cisplatin, Eptaplatin, Nedaplatin, Oxaliplatin, Picoplatin,Satraplatin, Bortezomib (Velcade), Metformin, Halofuginone,Metformin, N-acetyl-cysteine (NAC), RTA 402 (Bardoxolone methyl),Auranofin, BMS-345541, IMD-0354, PS-1145, TPCA-1, Wedelolactone,Echinomycin, 2-deoxy-D-glucose (2-DG), 2-bromo-D-glucose,2-fluoro-D-glucose, and 2-iodo-D-glucose, dichloro-acetate (DCA),3-chloro-pyruvate, 3-Bromo-pyruvate (3-BrPA),3-Bromo-2-oxopropionate, Oxamate, LY 294002, NVP-BEZ235, Rapamycin,Wortmannin, Quercetin, Resveratrol, N-acetyl-cysteine (NAC),N-acetyl-cysteine amide (NACA), Ascomycin, CP690550, Cyclosporin A,Everolimus, Fingolimod, FK-506, Mycophenolic Acid, Pimecrolimus,Rapamycin, Temsirolimus, Zotarolimus, Roscovitine, PD 0332991(CDK4/6 inhibitor), Chloroquine, BSI-201, Olaparib, DR 2313, and NU1025.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

[0037] The invention will be described in conjunction with thefollowing drawings wherein:

[0038] FIG. 1 shows Evidence for Oxidative Stress and MitochondrialDysfunction in Cav-1 (-/-) Null Mouse Tissues: ADMA and Ketones.Note that both 3-hydroxybuty, rate (BHBA) and asymmetric dimethylarginine (ADMA) are increased .about.3-4 fold in Cav-1 (-/-)mammary fat pads. Similar results were obtained with lung tissueharvested from Cav-1 (-/-) mice. Importantly, ADMA is a marker ofendothelial dysfunction and oxidative stress; it can also driveoxidative stress, as it functions as an uncoupler of NOS familymember, inhibiting the production NO and producing superoxideinstead. In addition, BHBA is a ketone body known to be a marker ofmitochondrial dysfunction. Oxidative stress induces mitochrondrialdysfunction, and visa versa, driving autophagy and mitophagy.

[0039] FIG. 2 shows Upregulation of Anti-Oxidants in Cav-1 (-/-)Mammary Fat Pads. One compensatory response to oxidative stress isthe over-production of anti-oxidants. Note that in Cav-1 (-/-)mammary fat pads an 11-fold increase in Vitamin C (ascorbic acid)and a near 3-fold increase in Vitamin E (alpha-tocopherol) wereobserved.

[0040] FIG. 3 shows Venn Diagrams for the Transcriptional OverlapBetween Autophagy and Tumor Stroma from Breast Cancer Patients.Upper panel, Overlap with tumor stroma. Note the overlap of 93genes with a p-value of 2.65.times.10-6. Middle panel, Overlap with"recurrence-prone" stroma. Note the overlap of 47 genes with ap-value of 2.22.times.10-3. Lower panel, Overlap with"metastasis-prone" stroma. Note the overlap of 17 genes with ap-value of 5.32.times.10-2.

[0041] FIG. 4 shows Venn Diagrams for the Transcriptional OverlapBetween Lysosomes and Telomere-related Genes, with Tumor Stromafrom Breast Cancer Patients. Upper panel, A, Overlap with tumorstroma. Note the overlap of 175 genes with a p-value of1.23.times.10-15. Middle panel, Overlap with "recurrence-prone"stroma. Note the overlap of 74 genes with a p-value of2.10.times.10-3. Lower panel, B, Overlap with "metastasis-prone"stroma. Note the overlap of 38 genes with a p-value of9.67.times.10-5.

[0042] FIG. 5 shows Venn Diagrams for the Transcriptional OverlapBetween Peroxisomes and Tumor Stroma from Breast Cancer Patients.Upper panel, Overlap with tumor stroma. Note the overlap of 204genes with a p-value of 4.25.times.10-12. Lower panel, Overlap with"recurrence-prone" stroma. Note the overlap of 101 genes with ap-value of 2.76.times.10-5.

[0043] FIG. 6 shows Over-Expression of Autophagy and MitophagyMarkers in Cav-1 (-/-) Null Mammary Fat Pads: Cathepsin B andBNIP3. To validate the idea that a loss of Cav-1 drives the onsetof autophagy, the expression of established autophagy markers wasassessed, namely cathepsin B and BNIP3, in Cav-1 (-/-) mammary fatpads. Cathepsin B is a well-known lysosomal protease. BNIP3 is amarker of autophagy that mediates the autophagic destruction ofmitochondria. Note that both cathepsin B (the pro-enzyme andactivated form) and BNIP3 are significantly over-expressed in Cav-1(-/-) null mammary fat pads (KO), relative wild-type controls (WT).Immuno-blotting with Cav-1 and beta-actin are shown for comparisonpurposes.

[0044] FIG. 7 shows A Lethal Tumor Micro-Environment: OxidativeStress Drives Stromal Autophagy/Mitophagy, ProvidingStromal-Derived Nutrients for Epithelial Cancer Cells. Here, usingmetabolic, transcriptional mRNA, and miR profiling, it was foundthat loss of stromal Cav-1 induces oxidative stress, mitochondrialdysfunction, and autophagy/mitophagy in the tumormicro-environment. This model would then provide recycled chemicalbuilding blocks (nutrients, amino acids, energy-rich metabolites,nucleotides) derived from stromal cells (fibroblasts) that thencould be harnessed by epithelial cancer cells to promote tumorgrowth. Mitochondrial dysfunction and mitophagy would result inaerobic glycolysis in stromal cells, explaining our previousobservations on the "Reverse Warburg Effect". Many of the keycomponents identified here through metabolic and micro-RNAprofiling are shown in BOLD: miR-31, miR-34c, ADMA, essential aminoacids (AA's), nucleotides, pyruvate, BHB, and TCA cycleintermediates.

[0045] FIG. 8 shows Ketones Can Fuel Tumor Growth. Ketones producedin the tumor micro-environment (in cancer associated fibroblasts)could fuel the growth of adjacent epithelial cancer cells. Ketoneproducing enzymes (in the fibroblasts) and ketone re-utilizingenzymes (in the epithelial cancer cells) are shown in BOLD.Transfer of ketones would be accomplished by monocarboxylatetransporters (MCTs). Normally the same scheme is used by the liver(for ketone production) and the brain (for ketone reutilization)during extreme fasting or starvation, to maintain neuronalfunction. Thus, the liver cells are the cancer fibroblasts, and theepithelial cells are the neurons. Interestingly, Cav-1 (-/-)stromal cells and the tumor stroma both show a shift towardsliver-specific gene and protein expression. For example, Cav-1(-/-) stromal cells produce alpha-fetoprotein and albumin, as seenby proteomics. Alpha-fetoprotein expression has been previouslylocalized to cancer-associated fibroblasts in human breast cancers.The enzymes involved in ketone metabolism are as follows: ACYL, ATPcitrate lyase (cytosolic); HMGCS1/2,3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (cytosolic)/2(mitochondrial); HMGCL, 3-hydroxymethyl-3-methylglutaryl-Coenzyme Alyase; HMGCLL1, 3-hydroxymethyl-3-methylglutaryl-Coenzyme Alyase-like 1; BDH1/2, 3-hydroxybutyrate dehydrogenase, type 1(mitochondrial)/type 2 (cytosolic); ACAT1/2, acetyl-Coenzyme Aacetyltransferase 1 (mitochondrial)/2 (cytosolic); OXCT1/2,3-oxoacid CoA transferase 1 (mitochondrial)/2 (testis-specific).The production of ketone bodies results from Acetyl-CoA derivedfrom pyruvate, via pyruvate dehydrogenase (PDH), and not from thebeta-oxidation of fatty acids, because Cav-1 (-/-) null mice have adefect in the beta-oxidation of fatty acids. This would alsomechanistically explain why lactate does not accumulate.Interestingly, ACLY (a cytosolic enzyme) may also contribute toketone production by converting citrate (a TCA metabolite) toAcetyl-CoA. This also results in the production of oxaloacetate,another TCA metabolite.

[0046] FIG. 9 shows Resolving the Autophagy Paradox in CancerTherapy, Autophagy/mitophagy (AM) in the tumor stroma may besustaining tumor growth. The large black arrow signifies energytransfer (E.T.) from the stromal cancer associated fibroblasts(CAFs) to the epithelial cancer cells, via stromalautophagy/mitophagy. Thus, inhibition of autophagy in the tumorstroma would be expected to halt or reverse tumor growth. Thiscould explain the effectiveness of known autophagy inhibitors asanti-tumor agents 50, such as chloroquine and 3-methyladenine(Upper panel). Conversely, induction of autophagy in epithelialcancer cells would also be expected to block or inhibit tumorgrowth (Lower panel). This idea would also explain the anti-tumoractivity of agents that activate autophagy, such as mTORinhibitors. Thus, using this model, compounds that eithersystemically block or activate autophagy would both have the samenet effect, which is to disrupt the metabolic coupling between theepithelial cancer cells and the tumor stromal fibroblasts. Thismodel directly resolves the long-lived "autophagy paradox", thatboth systemic inhibition of autophagy and systemic stimulation ofautophagy have the same net effect, which is to inhibit tumorgrowth. E.T., energy transfer; AM+, increased autophagy/mitophagy;AM-, decreased autophagy/mitophagy; Rx, therapy with autophagypromoters or inhibitors.

[0047] FIG. 10 shows Ketones Promote Mammary Tumor Growth. Axenograft model was employed in which MDA-MB-231 breast cancercells injected into the flanks of athymic nude mice to evaluate thepotential tumor promoting properties of the products of aerobicglycolysis (such as 3-hydroxy-butyrate and L-lactate). Tumor growthwas assessed by measuring tumor volume, at 3-weeks post tumor cellinjection. During this time period, mice were administered eitherPBS alone, or PBS containing 3-hydroxy-butyrate (500 mg/kg) orL-lactate (500 mg/kg), via daily intra-peritoneal (i.p.)injections. Note that 3-hydroxy-butyrate is sufficient to promotean .about.2.5-fold increase in tumor growth, relative to thePBS-alone control. Under these conditions. L-lactate had nosignificant effect on tumor growth. *p<0.05, PBS alone versus3-hydroxy-butyrate (Student's t-test).

[0048] FIG. 11 shows Ketones Promote Tumor Growth, Without AnyIncrease in Angiogenesis. Tumor angiogenesis could account for thetumor-promoting properties of 3-hydroxy-butyrate. Thus, the statusof tumor vascularity was evaluated using antibodies directedagainst CD31. However, the vascular density (number of vessels perfield) was not increased by the administration of either3-hydroxy-butyrate or L-lactate. Thus, other mechanisms, such asthe "Reverse Warburg Effect" may be operating to increase tumorgrowth. n.s., not significant.

[0049] FIG. 12 shows Ketones Promote Tumor Growth, Without AnyIncrease in Angiogenesis. Representative images of CD31immuno-staining in primary tumor samples.

[0050] FIG. 13 shows Ketones and Lactate Function asChemo-attractants, Stimulating Cancer Cell Migration.3-hydroxy-butyrate or L-lactate can function as chemo-attractants.shown using a modified "Boyden Chamber" assay, employing Transwellcell culture inserts. MDA-MB-231 cells were placed in the upperchambers, and 3-hydroxy-butyrate (10 mM) or L-lactate (10 mM) wereintroduced into the lower chambers. Note that both3-hydroxy-butyrate and L-lactate promoted cancer cell migration bynearly 2-fold. *p<0.05, control (vehicle alone) versus3-hydroxy-butyrate or L-lactate (Student's t-test).

[0051] FIG. 14 shows Lactate Fuels Lung Metastasis. To examine theeffect of 3-hydroxy-butyrate and L-lactate on cancer cellmetastasis, a well-established lung colonization assay was used,where MDA-MB-231 cells are injected into the tail vein of athymicnude mice. After 7 weeks post-injection, the lungs were harvestedand the metastases were visualized with India ink staining. Usingthis approach, the lung parenchyma stains black, while the tumormetastatic foci remain unstained, and appear white. The number ofmetastases per lung lobe was scored. Note that relative toPBS-alone, the administration of L-lacate stimulated the formationof metastatic foci by .about.10-fold. Under these conditions,3-hydroxy-butryate had no effect on metastasis formation.*p<0.01, PBS alone versus L-lactate (Student's t-test andANOVA); *p<0.05, PBS alone versus L-lactate (Mann-Whitneytest).

[0052] FIG. 15 shows Lactate Fuels Lung Metastasis. Images of LungMetastases. Representative examples of lung metastasis in PBS-alonecontrols and L-lactate-treated animals are shown. Note that themetastatic foci formed in L-lactate treated animals are morenumerous, and are also larger in size.

[0053] FIG. 16 shows Cav-1 knock-down is sufficient to promoteautophagy/mitophagy. Acute loss of Cav-1 increases the expressionof autophagic markers. hTERT-fibroblasts were treated with Cav-1siRNA or control (CTR) siRNA. Western blot analysis. Cells wereanalyzed by Western blot analysis using antibodies against theindicated autophagic markers. .beta.-tubulin was used as equalloading control.

[0054] FIG. 17 shows Cav-1 knock-down is sufficient to promoteautophagy/mitophagy. Immunofluorescence. Cells were fixed andimmuno-stained with antibodies against beclin 1, BNIP3, and BNIP3L.DAPI was used to visualize nuclei. Importantly, paired images wereacquired using identical exposure settings. Original magnification,40.times.. Note that acute Cav-1 knockdown is sufficient to greatlyincrease the expression levels of all the autophagy/mitophagymarkers examined.

[0055] FIG. 18 shows Human breast cancers lacking stromal Cav-1display increased stromal BNIP3L. BNIP3L is highly increased in thestroma of human breast cancers that lack stromal Cav-1.Paraffin-embedded sections of human breast cancer samples lackingstromal Cav-1 were immuno-stained with antibodies directed againstBNIP3L. Slides were then counter-stained with hematoxylin. Notethat BNIP3L is highly expressed in the stromal compartment of humanbreast cancers that lack stroma Cav-1. The boxed area shown athigher magnification reveals punctate staining, consistent withmitochondrial and/or lysosomal localization. Originalmagnification, 40.times. and 60.times., as indicated.

[0056] FIG. 19 shows the Reactions involved in Ketone Production(Ketogenesis).

[0057] FIG. 20 shows Reactions involved in Ketone (Re-)Utilization(Ketolysis).

[0058] FIGS. 21A through 21C showTable 1, Metabolomic Analysis ofMammary Fat Pads from Cav-1 (-/-) Deficient Mice.

[0059] FIG. 22 shows Table 2. Metabolomic Analysis of Mammary FatPads and Lung Tissue from Cav-1 (-/-) Deficient Mice.

[0060] FIGS. 23A and 23B show Table 3. Upregulation ofAutophagy/Mitophagy Related Gene Transcripts in Cav-1 (-/-) StromalCells.

[0061] FIGS. 24A and 24B show Table 4. Upregulation of GeneTranscripts Encoding Lysosomal Proteins in Cav-1 (-/-) StromalCells.

[0062] FIGS. 25A through 25D show Table 5. Upregulation ofTelomerase and Selected Redox-Related Gene Transcripts in Cav-1(-/-) Stromal Cells.

[0063] FIGS. 26A and 26B show Table 6. Upregulation ofAutophagy/Mitophagy Related Gene Transcripts in the Tumor Stromafrom Human Breast Cancer Patients.

[0064] FIGS. 27A and 27B show Table 7. Upregulation of GeneTranscripts Encoding Lysosomal Proteins in the Tumor Stroma fromHuman Breast Cancer Patients.

[0065] FIGS. 28A and 28B show Table 8. Upregulation of Telomeraseand Selected Redox-Related Gene Transcripts in the Tumor Stromafrom Human Breast Cancer Patients.

[0066] FIGS. 29A and 29B show Table 9. Transcriptional Profiling ofHuman Breast Cancer Tumor Stroma: ADMA and BHB Metabolism.

[0067] FIG. 30 shows Table 10. Up-regulation of miR's in Cav-1(-/-) null stromal cells.

[0068] FIGS. 31A through 31D show Table 11, Breast CancerEpithelial Cells Show a Transcriptional Shift Towards OxidativeMitochondrial Metabolism, Relative to Adjacent Stromal Tissue.

DETAILED DESCRIPTION OF THE INVENTION

[0069] The mammary fat pad of Cav-1 (-/-) null mice as apre-clinical model for a "lethal tumor-microenvironment", i.e., thetumor stroma without the tumor. The inventors have previouslydocumented that a loss of stromal Cav-1 in the fibroblastcompartment of human breast cancer, DCIS, and prostate cancer isassociated with a poor clinical outcome. In breast cancer, a lossof stromal Cav-1 is a single independent predictor of early tumorrecurrence, lymph node metastasis, and tamoxifen-resistance. InDCIS, a loss of stromal Cav-1 predicts both early recurrence andprogression to invasive breast cancer. Witkiewicz A K, Dasgupta A,Nguyen K H, Liu C, Kovatich A J, Schwartz G F, Pestell R G, SotgiaF, Rui H, Lisanti M P. Stromal caveolin-1 levels predict early DCISprogression to invasive breast cancer. Cancer Biol Ther 2009;8:1167-75. Finally, in prostate cancer patients, a loss of stromalCav-1 is associated with advanced prostate cancer and tumorprogression/metastasis, and high Gleason score, indicative of apoor prognosis. Witkiewicz A K, Dasgupta A, Sotgia F, Mercier I,Pestell R G, Sabel M, Kleer C G, Brody J R, Lisanti M P. An Absenceof Stromal Caveolin-1 Expression Predicts Early Tumor Recurrenceand Poor Clinical Outcome in Human Breast Cancers. Am J Pathol2009; 174:2023-34. Witkiewicz A K, Dasgupta A, Nguyen K H, Liu C,Kovatich A J, Schwartz G F, Pestell R G, Sotgia F, Rui H, Lisanti MP. Stromal caveolin-1 levels predict early DCIS progression toinvasive breast cancer. Cancer Biol Ther 2009; 8:1167-75. Di VizioD, Morello M, Sotgia F, Pestell R G, Freeman M R, Lisanti M P. AnAbsence of Stromal Caveolin-1 is Associated with Advanced ProstateCancer, Metastatic Disease and Epithelial Akt Activation. CellCycle 2009; 8:2420-4. Witkiewicz A K, Casimiro M C, Dasgupta A,Mercier I, Wang C, Bonuccelli G, Jasmin J F, Frank P G, Pestell RG, Kleer C G, Sotgia F, Lisanti M P. Towards a new "stromal-based"classification system for human breast cancer prognosis andtherapy. Cell Cycle 2009; 8:1654-8. Witkiewicz A K, Dasgupta A,Sammons S, Er O, Potoczek M B, Guiles F, Sotgia F, Brody J R,Mitchell E P, Lisanti M P. Loss of stromal caveolin-1 expressionpredicts poor clinical outcome in triple negative and basal-likebreast cancers. Cancer Biol Ther 2010; 10: In Press.

Oxidative Stress and Autophagy/Mitophagy in the TumorMicro-Environment

[0070] To mechanistically understand the lethality of a loss ofCav-1 in the tumor stromal compartment, an unbiased screeningapproach was used, by performing a metabolomics analysis on freshtissue harvested from the mammary fat pads of Cav-1 (-/-) nullmice, a robust animal model for a Cav-1 deficiency. Based on thisanalysis, the evidence for a series of severe metabolic defects inCav-1 deficient tissues is provided. More specifically, theinventors show that nearly 100 metabolites are elevated in Cav-1(-/-) null mammary fat pads. An analysis of these data isconsistent with the onset of oxidative stress phenotype, combinedwith mitochondrial dysfunction, and autophagy. The two mostsignificant metabolites that are elevated are ADMA and3-hydroxybutyrate. Also, several energy-rich metabolites, such aspyruvate and metabolic components of the TCA cycle are increased.These phenotypic changes provide a logical and intriguingexplanation for the lethality of a Cav-1 deficient tumormicro-environment, as oxidative stress is known to drive bothmitochondrial dysfunction and autophagy/mitophagy, and this would aset-up a situation in which catabolism of the tumor stroma could beused to directly "feed" the tumor epithelial cancer cells. This isan exceptionally ingenious parasitic strategy that could promotetumor progression and metastasis (Summarized in FIG. 7).

[0071] To independently validate these assertions, an informaticsapproach to reinterrogate the transcriptional profiling dataobtained from the analysis of Cav-1 (-/-) deficient stromal cellswas used, isolated from the bone marrow of Cav-1 knockout mice.Importantly, bone marrow mesenchymal stem cells are thought to bethe precursors of cancer associated fibroblasts that are recruitedby epithelial tumor cells to cancerous lesions. Based on ourre-analysis of this data set, the inventors provide evidence forthe upregulation of numerous gene transcripts specificallyassociated with authophagy/mitophagy, lysosomal biogenesis,oxidative stress, the glutathione pathway, and the compensatoryupregulation of anti-oxidant enzymes. These results provide directindependent validation of the metabolic profiling studies.

[0072] To directly assess the relevance of the findings for humanbreast cancers, evidence of the same transcriptional profiles inthe tumor stromal that was laser capture micro-dissected from theprimary human tumors of patients with breast cancer was examined.Importantly, re-interrogation of these data sets indicated that thefollowing biological processes are well-represented in the tumorstroma: authophagy/mitophagy, lysosomal biogenesis, oxidativestress, the glutathione pathway, and the upregulation ofanti-oxidant enzymes. Many of the transcripts associated with theseprocesses were also related to tumor recurrence, and lymph-nodemetastasis.

Identification of ADMA and Ketones as Key Metabolites: Implicationsfor Diagnosis and Drug Discovery

[0073] Since ADMA and 3-hydroxybutyrate emerged as the two mostimportant metabolites that were increased in metabolomic analysis,the enzymes responsible for their production were transcriptionallyincreased both in Cav-1 (-/-) stromal cells and the tumor stromaisolated from human breast cancers was validated. Thus, these newobservations now provide an opportunity for both diagnosticstratification of patients and the design of new drug therapies, toboth identify and combat the lethality of an aggressive tumormicroenvironment. In the case of ADMA, it is a catabolic breakdownproduct released from methylated proteins after their proteolyticdegradation. It is known to be strongly associated with endothelialcell dysfunction and oxidative stress. In addition, it also hasbiologically activity and can enhance and propagate the effects ofoxidative stress. For example, it is known to function as a naturalendogenous inhibitor of nitric oxide synthase (NOS) enzymes,halting the production of nitric oxide (NO). However, it alsochanges the specificity of the NOS enzymes, allowing them toconstitutively produce superoxide instead. Teerlink T, Luo Z, PalmF, Wilcox C S. Cellular ADMA: Regulation and action. Pharmacol Res2009; 60:448-60. 20. Yildirim A O, Bulau P, Zakrzewicz D, KitowskaK E, Weissmann N, Grimminger F, Morty R E, Eickelberg O. Increasedprotein arginine methylation in chronic hypoxia: role of proteinarginine methyltransferases. Am J Respir Cell Mol Biol 2006;35:436-43. Sud N, Wells S M, Sharma S. Wiseman D A, Wilham J, BlackS M. Asymmetric dimethylarginine inhibits HSP90 activity inpulmonary arterial endothelial cells: role of mitochondrialdysfunction. Am J Physiol Cell Physiol 2008; 294:C1407-18. Thus,ADMA is both a marker of oxidative stress and a producer of moreoxidative stress. Furthermore, ADMA changes the location of eNOSand directly targets the enzyme to mitochodria, where it themproduces superoxide. Thus, ADMA is a mitochondrial "time-bomb" thatleads to irreversible oxidative damage within mitochondria,necessitating their destruction by mitophagy. This, in turn,provides a mechanism for turning on aerobic glycolysis, so that thestromal cells will produce energy to ensure their own survival.However, aerobic glycolysis in the stroma releases both lactate andpyruvate, that can be used by epithelial cancer cells undergoingTCA based oxidative metabolism, thereby providing paracrine energyfor tumor growth. Stromal ketone production also likely plays astrong pathogenic role. Ketone production is a well-establishedmarker of mitochondrial dysfunction. Kennaway N G, Buist N R,Darley-Usmar V M, Papadimitriou A, Dimauro S, Kelley R I, Capaldi RA, Blank N K, D'Agostino A. Lactic acidosis and mitochondrialmyopathy associated with deficiency of several components ofcomplex III of the respiratory chain. Pediatr Res 1984; 18:991-9.Robinson B H, McKay N, Goodyer P, Lancaster G. Defectiveintramitochondrial NADH oxidation in skin fibroblasts from aninfant with fatal neonatal lacticacidemia. Am J Hum Genet 1985;37:938-46, consistent with our assertions regarding ADMA, oxidativestress, and autophagy/mitophagy. Ketones are normally produced bythe liver and virtually every other organ system in the body duringperiods of fasting and starvation, and they are then transferred tothe brain to maintain survival of the organism. Just as pyruvate,and lactate can be secreted and taken up by monocarboxylic acidtransporters (MCTs), the ketones 3-hydroxybutyrate andacetoacetate, both follow the same principles. Cahill G F, Jr.,Veech R L. Ketoacids? Good medicine? Trans Am Clin Climatol Assoc2003; 114:149-61; discussion 62-3. Veech R L. The therapeuticimplications of ketone bodies: the effects of ketone bodies inpathological conditions: ketosis, ketogenic diet, redox states,insulin resistance, and mitochondrial metabolism. ProstaglandinsLeukot Essent Fatty Acids 2004; 70:309-19. Veech R L, Chance B,Kashiwaya Y, Lardy H A, Cahill G F, Jr. Ketone bodies, potentialtherapeutic uses. IUBMB Life 2001; 51:241-7. So, ketone bodies canbe transferred directly from stromal cancer-associated fibroblaststo epithelial cancer cells via MCTs, without any energyexpenditure. Moreover, ketones are a "super-fuel" for mitochondria,producing more energy than lacate/pyruvate, and simultaneouslydecreasing oxygen consumption. In fact, because of theseproperties, ketones have been used to prevent ischemic tissuedamage, in animal models undergoing either myocardial infarctionsor stroke, leading to dramatically smaller ischemic/necrotic lesionarea. So, just as ketones are a "super-fuel" under conditions ofischemia in the heart and in the brain, they could fulfill asimilar function during tumorigenesis, as the tumor exceeds itsblood supply. So, stromal ketone production could obviate the needfor tumor angiogenesis. Once ketones are produced and released fromstromal cells, they could then be re-utilized by epithelial cancercells, where they could directly enter the TCA cycle, just likelactate and pyruvate. In this sense, ketones are a more powerfulmitochondrial fuel, as compared with lactate and pyruvate. As aconsequence, the "Reverse Warburg Effect" includes ketones as aparacrine energy source (Summarized in FIG. 8). In this scheme, theproduction of ketone bodies results from Acetyl-CoA derived frompyruvate, via pyruvate dehydrogenase (PDH), and not from thebetaoxidation of fatty acids, because Cav-1 (-/-) null mice have adefect in the beta-oxidation of fatty acids (discussed within 16).This would mechanistically explain why lactate does not accumulate.Interestingly, ACLY (a cytosolic enzyme) may also contribute toketone production by converting citrate (a TCA metabolite) toAcetyl-CoA.

[0074] Thus, ADMA and ketone bodies(3-hydroxybutyrate/acetoacetate) levels can be used as diagnostictools to assess patient outcome. ADMA and ketone levels couldeither be measured in patient serum/plasma, or directly determinedfrom homogenates of fresh tumor tissue. High AMDA and ketone levelsin cancer patient serum or human tumor samples will strictlycorrelate with poor clinical outcome. These simple diagnostic testscould be performed rapidly, and quantitatively, allowing us toidentify and monitor high-risk cancer patients, both at diagnosisand during therapy. They could also be used for treatmentstratification.

[0075] There is also a new opportunity here for new drugdevelopment via targeted therapies. Inhibition of ADMA productionor ketone production/re-utilization should halt tumor growth,leading to tumor regression. As such, the enzymes associated withi) ADMA production (all PRMT family members), ii) ketone production(ACYL, 18 HMGCS1/2, HMGCL, HMGCLL1, and BDH1/2) and iii) ketonere-utilization (ACAT1/2 and OXCT1/2) should now all be consideredas "druggable targets" for cancer chemotherapy and prevention. Infact, a number of known anti-oxidants have already been shown tohave anti-tumor activity 48, such as N-acetyl cysteine (NAC),vitamin C, quercetin, and curcumin. In this regard, NAC acts bothas a free radical scavenger, and directly feeds into theglutathione pathway, increasing the amounts of cellularglutathione; NAC is the known to be the most promising anti-oxidantfor inhibiting mitophagy. Deffieu M, Bhatia-Kissova I, Salin B,Galinier A, Manon S, Camougrand N. Glutathione participates in theregulation of mitophagy in yeast. J Biol Chem 2009; 284:14828-37.Furthermore, also anti-lysosomal drugs that inhibit autophagy, suchas chloroquine, are known to have very significant anti-tumoractivity. This may be due to their ability to inhibit autophagy inthe fibroblastic stromal tumor compartment. Cancer Connections withSystemic Sclerosis, Diabetes, and Fasting Interestingly, a varietyof human diseases are also associated with high levels of ADMA. Onesuch disease is systemic sclerosis (Sec; scleroderma), and Secpatients have a higher incidence of cancer. Similarly, diabeticpatients show both high serum levels of ADMA and ketones. Thus, ourcurrent observations may also explain the close and emergingassociation between diabetes and cancer susceptibility 60. A numberof elegant studies have been carried out in mouse animal models toassess this association, and chemical induction of diabetes in ratswith streptozocin is sufficient to enhance tumor growth 61.Similarly, acute fasting in rodent animal models is also sufficientto dramatically increase tumor growth. Both of these experimentalconditions (diabetes and fasting/starvation) are known to be highlyketogenic, and, thus, are consistent with our current hypothesisthat ketone production fuels tumor growth and metastasis. Thus, thecombination of ADMA and ketones plays a crucial and causal role inpromoting tumorigenesis, by providing oxidative stress and thesimultaneous release of high-energy nutrients from the tumormicro-environment. Of course, this would be complemented byoxidative stress induced autophagy/mitophagy in the tumormicroenvironment, thus providing the necessary recycled chemicalbuilding blocks (amino acids, nucleotides, TCA cycle intermediates,etc.) in a paracrine fashion to cancer epithelial cells, to promotetumor growth. Hu C, Solomon V R, Ulibarri G, Lee H. The efficacyand selectivity of tumor cell killing by Akt inhibitors aresubstantially increased by chloroquine. Bioorg Med Chem 2008;16:7888-93. Hiraki K, Kimura I. Studies on the Treatment ofMalignant Tumors with Fibroblast-Inhibiting Agent. 1.Fibroblast-Inhibiting Action of Chloroquine. Acta Med Okayama 1963;17:231-8. Hiraki K, Kimura I. Studies on the Treatment of MalignantTumors with Fibroblast-Inhibiting Agent. Ii. Effects of Chloroquineon Animal Tumors. Acta Med Okayama 1963; 17:239-52. Hiraki K,Kimura I. Studies on the Treatment of Malignant Tumors withFibroblast-Inhibiting Agent. Iv. Effects of Chloroquine onMalignant Lymphomas. Acta Med Okayama 1964; 18:87-92. Hiraki K,Kimura I. Studies on the Treatment of Malignant Tumors withFibroblast-Inhibiting Agent. 3. Effects of Chloroquine on HumanCancers. Acta Med Okayama 1964; 18:71-85. Dooley A, Gao B, BradleyN, Abraham D J, Black C M, Jacobs M, Bruckdorfer K R. Abnormalnitric oxide metabolism in systemic sclerosis: increased levels ofnitratedproteins and asymmetric dimethylarginine. Rheumatology(Oxford) 2006; 45:676-84. Dimitroulas T, Giannakoulas G, SfetsiosT, Karvounis H, Dimitroula H, Koliakos G, Settas L. Asymmetricaldimethylarginine in systemic sclerosis-related pulmonary arterialhypertension. Rheumatology (Oxford) 2008; 47:1682-5. Marasini B,Conciato L, Belloli L, Massarotti M. Systemic sclerosis and cancer.Int J Immunopathol Pharmacol 2009; 22:573-8. Anderssohn M,Schwedhelm E, Luneburg N, Vasan R S, Boger R H. Asymmetricdimethylarginine as a mediator of vascular dysfunction and a markerof cardiovascular disease and mortality: an intriguing interactionwith diabetes mellitus. Diab Vasc Dis Res 2010; 7:105-18. PitoccoD, Zaccardi F, Di Stasio E, Romitelli F, Martini F, Scaglione G L,Speranza D, Santini S, Zuppi C, Ghirlanda G. Role ofasymmetric-dimethyl-L-arginine (ADMA) and nitrite/nitrate (NOx) inthe pathogenesis of oxidative stress in female subjects withuncomplicated type 1 diabetes mellitus. Diabetes Res Clin Pract2009; 86:173-6. Nicolucci A. Epidemiological aspects of neoplasmsin diabetes. Acta Diabetol 2010; 47:87-95.

[0076] In support of these ideas linking fasting/autophagy, withcancer susceptibility and diabetes, adipocytes from obese patientswith type 2 diabetes show decreased mTOR signaling andsubstantially enhanced autophagy. Similarly, hypoxia, inflammation,and michondrial dysfunction all inactivate mTOR signaling, leadingto autophagy. Ost A, Svensson K, Ruishalme I, Brannmark C, FranckN, Krook H, Sandstrom P, Kjolhede P, Stralfors P. Attenuated mTORsignaling and enhanced autophagy in adipocytes from obese patientswith type 2 diabetes. Mol Med 2010.

Autophagy in the Tumor Micro-Environment can Substitute forAngiogenesis in Promoting Tumor Growth

[0077] The combination of oxidative stress, mitochondrialdysfunction, and autophagy/mitophagy in cancer-associatedfibroblasts reduces the dependence of tumor growth and survival onneo-angiogenesis and vascularization. This explains why many of thenew angiogenesis inhibitors have not been as promising as expectedin ongoing clinical trials, as our current observations suggestthat a Cav-1 negative fibroblastic tumor micro-environment couldactually subsume the role of tumor angiogenesis, without the needfor increased tumor vascularization. This is particularly relevantin the case of pancreatic cancers, which are known to be a highlyfibrotic class of relatively avascular tumors which areexceptionally lethal. Genetically modified glycolytic fibroblasts,that lack Cav-1 expression, were used to assess their affects onhuman xenograft tumor growth using co-injections with a breastcancer cell line, namely MDA-MB-231 cells. In these xenograftmodels, the genetically modified fibroblasts, lacking Cav-1expression, increased tumor weight by .about.4-fold, and tumorvolume by nearly 8-fold, without a measurable increase in tumorangiogenesis. Migneco G, Whitaker-Menezes D, Chiavarina B,Castello-Cros R, Pavlides S, Pestell R G, Fatatis A, Flomenberg N,Tsirigos A, Howell A, Martinez-Outschoom U E, Sotgia F, Lisanti MP. Glycolytic cancer associated fibroblasts promote breast cancertumor growth, without a measurable increase in angiogenesis:Evidence for stromalepithelial metabolic coupling. Cell Cycle 2010;9.

Micro-RNA Profiling: Associations with Oxidative Stress andAutophagy/Mitophagy.

[0078] Micro-RNA (miR) profiling on Cav-1 (-/-) deficient stromalcells was performed to gain mechanistic insight into how a loss ofCav-1 may drive oxidative stress, mitochondrial dysfunction, andautophagy/mitophagy. Using this approach, 20 two miR species wereidentified that were highly over-expressed in Cav-1 null stromalcells, namely miR-31 and miR-34c.

[0079] The upregulation of miR-34c is consistent with results fromboth metabolomics and transcriptional profiling, as it is normallyupregulated by oxidative stress, and is also associated with DNAdamage and senescence, which are known down-stream effects ofoxidative stress. Similarly, the upregulation of miR-31 provides ameans for the transcriptional activation of HIF1-alpha 18, which isknown to induce both autophagy, and mitophagy, and to inhibitmitochondrial biogenesis. The transcriptional activation ofHIF1-alpha by miR-31 is indirectly mediated by FIH-1 (factorinhibiting HIF), which is the direct target of miR-31 18. Thus,over-expression of miR-31 blocks the transcriptional expression ofa HIF inhibitory factor, FIH-1, leading to HIF activation. TheAutophagic Tumor Stroma Model of Cancer: CompartmentalizedAutophagy. Based on current observations, the inventors havedeveloped a new model for cancer pathogenesis. In this model, tumorcells activate autophagy in the tumor stromal compartment viaparacrine mechanisms. Autophagy in the tumor stroma, especially incancer-associated fibroblasts, then provides epithelial cancercells with a steady stream of recycled nutrients and energy-richmetabolites which could then be re-used by cancer cells to driveincreases in tumor growth and metastasis. Additional mesenchymalstem cells from the bone marrow can be recruited to the tumor andinduced to undergo autophagy, to satisfy the tumor's appetite. Theextension of this scheme from a local to a systemic phenomenon, canexplain the onset of anorexia, cachexia, insulin-resistance, andmetabolic syndrome, all features that are known to be associatedwith chronic malignancy, and this would provide the tumor withautophagic/catabolic-based nutrients (including ketonebodies)--from distant systemic sources. Tumor cells might evenmetastasize to the major sites of ketone production (the liver oradipose-tissue-rich bone marrow) or ketone re-utilization (thebrain), in search of energy-rich metabolites. Cannell I G, Kong YW, Johnston S J, Chen M L, Collins H M, Dobbyn H C, Elia A, Kress TR, Dickens M, Clemens M J, Heery D M, Gaestel M, Eilers M, Willis AE, Bushell M. p38 MAPK/MK2-mediated induction of miR-34c followingDNA damage prevents Myc-dependent DNA replication. Proc Natl AcadSci USA 2010; 107:5375-80. He X, He L, Hannon G J. The guardian'slittle helper: microRNAs in the p53 tumor suppressor network.Cancer Res 2007; 67:11099-101. Lafferty-Whyte K, Cairney C J,Jamieson N B, Oien K A, Keith W N. Pathway analysis ofsenescence-associated miRNA targets reveals common processes todifferent senescence induction mechanisms. Biochim Biophys Acta2009; 1792:341-52. Mazure N M, Pouyssegur J. Hypoxia-inducedautophagy: cell death or cell survival? Curr Opin Cell Biol 2010;22:177-80. Bellot G, Garcia-Medina R, Gounon P, Chiche J, Roux D,Pouyssegur J, Mazure N M. Hypoxia-induced autophagy is mediatedthrough hypoxia-inducible factor induction of BNIP3 and BNIP3L viatheir BH3 domains. Mol Cell Biol 2009; 29:2570-81. Chan S Y,Loscalzo J. MicroRNA-210: A unique and pleiotropic hypoxamir. CellCycle 2010; 9. 39. Liu C J, Kao S Y, Tu H F, Tsai M M, Chang K W,Lin S C. Increase of microRNA miR-31 level in plasma could be apotential marker of oral cancer. Oral Dis 2010. Wang C J, Zhou Z G,Wang L, Yang L, Zhou B, Gu J, Chen H Y, Sun X F.Clinicopathological significance of microRNA-31, -143 and -145expression in colorectal cancer. Dis Markers 2009; 26:27-34.

[0080] This model also provides a rationale basis for designing newtherapeutic intervention(s), as autophagy in the tumor stroma maybe sustaining tumor growth. Thus, inhibition of autophagy in thetumor stroma halts or reverses tumor growth. This can explain theeffectiveness of known autophagy inhibitors as anti-tumor agents,such as chloroquine and 3-m ethyladenine. Conversely, induction ofautophagy in epithelial cancer cells would also be expected toblock or inhibit tumor growth. This idea would also explain theanti-tumor activity of agents that activate autophagy, such as mTORinhibitors. Thus, using this model, compounds that eithersystemically block or activate autophagy would both have the samenet effect, which is to disrupt the metabolic coupling between theepithelial cancer cells and the tumor stromal fibroblasts (Figure______). This model directly resolves the long-lived "autophagyparadox", that both systemic inhibition of autophagy and systemicstimulation of autophagy have the same net effect, which is toinhibit tumor growth. This new model provides a new paradigm andrationale basis for drug development, driving new metabolictherapeutic interventions.

Clinical Connections with Malignancy: ADMA, ATG16L, and SPARC

[0081] The inventors have found an association of ADMA and ketoneproduction with malignancy, based on metabolomics analysis of amouse model of a "lethal tumor-microenvironment". In addition, theinventors have elucidated the role of autophagy in the tumormicro-environment. With regard to An autophagic marker, ATG16L,high levels of stromal ATG16L were associated with i) thelympho-vascular invasion of tumor cells and ii) positive lymph nodestatus--consistent with our proposed model. Unfortunately, no dataon clinical outcome were presented. The inventors have found thatATG16L was transcriptionally over-expressed in Cav-1 (-/-) stromalcells and the tumor stroma of human breast cancer patients, and itsexpression was associated with tumor recurrence (See Tables 3 and6, Figures ______ and ______). Thus, high expression ofautophagy-associated biomarkers in the tumor stroma are a generalfeature of human epithelial cancers and are associated with poorclinical outcome. SPARC is a multi-functional extracellular matrixprotein that is associated with the tumor stroma. Recently, SPARCover-expression has been shown to be sufficient to induce autophagyin cells in culture via the up-regulation of cathepsin B.Similarly, the inventors have previously demonstrated that Cav-1(-/-) deficient stromal cells over-express SPARC, as evidenced byboth unbiased proteomic and genome-wide transcriptional profiling.Also, it was shown that the stromal expression of SPARC accuratelypredicts DCIS recurrence and/or progression. Taken together, thesefinding are consistent with the idea that a loss of stromal Cav-1induces SPARC and autophagy in the tumor microenvironment, therebypromoting tumor progression in DCIS patients. In accordance withthis idea, a loss of stromal Cav-1 is strongly associated withprogression to invasive breast cancer in DCIS patients.

[0082] The inventors provide the first evidence that theend-products of aerobic glycolysis (namely, 3-hydroxy-butyrate andL-lactate) can fuel tumor growth and metastasis, when administeredsystemically in a human tumor xenograft model. More specifically,3-hydroxy-butyrate is sufficient to promote a 2.5-fold increase intumor volume, without any significant increases in angiogenesis.Although L-lactate did not increase tumor growth, it had asignificant effect on lung colonization/metastasis, resulting in a10-fold increase in the formation of metastatic tumor foci. Theresults are consistent with the idea that human breast cancer cellscan re-utilize the energy-rich end-products of glycolysis foroxidative mitochondrial metabolism. Consistent with thesefunctional xenograft data, the inventors also show, oxidativemitochondrial metabolism is indeed up-regulated in human breastcancer cells, relative to adjacent stromal tissue, measured using atranscriptional informatics analysis.

Ketones and Tumor Growth: the Diabetes-Cancer Connection.

[0083] Ketones are a "super-fuel" for mitochondria, producing moreenergy than lacate, and simultaneously decreasing oxygenconsumption. In fact, because of these properties, ketones havebeen used to prevent ischemic tissue damage, in animal modelsundergoing either myocardial infarctions or stroke, leading todramatically smaller ischemic/necrotic lesion area. So, just asketones are a "super-fuel" under conditions of ischemia in theheart and in the brain, the inventors found they fill a similarfunction during tumorigenesis, as the hypoxic tumor exceeds itsblood supply. Stromal ketone production obviates the need for tumorangiogenesis. Once ketones are produced and released from stromalcells, they could then be re-utilized by epithelial cancer cells,where they could directly enter the TCA cycle, just like lactate.In this sense, ketones are a more powerful mitochondrial fuel, ascompared with lactate. Cahill G F, Jr., Veech R L. Ketoacids? Goodmedicine? Trans Am Clin Climatol Assoc 2003; 114:149-61; discussion62-3. Veech R L. The therapeutic implications of ketone bodies: theeffects of ketone bodies in pathological conditions: ketosis,ketogenic diet, redox states, insulin resistance, and mitochondrialmetabolism. Prostaglandins Leukot Essent Fatty Acids 2004;70:309-19. Veech R L, Chance 13, Kashiwaya Y, Lardy H A, Cahill GF, Jr. Ketone bodies, potential therapeutic uses. IUBMB Life 2001;51:241-7. Zou Z, Sasaguri S, Rajesh K G, Suzuki R.dl-3-Hydroxybutyrate administration prevents myocardial damageafter coronary occlusion in rat hearts. Am J Physiol Heart CircPhysiol 2002; 283:H1968-74. Puchowicz M A, Zechel J L, Valerio J,Emancipator D S, Xu K, Pundik S, LaManna J C, Lust W D.Neuroprotection in diet-induced ketotic rat brain after focalischemia. J Cereb Blood Flow Metab 2008; 28:1907-16.

[0084] Thus, our current observations may also explain the closeand emerging association between diabetes and cancersusceptibility. A number of elegant studies have been carried outin mouse animal models to assess this association, and chemicalinduction of diabetes in rats with streptozocin is sufficient toenhance tumor growth. Similarly, acute fasting in rodent animalmodels is also sufficient to dramatically increase tumor growth.Both of these experimental conditions (diabetes andfasting/starvation) are known to be highly ketogenic, and, thus,are consistent with the model wherein ketone production fuels tumorgrowth. Finally, given our current findings that ketones increasetumor growth, cancer patients and their dieticians may want tore-consider the use of a "ketogenic diet" as a form of anti-cancertherapy. Nicolucci A. Epidemiological aspects of neoplasms indiabetes. Acta Diabetol 2010; 47:87-95. Sauer L A, Dauchy R T.Stimulation of tumor growth in adult rats in vivo during acutestreptozotocin-induced diabetes. Cancer Res 1987; 47:1756-61.Goodstein M L, Richtsmeier W J, Sauer L A. The effect of an acutefast on human head and neck carcinoma xenograft. Growth effects onan `isolated tumor vascular pedicle` in the nude rat. ArchOtolaryngol Head Neck Surg 1993; 119:897-902.

Lactate Drives Metastatic Disease Progression: Quercetin andLactated Ringers Solution.

[0085] Tumor lactate production, serum lactate levels, and serumLDH levels have long been known as biomarkers for poor clinicaloutcome in many different types of human epithelial cancers,including breast cancer. In fact, lactic-acidosis (due to theover-production and/or accumulation of serum lactate) is often thecause of death in patients with metastatic breast cancer, or othertypes of metastatic cancer 34-49. However, a causative role forL-lactate production in tumor metastatic progression has not yetbeen suggested or demonstrated. Koukourakis M I, Kontomanolis E,Giatromanolaki A, Sivridis E, Liberis V. Serum and tissue LDHlevels in patients with breast/gynaecological cancer and benigndiseases. Gynecol Obstet Invest 2009; 67:162-8. Ryberg M, NielsenD, Osterlind K, Andersen P K, Skovsgaard T, Dombernowsky P.Predictors of central nervous system metastasis in patients withmetastatic breast cancer. A competing risk analysis of 579 patientstreated with epirubicin-based chemotherapy. Breast Cancer Res Treat2005; 91:217-25. Nisman B, Barak V, Hubert A, Kaduri L, Lyass O,Baras M, Peretz T. Prognostic factors for survival in metastaticbreast cancer during first-line paclitaxel chemotherapy. AnticancerRes 2003; 23:1939-42. Ryberg M, Nielsen D, Osterlind K, SkovsgaardT, Dombernowsky P. Prognostic factors and long-teen survival in 585patients with metastatic breast cancer treated withepirubicin-based chemotherapy. Ann Oncol 2001; 12:81-7. Vigano A,Bruera E, Jhangri G S, Newman S C, Fields A L, Suarez-Almazor M E.Clinical survival predictors in patients with advanced cancer. ArchIntern Med 2000; 160:861-8. Kher A, Moghe G. Deshpande A.Significance of serum ferritin and lactate dehydrogenase in benignand malignant disease of breast. Indian J Pathol Microbiol 1997;40:321-6. Khan N, Tyagi S P, Salahuddin A. Diagnostic andprognostic significance of serum cholinesterase and lactatedehydrogenase in breast cancer. Indian J Pathol Microbiol 1991;34:126-30. Yeshowardhana, Gupta M. M, Bansal G, Goyal S, Singh V S,Jain S. Sangita Jain K. Serum glycolytic enzymes in breastcarcinoma. Tumori 1986; 72:35-41.

[0086] Here, the inventors have directly demonstrated thatL-lactate can play a causative role in breast cancer cellmetastasis, by increasing the number of lung metastatic foci by.about.10-fold. This provides the necessary evidence thatmitochondrial oxidative metabolism can also fuel cancer cellmetastasis. This may have important clinical implications, asMCT/lactate transport inhibitors could be used to therapeuticallyto suppress tumor metastasis. Our findings also explain themultiple therapeutic activities of quercetin. Quercetin is anaturally occurring dietary flavenoid (available as anover-the-counter supplement) that functions both as an MCT/lactatetransport inhibitor, and inhibitor of TGF-beta signaling. Oneexplanation for these dual activities is that L-lactate uptake intotumor cells somehow metabolically activates TGF-beta signaling. Assuch, the inhibitory effects of quercetin on TGF-beta signaling maybe due to its ability to inhibit the uptake of L-lactate into tumorcells, presumably resulting in reduced cell migration andmetastasis. Further studies will be necessary to address thisattractive possibility. Belt J A, Thomas J A, Buchsbaum R N, RackerE. Inhibition of lactate transport and glycolysis in Ehrlichascites tumor cells by bioflavonoids. Biochemistry 1979;18:3506-11. Hu Q, Noor M, Wong Y F, Hylands P J, Simmonds M S, XuQ, Jiang D, Hendry B M. In vitro anti-fibrotic activities of herbalcompounds and herbs. Nephrol Dial Transplant 2009. Phan T T, Lim IJ, Chan S Y, Tan E K, Lee S T, Longaker M T. Suppression oftransforming growth factor beta/smad signaling in keloid-derivedfibroblasts by quercetin: implications for the treatment ofexcessive scars. J Trauma 2004; 57:1032-7. Subramanian A, Tamayo P,Mootha V K, Mukherjee S, Ebert B L, Gillette M A, Paulovich A,Pomeroy S L, Golub T R, Lander E S, Mesirov J P. Gene setenrichment analysis: a knowledge-based approach for interpretinggenome-wide expression profiles. Proc Natl Acad Sci USA 2005;102:15545-50.

[0087] Finally, given the pro-metastatic activity of L-lactate, itsmedical use in cancer patients should be restricted. However,nearly every oncology surgeon world-wide uses "Lactated Ringers"(which contains 25 mM L-lactate) as an intravenous (i.v.) solutionin cancer patients, before, during, and after tumor excision, andpossibly during the entire extended post-operative hospital stay.Based on our current studies, the use of "Lactated Ringers" incancer patients may unnecessarily increase their risk forprogression to metastatic disease. Thus, oncology surgeons may wishto re-consider using "Lactated Ringers" solution in cancerpatients.

Loss of Cav-1 is Sufficient to Induce Autophagy.

[0088] Loss of Cav-1 is sufficient to activate the autophagy and/ormitophagy program in fibroblasts. Western blot analysis wasperformed on hTERT-fibroblasts treated with Cav-1 siRNA or controlsiRNA, using antibodies directed against a panel of autophagymarkers. FIG. 16 shows that acute Cav-1 knock-down in fibroblastsdrives the increased expression of six autophagy markers, includingCathepsin B (active form), LAMP-1, LC3B, Beclin 1, ATG16L, andBNIP3.

[0089] To independently validate these results, hTERT-fibroblaststreated with Cav-1 siRNA or control siRNA were also immuno-stainedwith a subset of autophagy/mitophagy markers. FIG. 17 shows thatBeclin 1, BNIP3 and BNIP3L are greatly increased in Cav-1knock-down cells, indicating that an acute loss of Cav-1 issufficient to promote autophagy. Taken together, our currentfindings indicate that oxidative stress and hypoxia induce theautophagy-mediated loss of Cav-1 in fibroblasts, and that loss ofCav-1 further promotes autophagy/mitophagy in fibroblasts, via afeed-forward mechanism.

Human Breast Cancers Lacking Stromal Cav-1 Display IncreasedStromal BNIP3L.

[0090] The inventors have shown that a loss of Cav-1 promotesautophagy and that Cav-1 is degraded via an autophagic mechanism.To evaluate the translational significance of our findings, loss ofstromal Cav-1 in human breast cancer was evaluated and correlateswith increased autophagy. To this end, a number of human breastcancer samples were selected that lack Cav-1 in the stroma toperform immuno-staining with the autophagy/mitophagy markerBNIP3L.

[0091] FIG. 18 shows that BNIP3L is highly expressed in the stromalcompartment of human breast cancers that lack Cav-1. These resultsdirectly support the "Autophagic Tumor Stroma Model of CancerMetabolism". As a loss of Cav-1 is a powerful predictor of poorclinical outcome in breast cancers, our findings indicate that inhuman breast cancer a loss of Cav-1 promotes autophagy/mitophagy inthe stroma, to support the growth and aggressive behavior ofadjacent cancer cells.

[0092] Biomarkers

[0093] The present invention relates to biomarkers that aredifferentially expressed in neoplastic disease compared to normalpatients, and various methods, reagents and kits for diagnosis,staging, prognosis, monitoring and treatment of neoplastic disease,including, e.g., breast cancer.

[0094] In one aspect, the present invention provides biomarkerswhich are, for example, as set forth in FIG. 29. In another aspect,the biomarker is selected from the group consisting of ACLY,HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, miR-31,miR-34c, ACAT1, ACAT2, OXCT1, OXCT2, ADMA, 3-hydroxybutyrate, andcombinations thereof.

[0095] In one aspect, the present invention provides methods fordetermining the expression levels of individual and/or combinationsof the differentially expressed biomarker sequences in a biologicalsample that are indicative of the presence, or stage of thedisease, or the efficacy of therapy. The method comprisescontacting said sample with a polynucleotide probe or a polypeptideligand under conditions effective for said probe or ligand tohybridize specifically to a nucleic acid or a polypeptide in saidsample, and detecting the presence or absence of biomarker. In oneembodiment, methods are provided to determine the amounts and/orthe differentially expressed levels at which the marker sequencesof the present invention are expressed in samples. Such methods cancomprise contacting said sample with a polynucleotide probe or apolypeptide ligand under conditions effective for said probe tohybridize specifically to the nucleic acids in said sample, anddetecting the amounts or differentially expressed level of themarker sequences. In one preferred embodiment, said polynucleotideprobe is a polynucleotide designed to identify one of the markersequences in selected from the group consisting of ACLY, HMGCS1,HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, miR-31, miR-34c,ACAT1, ACAT2, OXCT1, OXCT2, ADMA, 3-hydroxybutyrate, andcombinations thereof. See FIG. 29. In another preferred embodiment,said polypeptide ligand is an antibody.

[0096] In another aspect, the present invention providespolypeptides encoded by the marker sequences, biologically activeportions thereof, and polypeptide fragments suitable for use asimmunogens to raise antibodies directed against polypeptides of themarker sequences of the present invention.

[0097] In another aspect, the present invention provides ligandsdirected to polypeptides and fragments thereof of the markersequences of the present invention. Preferably, said polypeptideligands are antibodies. Antibodies of the invention include, butare not limited to, polyclonal, monoclonal, multispecific, human,humanized, or chimeric antibodies, single chain antibodies, Fabfragments, Fv fragments F(ab') fragments, fragments produced by aFab expression library, anti-idiotypic antibodies, or other epitopebinding polypeptide. Preferably, an antibody, useful in the presentinvention for the detection of the individual marker sequences (andoptionally at least one additional neoplastic disease-specificmarker), is a human antibody or fragment thereof, including scFv,Fab, Fab', F(ab'), Fd, single chain antibody, of Fv. Antibodies,useful in the invention may include a complete heavy or light chainconstant region, or a portion thereof, or an absence thereof.

[0098] Another aspect of the present invention provides a method ofassessing whether a subject is suffering from or at risk ofdeveloping neoplastic disease including colon neoplastic disease bydetecting the differential expression of the marker sequences ofthe present invention. In one embodiment, the diagnostic methodcomprises determining whether a subject has an abnormal mRNA orcDNA and/or protein level of the marker sequences. The methodcomprises detecting the expression level of the individual and/orthe combinations of the marker sequences in a biological sampleobtained from a patient.

[0099] In some embodiments, the present invention provides methodsfor detection of expression of biomarkers in some embodiments,expression is measured directly (e.g., at the nucleic acid orprotein level). In some embodiments, expression is detected intissue samples (e.g., biopsy tissue). In other embodiments,expression is detected in bodily fluids (e.g., including but notlimited to, plasma, serum, whole blood, mucus, and urine). Thepresent invention further provides panels and kits for thedetection of biomarkers. In preferred embodiments, the presence ofa biomarker is used to provide a prognosis to a subject. Forexample, the detection of a biomarker in neoplastic disease tissuesmay be indicative of a neoplastic disease that is or is not likelyto metastasize. In addition, the expression level of a biomarkermay be indicative of a transformed cell, cancerous tissue or aneoplastic disease likely to metastasize.

[0100] The information provided can also be used to direct thecourse of treatment. For example, if a subject is found to possessor lack a biomarker that is likely to metastasize, therapies can bechosen to optimize the response to treatment (e.g., for subjectswith a high probability of possessing a metastatic neoplasticdisease more aggressive forms of treatment can be used).

[0101] Biomarkers may be measured as up or down-regulated using themethods of the present invention, and can be further characterizedusing microarray (e.g., nucleic acid or tissue microarray),immunohistochemistry, Northern blot analysis, siRNA or antisenseRNA inhibition, mutation analysis, investigation of expression withclinical outcome, as well as other methods disclosed herein.

[0102] In some embodiments, the present invention provides a panelfor the analysis of a plurality of biomarkers. The panel allows forthe simultaneous analysis of multiple biomarkers correlating withcarcinogenesis, metastasis and/or angiogenesis associated withneoplastic disease. For example, a panel may include biomarkersidentified as correlating with cancerous tissue, metastatic cancer,localized neoplastic disease that is likely to metastasize,pre-cancerous tissue that is likely to become cancerous,pre-cancerous tissue that is not likely to become cancerous, andcancerous tissues or cells likely or not likely to respond totreatment. Depending on the subject, panels may be analyzed aloneor in combination in order to provide the best possible diagnosisand prognosis. Markers for inclusion on a panel are selected byscreening for their predictive value using any suitable method,including but not limited to, those described in the illustrativeexamples below.

[0103] In other embodiments, the present invention provides anexpression profile map comprising expression profiles of s (e.g.,of various stages or progeny) or prognoses (e.g., likelihood torespond to treatment or likelihood of future metastasis). Such mapscan be used for comparison with patient samples. Any suitablemethod may be utilized, including but not limited to, by computercomparison of digitized data. The comparison data is used toprovide diagnoses and/or prognoses to patients.

[0104] In some preferred embodiments, biomarkers (e.g., includingbut not limited to, those disclosed herein) are detected bymeasuring the levels of the biomarker in cells and tissue (e.g.,cancer cells and tissues). For example, in some embodiments, abiomarker are monitored using antibodies (e.g., antibodiesgenerated according to methods described below) or by detecting abiomarker protein. In some embodiments, detection is performed oncells or tissue after the cells or tissues are removed from thesubject. In other embodiments, detection is performed byvisualizing the biomarker in cells and tissues residing within thesubject.

[0105] In some preferred embodiments, biomarkers of the inventionare detected by measuring the expression of corresponding mRNA in atissue sample (e.g., cancerous tissue).

[0106] In some embodiments, RNA is detected by Northern blotanalysis. Northern blot analysis involves the separation of RNA andhybridization of a complementary labeled probe.

[0107] In still further embodiments, RNA (or corresponding cDNA) ofthe biomarkers of the invention is detected by hybridization to aoligonucleotide probe. A variety of hybridization assays using avariety of technologies for hybridization and detection areavailable. For example, in some embodiments, a TaqMan assay (PEBiosystems, Foster City, Calif.; See e.g., U.S. Pat. Nos. 5,962,233and 5,538,848, each of which is herein incorporated by reference)is utilized. The assay is performed during a PCR reaction. TheTaqMan assay exploits the 5'-3' exonuclease activity of theAMPLITAQ GOLD DNA polymerase. A probe consisting of anoligonucleotide with a 5'-reporter dye (e.g., a fluorescent dye)and a 3'-quencher dye is included in the PCR reaction. During PCR,if the probe is bound to its target, the 5'-3' nucleolytic activityof the AMPLITAQ GOLD polymerase cleaves the probe between thereporter and the quencher dye. The separation of the reporter dyefrom the quencher dye results in an increase of fluorescence. Thesignal accumulates with each cycle of PCR and can be monitored witha fluorimeter.

[0108] In yet other embodiments, reverse-transcriptase PCR (RT-PCR)is used to detect the expression of RNA of the biomarkers of theinvention. In RT-PCR, RNA is enzymatically converted tocomplementary DNA or "cDNA" using a reverse transcriptase enzyme.The cDNA is then used as a template for a PCR reaction. PCRproducts can be detected by any suitable method, including but notlimited to, gel electrophoresis and staining with a DNA specificstain or hybridization to a labeled probe. In some embodiments, thequantitative reverse transcriptase PCR with standardized mixturesof competitive templates method described in U.S. Pat. Nos.5,639,606, 5,643,765, and 5,876,978 (each of which is hereinincorporated by reference) is utilized.

[0109] In other embodiments, gene expression of a biomarker of theinvention is detected by measuring the expression of thecorresponding protein or polypeptide. Protein expression may bedetected by any suitable method. In some embodiments, proteins aredetected by immunohistochemistry. In other embodiments, proteinsare detected by their binding to an antibody raised against theprotein. The generation of antibodies is described below.

[0110] Antibody binding is detected by techniques known in the art(e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),"sandwich" immunoassays, immunoradiometric assays, gel diffusionprecipitation reactions, immunodiffusion assays, in situimmunoassays (e.g., using colloidal gold, enzyme or radioisotopelabels, for example), Western blots, precipitation reactions,agglutination assays (e.g., gel agglutination assays,hemagglutination assays, etc.), complement fixation assays,immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.

[0111] In one embodiment, antibody binding is detected by detectinga label on the primary antibody. In another embodiment, the primaryantibody is detected by detecting binding of a secondary antibodyor reagent to the primary antibody. In a further embodiment, thesecondary antibody is labeled. Many methods are known in the artfor detecting binding in an immunoassay and are within the scope ofthe present invention.

[0112] In some embodiments, an automated detection assay isutilized. Methods for the automation of immunoassays include thosedescribed in U.S. Pat. Nos. 5,885,530, 4,981,785, 6,159,750, and5,358,691, each of which is herein incorporated by reference. Insome embodiments, the analysis and presentation of results is alsoautomated. For example, in some embodiments, software thatgenerates a prognosis based on the presence or absence of a seriesof proteins corresponding to neoplastic disease markers isutilized.

[0113] In other embodiments, the immunoassay described in U.S. Pat.Nos. 5,599,677 and 5,672,480; each of which is herein incorporatedby reference.

[0114] In yet other embodiments, the present invention provideskits for the detection and characterization of biomarkers of theinvention. In some preferred embodiments, the kit contains s. Insome embodiments, the kits contain antibodies specific for abiomarker of the invention, in addition to detection reagents andbuffers. In other embodiments, the kits contain reagents specificfor the detection of mRNA or cDNA (e.g., oligonucleotide probes orprimers). In preferred embodiments, the kits contain all of thecomponents necessary to perform a detection assay, including allcontrols, directions for performing assays, and any necessarysoftware for analysis and presentation of results.

[0115] In some embodiments, in vivo imaging techniques are used tovisualize the expression of a biomarker of the invention in ananimal (e.g., a human or non-human mammal). For example, in someembodiments, a biomarker mRNA or protein is labeled using anlabeled antibody specific for the biomarker. A specifically boundand labeled antibody can be detected in an individual using an invivo imaging method, including, but not limited to, radionuclideimaging, positron emission tomography, computerized axialtomography, X-ray or magnetic resonance imaging method,fluorescence detection, and chemiluminescent detection. Methods forgenerating antibodies to the biomarkers of the present inventionare described herein.

[0116] The in vivo imaging methods of the present invention areuseful in the diagnosis of cancers that express a biomarker of theinvention of the present invention (e.g., cancerous cells ortissue). In vivo imaging is used to visualize the presence of abiomarker. Such techniques allow for diagnosis without the use of abiopsy. The in vivo imaging methods of the present invention arealso useful for providing prognoses to neoplastic disease patients.For example. the presence of a biomarker indicative of anaggressive neoplastic disease likely to metastasize or likely torespond to a certain treatment can be detected. The in vivo imagingmethods of the present invention can further be used to detect a(e.g., one that has metastasized) in other parts of the body.

[0117] In some embodiments, reagents (e.g., antibodies) specificfor biomarkers of the present invention are fluorescently labeled.The labeled antibodies are introduced into a subject (e.g., orallyor parenterally). Fluorescently labeled antibodies are detectedusing any suitable method (e.g., using the apparatus described inU.S. Pat. No. 6,198,107, herein incorporated by reference).

[0118] In some embodiments, flow-cytometry is utilized to monitor(e.g., detect) a marker (e.g., a biomarker of the presentinvention) (See, e.g., Example 1). The use of flow-cytometry toidentify and/or isolate and/or purify cell populations is wellknown in the art (See, e.g., Givan, Methods Mol Biol 263, 1-32(2004)).

[0119] In other embodiments, antibodies are radioactively labeled.The use of antibodies for in vivo diagnosis is well known in theart. Sumerdon et al., (Nucl. Med. Biol 17:247-254 (1990) havedescribed an optimized antibody-chelator for theradioimmunoscintographic imaging of tumors using Indium-111 as thelabel. Griffin et al., (J Clin One 9:631-640 (1991)) have describedthe use of this agent in detecting tumors in patients suspected ofhaving recurrent neoplastic disease. The use of similar agents withparamagnetic ions as labels for magnetic resonance imaging is knownin the art (Lauffer, Magnetic Resonance in Medicine 22:339-342(1991)). The label used will depend on the imaging modality chosen.Radioactive labels such as Indium-111, Technetium-99m, orIodine-131 can be used for planar scans or single photon emissioncomputed tomography (SPECT). Positron emitting labels such asFluorine-19 can also be used for positron emission tomography(PET). For MRI, paramagnetic ions such as Gadolinium (III) orManganese (II) can be used.

[0120] Radioactive metals with half-lives ranging from 1 hour to3.5 days are available for conjugation to antibodies, such asscandium-47 (3.5 days) gallium-67 (2.8 days), gallium-68 (68minutes), technetiium-99m (6 hours), and indium-111 (3.2 days), ofwhich gallium-67, technetium-99m, and indium-111 are preferable forgamma camera imaging, gallium-68 is preferable for positronemission tomography.

[0121] The present invention relates to methods using thebiomarkers of the invention as an assayable biomarker in cell,fluid, and/or tissue samples obtained from individuals havingneoplastic disease or suspected to have neoplastic disease,including e.g., various breast neoplasms, cancers and tumors, andalso including primary disease samples. In this embodiment, thepresent invention provides substrate specific enzyme assays thatare performed on samples, e.g. a specimen obtained from a breastbiopsy or aspiration, to determine the enzyme level and activity ofthe biomarkers of the invention in the samples.

[0122] Therapeutic Compounds

[0123] The markers and marker sets of the present invention assessthe likelihood of short or long term survival in neoplastic diseasepatients, e.g., patients having breast neoplastic disease. Usingthis prediction, neoplastic disease therapies can be evaluated todesign a therapy regimen best suited for patients.

[0124] Known angiogenesis inhibitors that may used in methods ofthe invention include, but are not limited to, both direct andindirect angiogenesis inhibitors such as Angiostatin, bevacizumab(Avastin), Arresten, Canstatin, Combretastatin, Endostatin, NM-3,Thrombospondin, Tumstatin, 2-methoxyestradiol, and Vitaxin, ZD1839(Iressa; getfitinib), ZD6474, OS1774 (tarceva), CI1033, PKI1666,IMC225 (Erbitux), PTK787, SU6668, SU11248, Herceptin, Marimastat,COL-3, Neovastat, 2-ME, SU6668, anti-VEGF antibody, Medi-522(Vitaxin II), tumstatin, arrestin, recombinant EPO, troponin I,EMD121974, and IFN CELEBREX.RTM. (celecoxib), and THALOMID.RTM.(thalidomide), have also been recognized as angiogenesis inhibitors(Kerbel et al., Nature Reviews, Vol. 2, October 2002, pp. 727). Afurther example of an anti-angiogenic compound includes, but is notlimited to PD 0332991 (see Fry, D. W. et al. Specific inhibition ofcyclin-dependent kinase 4/6 by PD 0332991 and associated antitumoractivity in human tumor xenografts. Mol Cancer Ther.2004;3:1427-1438). Suitable antiangiogenic compositions include,but are not limited to Galardin (GM6001, Glycomed, Inc., Alameda,Calif.), endothelial response inhibitors (e.g., agents such asinterferon alpha, TNP-470, and vascular endothelial growth factorinhibitors), agents that prompt the breakdown of the cellularmatrix (e.g., Vitaxin (human LM-609 antibody, Ixsys Co., San Diego,Calif.; Metastat, CollaGenex, Newtown, Pa.; and Marimastat BB2516,British Biotech), and agents that act directly on vessel growth(e.g., CM-101, which is derived from exotoxin of Group AStreptococcus antigen and binds to new blood vessels inducing anintense host inflammatory response; and Thalidomide). Preferredanti-angiogenic inhibitors include, for example, bevacizumab,getfitinib thalidomide, tarceva, celecoxib, erbitux, arrestin,recombinant EPO, troponin I, herceptin. Dosages and routes ofadministration for these Food and Drug Administration (FDA)approved therapeutic compound are known to those of ordinary skillin the art as a matter of the public record.

[0125] Several kinds of steroids have also been noted to exertantiangiogenic activity. In particular, several reports haveindicated that medroxyprogesterone acetate (MPA), a syntheticprogesterone, potently inhibited neovascularization in the rabbitcorneal assay (Oikawa (1988) Cancer Lett. 43: 85). A pro-drug of5FU, 5'-deoxy-5-fluorouridine (5DFUR), might be also characterizedas an antiangiogenic compound, because 5'DFUR is converted to 5-FUby the thymidine phosphorylase activity of PD-ECGF/TP. 5'DFUR mightbe selectively active for PD-ECGF/TP positive tumor cells with highangiogenesis potential. Recent clinical investigations in showedthat 5DFUR is likely to be effective for PD-ECGF/TP-positivetumors. It was showed that a dramatic enhancement of antitumoreffect of 5'DFUR appeared in PD-ECGF/TP transfected cells comparedwith untransfected wild-type cells (Haraguchi (1993) Cancer Res.53: 5680 5682). In addition, combined 5DFUR+MPA compounds are alsoeffective antiangiogenics (Yayoi (1994) Int J Oncol. 5: 27 32). Thecombination of the 5'DFUR+MPA might be categorized as a combinationof two angiogenesis inhibitors with different spectrums, anendothelial growth factor inhibitor and a protease inhibitor.Furthermore, in in-vivo experiments using DMBA-induced rat mammarycarcinomas, 5'DFUR exhibited a combination effect with AGM-1470(Yamamoto (1995) Oncol Reports 2:793 796).

[0126] Another group of antiangiogenic compounds for use in thisinvention include polysaccharides capable of interfering with thefunction of heparin-binding growth factors that promoteangiogenesis (e.g., pentosan polysulfate).

[0127] Other modulators of angiogenesis include platelet factor IV,and AGM 1470. Still others are derived from natural sourcescollagenase inhibitor, vitamin D3-analogues, fumigallin, herbimycinA, and isoflavones.

[0128] Therapeutic agents for use in the methods of the inventioninclude, for example, a class of therapeutic agents known asproteosome inhibitors. As used herein, the term "proteasomeinhibitor" refers to any substance which directly inhibitsenzymatic activity of the 20S or 26S proteasome in vitro or invivo. In some embodiments, the proteasome inhibitor is a peptidylboronic acid. Examples of peptidyl boronic acid proteasomeinhibitors suitable for use in the methods of the invention aredisclosed in Adams et al., U.S. Pat. No. 5,780,454 (1998), U.S.Pat. No. 6,066,730 (2000), U.S. Pat. No. 6,083,903 (2000); U.S.Pat. No. 6,297,217 (2001), U.S. Pat. No. 6,465,433 (2002), U.S.Pat. No. 6,548,668 (2003), U.S. Pat. No. 6,617,317 (2003), and U.S.Pat. No. 6,747,150 (2004), each of which is hereby incorporated byreference in its entirety, including all compounds and formulaedisclosed therein. Preferably, the peptidyl boronic acid proteasomeinhibitor is selected from the group consisting of: N(4morpholine)carbonyl-.beta.-(1-naphthyl)-L-alanine-L-leucine boronicacid; N(8quinoline)sulfonyl-.beta.-(1-naphthyl)-L-alanine-L-alanine-L-leucineboronic acid; N(pyrazine)carbonyl-L-phenylalanine-L-leucine boronicacid, and N(4morpholine)-carbonyl-[O-(2-pyridylmethyl)]L-tyrosine-L-leucineboronic acid. In a particular embodiment, the proteasome inhibitoris N (pyrazine)carbonyl-L-phenylalanine-L-leucine boronic acid(bortezomib; VELCADE.RTM.; formerly known as MLN341 or PS-341).

[0129] Additional peptidyl boronic acid proteasome inhibitors aredisclosed in Siman et al., international patent publication WO99/30707; Bernareggi et al., international patent publication WO05/021558; Chatterjee et al., international patent publication WO05/016859; Furet et al., U.S. patent publication 2004/0167337;Furet et al., international patent publication 02/096933; Attwoodet al., U.S. Pat. No. 6,018,020 (2000); Magde et al., internationalpatent publication WO 04/022070; and Purandare and Laing,international patent publication WO 04/064755.

[0130] Additionally, proteasome inhibitors include peptide aldehydeproteasome inhibitors, such as those disclosed in Stein et al.,U.S. Pat. No. 5,693,617 (1997); Siman et al., international patentpublication WO 91/13904; Iqbal et al., J. Med. Chem. 38:2276-2277(1995); and linuma et al., international patent publication WO05/105826, each of which is hereby incorporated by reference in itsentirety.

[0131] Additionally, proteasome inhibitors include peptidyl epoxyketone proteasome inhibitors, examples of which are disclosed inCrews et al., U.S. Pat. No. 6,831,099; Smyth et al., internationalpatent publication WO 05/111008; Bennett et al., internationalpatent publication WO 06/045066; Spaltenstein et al. TetrahedronLett. 37:1343 (1996); Meng, Proc. Natl. Acad. Sci. 96: 10403(1999); and Meng, Cancer Res. 59: 2798 (1999), each of which ishereby incorporated by reference in its entirety.

[0132] Additionally, proteasome inhibitors include alpha-ketoamideproteasome inhibitors, examples of which are disclosed inChatterjee and Mallamo, U.S. Pat. No. 6,310,057 (2001) and U.S.Pat. No. 6,096,778 (2000); and Wang et al., U.S. Pat. No. 6,075,150(2000) and U.S. Pat. No. 6,781,000 (2004), each of which is herebyincorporated by reference in its entirety.

[0133] Additional proteasome inhibitors include peptidyl vinylester proteasome inhibitors, such as those disclosed in Marastoniet al., J. Med. Chem. 48:5038 (2005), and peptidyl vinyl sulfoneand 2-keto-1,3,4-oxadiazole proteasome inhibitors, such as thosedisclosed in Rydzewski et al., J. Med. Chem. 49:2953 (2006); andBogyo et al., Proc. Natl. Acad. Sci. 94:6629 (1997), each of whichis hereby incorporated by reference in its entirety.

[0134] Additional proteasome inhibitors include azapeptoids andhydrazinopeptoids, such as those disclosed in Bouget et al.,Bioorg. Med. Chem. 11:4881 (2003); Baudy-Floc'h et al.,international patent publication WO 05/030707; and Bonnemains etal., international patent publication WO 03/018557, each of whichis hereby incorporated by reference in its entirety.

[0135] Furthermore, proteasome inhibitors include peptidederivatives, such as those disclosed in Furet et al., U.S. patentpublication 2003/0166572, and efrapeptin oligopeptides, such asthose disclosed in Papathanassiu, international patent publicationWO 05/115431, each of which is hereby incorporated by reference inits entirety.

[0136] Further, proteasome inhibitors include lactacystin andsalinosporamide and analogs thereof, which have been disclosed inFenteany et al., U.S. Pat. No. 5,756,764 (1998), U.S. Pat. No.6,147,223 (2000), U.S. Pat. No. 6,335,358 (2002), and U.S. Pat. No.6,645,999 (2003); Fenteany et al., Proc. Natl. Acad. Sci. USA(1994) 91:3358; Fenical et al., international patent publication WO05/003137; Palladino et al., international patent publication WO05/002572; Stadler et al., international patent publication WO04/071382; Xiao and Patel, U.S. patent publication 2005/023162; andCorey, international patent publication WO 05/099687, each of whichis hereby incorporated by reference in its entirety.

[0137] Further, proteasome inhibitors include naturally occurringcompounds shown to have proteasome inhibition activity can be usedin the present methods. For example, TMC-95A, a cyclic peptide, andgliotoxin, a fungal metabolite, have been identified as proteasomeinhibitors. See, e.g., Koguchi, Antibiot. (Tokyo) 53:105 (2000);Kroll M, Chem. Biol. 6:689 (1999); and Nam S, J. Biol. Chem. 276:13322 (2001), each of which is hereby incorporated by reference inits entirety. Additional proteasome inhibitors include polyphenolproteasome inhibitors, such as those disclosed in Nam et al., J.Biol. Chem. 276:13322 (2001); and Dou et al., U.S. patentpublication 2004/0186167, each of which is hereby incorporated byreference in its entirety.

[0138] Preferred proteasome inhibitors include, for example,bortezomib. Dosages and routes of administration for Food and DrugAdministration (FDA) approved therapeutic compounds are known tothose of ordinary skill in the art as a matter of the publicrecord.

[0139] Preferred angiogenesis inhibitors and other anti-neoplasticdisease compounds, for use in the methods of the invention include,for example, 17-AAG, Apatinib, Ascomycin, Axitinib, Bexarotene,Bortezomib, Bosutinib, Bryostatin 1, Bryostatin 2, Canertinib,Carboplatin, Cediranib, Cisplatin, Cyclopamine, Dasatinib, 17-DMAG,Docetaxel, Doramapimod, Dovitinib, Erlotinib, Everolimus,Gefitinib, Geldanamycin, Gemcitabine, Imatinib, Imiquimod, Ingenol3-Angelate, Ingenol 3-Angelate 20-Acetate, Irinotecan, Lapatinib,Lestaurtinib, Nedaplatin, Masitinib, Mubritinib, Nilotinib,NVP-BEZ235, OSU-03012, Oxaliplatin, Paclitaxel, Pazopanib,Picoplatin, Pimecrolimus, PKC412, Rapamycin, Satraplatin,Sorafenib, Sunitinib, Tandutinib, Tivozanib, Thalidomide,Temsirolimus, Tozasertib, Vandetanib, Vargatef, Vatalanib,Zotarolimus, ZSTK474, Bevacizumab (Avasti). Cetuximab, Herceptin,Rituximab, Trastuzumab.

[0140] Preferred protein kinase inhibitors for use in the methodsof the invention include, for example, Apatinib, Axitinib,Bisindolylmaleimide I, Bisindolylmaleimide I, Bosutinib,Canertinib, Cediranib, Chelerythrine, CP690550, Dasatinib,Dovitinib. Erlotinib, Fasudil, Gefitinib, Genistein, Go 6976, H-89,HA-1077, Imatinib, K252a, K252c, Lapatinib, Di-p-Toluenesulfonate,Lestaurtinib, LY 294002, Masitinib, Mubritinib, Nilotinib,OSU-03012, Pazopanib, PD 98059, PKC412, Roscovitine, SB 202190, SB203580, Sorafenib, SP600125, Staurosporine, Sunitinib, Tandutinib,Tivozanib, Tozasertib, Tyrphostin AG 490, Tyrphostin AG 1478,U0126, Vandetanib, Vargatef Vatalanib, Wortmannin, ZSTK474.Preferred Hedgehog and Smoothened (Smo) Inhibitors for use in themethods of the invention include, for example, Cyclopamine.

[0141] Platinum-based Anti-Cancer Compounds for use in the methodsof the invention include, for example, Carboplatin, Cisplatin,Eptaplatin, Nedaplatin, Oxaliplatin, Picoplatin, Satraplatin.Proteasome Inhibitors for use in the methods of the inventioninclude, for example, Bortezomib (Velcade). Anti-Diabetes Drugs foruse in the methods of the invention include, for example,Metformin.

[0142] Fibrosis Inhibitors for use in the methods of the inventioninclude, for example, Halofuginone. Metformin, N-acetyl-cysteine(NAC). NfkB Inhibitors for use in the methods of the inventioninclude, for example, RTA 402 (Bardoxolone methyl), Auranofin,BMS-345541, IMD-0354, PS-1145, TPCA-1, Wedelolactone. HIFInhibitors for use in the methods of the invention include, forexample, Echinomycin. Glycolysis Inhibitors for use in the methodsof the invention include, for example, 2-deoxy-D-glucose (2-DG),2-bromo-D-glucose, 2-fluoro-D-glucose, and 2-iodo-D-glucose,dichloro-acetate (DCA), 3-chloro-pyruvate, 3-Bromo-pyruvate(3-BrPA), 3-Bromo-2-oxopropionate, Oxamate.

[0143] PI-3 Kinase, Akt, and mTOR inhibitors for use in the methodsof the invention include, for example, LY 294002, NVP-BEZ235,Rapamycin, Wortmannin. Isoflavones for use in the methods of theinvention include, for example, Quercetin, and Resveratrol.Anti-Oxidants for use in the methods of the invention include, forexample, N-acetyl-cysteine (NAC), N-acetyl-cysteine amide(NACA).

[0144] Immunosuppressants for use in the methods of the inventioninclude, for example, Ascomycin, CP690550, Cyclosporin A,Everolimus, Fingolimod, FK-506, Mycophenolic Acid, Pimecrolimus,Rapamycin, Temsirolimus, Zotarolimus, and AR-C117977, whichinhibits monocarboxylate transporter 1 (MCT1). Cyclin dependentkinase inhibitors (CDK) inhibitors for use in the methods of theinvention include, for example, Roscovitine, and PD 0332991 (CDK4/6inhibitor). Lysosomal acidification inhibitors for use in themethods of the invention include, for example, Chloroquine. PARPInhibitors for use in the methods of the invention include, forexample, BSI-201, Olaparib, DR 2313, NU 1025.

[0145] Abraxane.RTM. is an albumin-bound paclitaxel nanoparticlesformulation as an injectable suspension for the treatment ofmetastatic breast cancer. It contains albumin-bound paclitaxel forthe treatment of metastatic breast cancer. Schaumburg, III: AbraxisOncology, a Division of American Pharmaceutical Partners, Inc;January 2005). See O'Shaughnessy, J. A. et al. (2004). "WeeklyNanoparticle Albumin Paclitaxel (Abraxane) Results in Long-TermDisease Control in Patients With Taxane-Refractory MetastaticBreast Cancer," Breast Cancer Research and Treatment, 27.sup.thAnnual Charles A. Coltman San Antonio Breast Cancer Symposium, SanAntonio, Tex., Dec. 8-11, 2004, 88(1):S65, Abstract No. 1070.

[0146] Compounds described herein can be administered to a humanpatient per se, or in pharmaceutical compositions mixed withsuitable carriers or excipient(s). Techniques for formulation andadministration of the compounds of the instant application may befound in "Remington's Pharmaceutical Sciences," Mack PublishingCo., Easton, Pa., latest edition. Suitable routes of administrationmay, for example, include oral, rectal, transmucosal, or intestinaladministration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal,direct intraventricular, intravenous, intraperitoneal, intranasal,or intraocular injections. Pharmaceutical compositions suitable foruse in the present invention include compositions wherein theactive ingredients are contained in an amount effective to achieveits intended purpose. More specifically, a therapeuticallyeffective amount means an amount of compound effective to prevent,alleviate or ameliorate symptoms of disease or prolong the survivalof the subject being treated. Determination of a therapeuticallyeffective amount is well within the capability of those skilled inthe art, especially in light of the detailed disclosure providedherein.

[0147] Antibodies

[0148] The invention provides antibodies to biomarker proteins, orfragments of biomarker proteins. e.g., ACLY, HMGCS1, HMGCS2, HMGCL,HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, miR-31, miR-34c, ACAT1, ACAT2,OXCT1, OXCT2. ADMA, 3-hydroxybutyrate, and combinations thereof.The term "antibody" as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulin(Ig) molecules, i.e., molecules that contain an antigen bindingsite that specifically binds (immunoreacts with) an antigen. Suchantibodies include, but are not limited to, polyclonal, monoclonal,chimeric, single chain, Fab, Fab and F(ab)2 fragments, and an Fabexpression library, in general, an antibody molecule obtained fromhumans relates to any of the classes IgG, IgM, IgA, IgE and IgD,which differ from one another by the nature of the heavy chainpresent in the molecule. Certain classes have subclasses as well,such as IgG1, IgG2, and others. Furthermore, in humans, the lightchain may be a kappa chain or a lambda chain. Reference herein toantibodies includes a reference to all such classes, subclasses andtypes of human antibody species.

[0149] Predictive Medicine

[0150] The invention also pertains to the field of predictivemedicine in which diagnostic assays, prognostic assays,pharmacogenomics, and monitoring clinical trials are used forprognostic (predictive) purposes to thereby treat an individualprophylactically. Accordingly, one aspect of the invention relatesto diagnostic assays for determining biomarker protein expressionas well as biomarker activity, in the context of a biologicalsample (e.g., blood, serum, cells, tissue) to thereby determinewhether an individual is afflicted with a disease or disorder, oris at risk of developing a disorder, associated with aberrantbiomarker expression or activity. The disorders include cellproliferative disorders such as neoplastic disease. The inventionalso provides for prognostic (or predictive) assays for determiningwhether an individual is at risk of developing, a disorderassociated with biomarker protein expression or activity. Suchassays may be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of adisorder characterized by or associated with protein, nucleic acidexpression, or biological activity, wherein the biomarker is e.g.,ACLY, HMGCS1, HMGCS2, HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L,miR-31, miR-34c, ACAT1, ACAT2, OXCT1, OXCT2, ADMA,3-hydroxybutyrate, and combinations thereof.

[0151] Another aspect of the invention provides methods fordetermining biomarker protein expression or activity in anindividual to thereby select appropriate therapeutic orprophylactic agents for that individual (referred to herein as"pharmacogenomics"). Pharmacogenomics allows for the selection ofagents (e.g., drugs) for therapeutic or prophylactic treatment ofan individual based on the genotype of the individual (e.g., thegenotype of the individual examined to determine the ability of theindividual to respond to a particular agent.)

[0152] Yet another aspect of the invention pertains to monitoringthe influence of agents (e.g. drugs, compounds) on the expressionor activity of biomarker in clinical trials.

[0153] Diagnostic Assays

[0154] An exemplary method for detecting the presence or absence ofthe biomarkers of the invention in a biological sample involvesobtaining a biological sample from a test subject and contactingthe biological sample with a compound or an agent capable ofdetecting biomarker protein such that the presence of biomarker isdetected in the biological sample, wherein the biological sampleincludes, for example, cells, and/or physiological fluids.

[0155] An agent for detecting biomarker protein is an antibodycapable of binding to biomarker protein, preferably an antibodywith a detectable label. Antibodies can be polyclonal, or morepreferably, monoclonal. An intact antibody, or a fragment thereof(e.g., Fab or F(ab')2) can be used. The term "labeled", faithregard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physicallylinking) a detectable substance to the probe or antibody, as wellas indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirectlabeling include detection of a primary antibody using afluorescently-labeled secondary antibody and end-labeling of a DNAprobe with biotin such that it can be detected withfluorescently-labeled streptavidin. The term "biological sample" isintended to include tissues, cells and biological fluids isolatedfrom a subject, as well as tissues, cells and fluids present withina subject. That is, the detection method of the invention can beused to detect biomarker protein in a biological sample in vitro aswell as in vivo. For example, in vitro techniques for detection ofbiomarker protein include enzyme linked immunosorbent as (ELISA),Western blot, immunoprecipitation, and immunofluorescence.Furthermore, in vitro techniques for detection of biomarker proteininclude introducing into a subject a labeled anti-biomarkerantibody. For example the antibody can be labeled with aradioactive marker whose presence and location in a subject can bedetected by standard imaging techniques.

[0156] In another embodiment, the methods further involve obtaininga control biological sample from a control subject, contacting thecontrol sample with a compound or agent capable of detectingbiomarker protein, for example, ACLY, HMGCS1, HMGCS2, HMGCL,HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, miR-31, miR-34c, ACAT1, ACAT2,OXCT1, OXCT2, ADMA, 3-hydroxybutyrate, and combinations thereof, isdetected in the biological sample, and comparing the presence ofbiomarker protein, or lack thereof in cells, for example controlcells, compared to the control sample with the presence ofbiomarker protein, in the test sample.

[0157] The invention also encompasses kits for detecting thepresence of the biomarkers of the invention in a biological sample.For example, the kit can comprise: a labeled compound or agentcapable of detecting, for example, ACLY, HMGCS1, HMGCS2, HMGCL,HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, miR-31, miR-34c, ACAT1, ACAT2,OXCT1, OXCT2, ADMA, 3-hydroxybutyrate protein in a biologicalsample, for example cells; means for determining the amount of thebiomarkers of the invention in the sample; and means for comparingthe amount of the biomarkers of the invention in the sample with astandard. The compound or agent can be packaged in a suitablecontainer. The kit can further comprise instructions for using thekit to detect the biomarkers of the invention protein in, forexample, cells.

[0158] In yet other embodiments, the present invention provideskits for the detection, characterization, and/or treatment ofneoplastic disease. In some embodiments, the kits containantibodies specific for biomarkers (e.g., ACLY, HMGCS1, HMGCS2,HMGCL, HMGCLL1, BDH1, BDH2, BNIP3, BNIP3L, miR-31, miR-34c, ACAT1,ACAT2, OXCT1, OXCT2, ADMA, 3-hydroxybutyrate, or combinationsthereof). In some embodiments, the kits further contain detectionreagents and buffers. In other embodiments, the kits containreagents specific for the detection of nucleic acids (e.g., DNA,RNA, mRNA or cDNA, oligonucleotide probes or primers). In preferredembodiments, the kits contain all of the components necessaryand/or sufficient to perform a detection assay, including allcontrols, directions for performing assays, and any necessarysoftware for analysis and presentation of results.

[0159] Prognostic Assays

[0160] The diagnostic methods described herein can furthermore beutilized to identify subjects having or at risk of developing adisease or disorder associated with aberrant expression or activityof the biomarkers of the invention. For example, the assaysdescribed herein. Such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having orat risk of developing a disorder associated with protein, nucleicacid expression or activity of the biomarkers of the invention.Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing, a disease or disorder.Thus the invention provides method for identifying a disease ordisorder in which a test sample is obtained from a subject andbiomarker protein is detected, wherein the presence or absence ofbiomarker protein in cells is diagnostic for a subject having or atrisk of developing a disease or disorder such as neoplastic diseaseor disorder. As used herein, a "test sample" refers to a biologicalsample obtained from a subject of interest. For example, a testsample can be a biological fluid (e.g. serum), cell sample, and/ortissue, including but not limited to cells.

[0161] Furthermore, the prognostic assays described herein can beused to determine whether a subject can be administered an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide,nucleic acid, small molecule, or other drug candidate) to treat adisease or disorder. For example, such methods can be used todetermine whether a subject can be effectively treated with anagent for a disorder. Thus, the invention provides methods fordetermining whether a subject can be effectively treated with anagent for a disorder in which a test sample is obtained andbiomarker protein expression or activity is detected (e.g., hereinthe presence of biomarker protein is diagnostic for a subject thatcan be administered the agent to treat, for example, a neoplasticdisorder).

[0162] The methods described herein may be performed, for example,by utilizing pre-packaged diagnostic kits comprising at least oneantibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptomsor family history of a disease or illness.

[0163] The term "control" refers, for example, to a cell or groupof cells that is exhibiting common characteristics for theparticular cell type from which the cell or group of cells wasisolated. A normal cell sample does not exhibit tumorigenicpotential, metastatic potential, or aberrant growth in vivo or invitro. A normal control cell sample can be isolated from tissues ina subject that is not suffering from neoplastic disease. It may notbe necessary to isolate a normal control cell sample each time acell sample is tested for neoplastic disease as long as the normalcontrol cell sample allows for probing during the testingprocedure. In some embodiments, the levels of expression of theprotein markers in the cell sample are compared to the levels ofexpression of the protein markers in a normal control cell sampleof the same tissue type as the cell sample.

[0164] A "control" refers, for example, to a sample of biologicalmaterial representative of healthy, neoplastic disease-freeanimals, and/or cells or tissues. The level of the biomarkers ofthe invention in a control sample is desirably typical of thegeneral population of normal, neoplastic disease-free animals or ofa particular individual at a particular time (e.g. before, duringor after a treatment regimen), or in a particular tissue. Thissample can be removed from an animal expressly for use in themethods described in this invention, or can be any biologicalmaterial representative of normal, neoplastic disease-free animals,including neoplastic disease-free biological material taken from ananimal with neoplastic disease elsewhere in its body. A controlsample can also refer to an established level of the biomarkers ofthe invention, representative of the neoplastic disease-freepopulation, that has been previously established based onmeasurements from normal, neoplastic disease-free animals. In oneembodiment, the control may be adjacent normal tissue. In oneembodiment, the control may be any commonly used positive ornegative controls. In one embodiment, the control is anon-invasive,non-metastatic control sample. Kits may also comprise, for example,positive and negative control samples for quality controlpurposes.

[0165] In preferred embodiments, the level of activity of one ormore proteasomal peptidases in a test sample is used in conjunctionwith clinical factors other than proteasomal peptidase activity todiagnose a disease. In these embodiments, the level of proteasomeactivity measured in the test sample is compared to a referencevalue to determine if the levels of activity are elevated orreduced relative to the reference value. Preferably, the referencevalue is the proteasomal peptidase activity measured in acomparable sample from one or more healthy individuals. An increaseor decrease in proteasome activity may be used in conjunction withclinical factors other than proteasomal peptidase activity todiagnose a disease.

[0166] The term "elevated levels" or "higher levels" as used hereinrefers to levels of a proteasome peptidase activity, that arehigher than what would normally be observed in a comparable samplefrom control or normal subjects (i.e., a reference value). In someembodiments of the invention "control levels" (i.e. normal levels)refer to a range of biomarker or biomarker activity levels thatwould be normally be expected to be observed in a mammal that doesnot have a neoplastic disorder and "elevated levels" refer tobiomarker or biomarker activity levels that are above the range ofcontrol levels. The ranges accepted as "elevated levels" or"control levels" are dependant on a number of factors. For example,one laboratory may routinely determine absolute levels of anactivity of an enzyme in a sample that are different than theabsolute levels obtained for the same sample by another laboratory.Also, different assay methods may achieve different value ranges.Value ranges may also differ in various sample types, for exampledifferent body fluids or by different treatments of the sample. Oneof ordinary skill in the art is capable of considering the relevantfactors and establishing appropriate reference ranges for "controlvalues" and "elevated values" of the present invention. Forexample, a series of samples from control subjects and subjectsdiagnosed with neoplastic disorders can be used to establish rangesthat are "normal" or "control" levels and ranges that are"elevated" or "higher" than the control range.

[0167] Similarly, "reduced levels" or "lower levels" as used hereinrefer to levels of a biomarker or biomarker activity that are lowerthan what would normally be observed in a comparable sample fromcontrol or normal subjects (i.e., a reference value). In someembodiments of the invention "control levels" (i.e. normal levels)refer to a range of biomarker or biomarker activity levels thatwould be normally be expected to be observed in a mammal that doesnot have a hematological disorder and "reduced levels" refer tobiomarker or biomarker activity levels that are below the range ofsuch control levels.

[0168] Monitoring of Effects During Clinical Trials

[0169] Monitoring the influence of agents (e.g., drugs, compounds)on the expression or activity of the biomarkers of the invention(e.g., the ability to modulate aberrant cell proliferation and/ordifferentiation) can be applied not only in basic drug screening,but also in clinical trials. For example, the effectiveness of anagent determined by a screening assay as described herein toincrease biomarker gene expression, protein levels, or upregulatebiomarker activity, can be monitored in clinical trails of subjectsexhibiting, for example, increased biomarker expression, proteinlevels, or downregulated biomarker activity or expression, forexample in cells. Alternatively, the effectiveness of an agentdetermined by a screening assay to decrease biomarker expression,protein levels, or downregulate biomarker activity or expression,can be monitored in clinical trails of subjects exhibitingincreased biomarker expression, protein levels, or upregulatedbiomarker activity. In such clinical trials, the expression oractivity of biomarker and, preferably, other genes that have beenimplicated in, for example, a cellular proliferation or immunedisorder can be used as a "read out" or markers of the immuneresponsiveness of a particular cell.

[0170] In one embodiment, the invention provides a method formonitoring the effectiveness of treatment of a subject with anagent (e.g., an agonist, antagonist, protein, peptide,peptidomimetic, nucleic acid, small molecule, or other drugcandidate) comprising the steps of (i) obtaining apre-administration sample from a subject prior to administration ofthe agent: (ii) detecting the level of expression of a biomarkerprotein, in the preadministration sample; (iii) obtaining one ormore post-administration samples from the subject; (ill) detectingthe level of expression or activity of the biomarker protein, inthe post-administration samples; (v) comparing the level ofexpression or activity of the biomarker protein, in thepre-administration sample with the biomarker protein, in the postadministration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. Forexample, increased administration of the agent may be desirable toincrease the expression or activity of biomarker to higher levelsthan detected, i.e., to increase the effectiveness of the agent.Alternatively, decreased administration of the agent may bedesirable to decrease expression or activity of biomarker to lowerlevels than detected, i.e., to decrease the effectiveness of theagent.

[0171] Methods of Treatment

[0172] The invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) adisorder or having a disorder associated with aberrant expressionor activity of the biomarkers of the invention. The disordersinclude, but are not limited to cell proliferative disorders suchas neoplastic disease.

[0173] Prophylactic Methods

[0174] In one aspect, the invention provides a method forpreventing, in a subject, a disease or condition associated with anaberrant expression or activity of the biomarkers of the invention,by administering to the subject an agent that modulates biomarkersexpression or at least one biomarker activity, in for examplecells. Subjects at risk for a disease that is caused or contributedto by aberrant biomarker expression or activity can be identifiedby, for example, any or a combination of diagnostic or prognosticassays as described herein. Administration of a prophylactic agentcan occur prior to the manifestation of symptoms characteristic ofthe biomarker aberrancy, such that a disease or disorder isprevented or, alternatively, delayed in its progression. Dependingupon the type of biomarker aberrancy, for example, a biomarkeragonist or biomarker antagonist agent can be used for treating thesubject. The appropriate agent can be determined based on screeningassays described herein.

[0175] Therapeutic Methods

[0176] Another aspect of the invention pertains to methods ofmodulating expression or activity of the biomarkers of theinvention in, for example cells, for therapeutic purposes. Themodulatory method of the invention involves contacting a cell withan agent that modulates one or more of the activities of biomarkerprotein activity associated with the cell. An agent that modulatesbiomarker protein activity can be an agent as described herein,such as a nucleic acid or a protein, a naturally-occurring cognateligand of a biomarker protein, a peptide. a biomarkerpeptidomimetic, or other small molecule. In one embodiment, theagent stimulates one or more biomarker protein activity. Examplesof such stimulatory agents include active protein and a nucleicacid molecule encoding biomarker that has been introduced into thecell. In another embodiment, the agent inhibits one or morebiomarker protein activity. Examples of such inhibitory agentsinclude antisense biomarker nucleic acid molecules andanti-biomarker antibodies. These modulatory methods can beperformed in vitro (e.g. by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to asubject). As such, the invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant expression or activity of a biomarker protein molecule. Inone embodiment, the method involves administering an agent (e.g.,an agent identified by a screening assays described herein), orcombination of agents that modulates (e.g., up-regulates ordown-regulates) biomarker expression or activity.

[0177] Stimulation of biomarker activity is desirable in situationsin which biomarker is abnormally downregulated and/or in whichincreased biomarker activity is likely to have a beneficial effect.One example of such a situation is where a subject has a disordercharacterized by aberrant cell proliferation and/or differentiation(e.g., neoplastic disease or immune associated disorders).

[0178] Determination of the Biological Effect of theTherapeutic

[0179] In various embodiments of the invention, suitable in vitroor in vivo assays are performed to determine the effect of aspecific therapeutic and whether its administration is indicatedfor treatment of the affected tissue.

[0180] In various specific embodiments, in vitro assays may beperformed with representative cells of the type(s) involved in thepatient's disorder, to determine if a given Therapeutic exerts thedesired effect upon the cell type(s). Compounds for use in therapymay be tested in suitable animal model systems including, but notlimited to rats, mice, chicken, cows, monkeys, rabbits, and thelike, prior to testing in human subjects. Similarly, for in vivotesting, any of the animal model system known in the art may beused prior to administration to human subjects.

[0181] Kits

[0182] As used herein, the term "label" encompasses chemical orbiological molecules that are used in detecting the presence in asample of a target molecule which is capable of binding to orotherwise interact with the label so as to indicate its presence inthe sample, and the amount of the target molecule in the sample.Examples of such labels include, but not limited to, a nucleic acidprobe such as a DNA probe. or RNA probe, an antibody, aradioisotope, a fluorescent dye, and the like.

[0183] As used herein, the term "usage instruction" includesinstructions in the kit for carrying out the procedure fordetecting the presence of a target molecular such as the biomarkersof the invention in the sample to be tested. In the context of kitbeing used in the United States, the usage instruction comprisingthe statement of intended use required by the U.S. Food and DrugAdministration (FDA) in labeling in vitro diagnostic products. Itwould be apparent to one with ordinary skill in the art of medicaldiagnostic devices as to the format and content of these usageinstructions as required by the FDA.

[0184] As used in the present invention, an appropriate bindingassay for selecting specific biomarker-related angiogenesisinhibitor includes HPLC, immunoprecipitation, fluorescent-bindingassay, capillary electrophoresis, and so forth.

[0185] As used herein, an "anti-angiogenesis assay" is anexperiment where a pool of candidate molecules are screened inorder to discover the effectiveness of the candidate molecules ininhibiting angiogenesis. In order to discover whether a moleculehas anti-angiogenesis property, various methods can be applied tocarry out the present invention. For example, proteins and peptidesderived from these and other sources, including manual or automatedprotein synthesis, may be quickly and easily tested for endothelialproliferation inhibiting activity using a biological activity assaysuch as the bovine capillary endothelial cell proliferation assay.Other bioassays for inhibiting activity include the chick embryonicchorioallantoic membrane (CAM) assay, the mouse corneal assay, andthe effect of administering isolated or synthesized proteins onimplanted tumors. The chick CAM assay is described by O'Reilly, etal. in "Angiogenic Regulation of Metastatic Growth", Cell, vol.79(2), Oct. 21, 1994, pp. 315-328, which is hereby incorporated byreference in its entirety. Additional anti-angiogenesis assays forscreening for angiogenesis inhibitors can be found in Yu, et al.,PNAS, Vol. 101, No. 21, pp 8005-8010 (2004), which is herebyincorporated by reference in its entirety.

[0186] In some embodiments of the invention, methods such as flowcytometry as well as Enzyme-linked Immunosorbent Assay (ELISA)techniques are used for quantification of the biomarkers of theinvention.

[0187] Detection of the protein molecule of the biomarkers of theinvention can be performed using techniques known in the art (e.g.,radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),"sandwich" immunoassays, immunoradiometric assays, gel diffusionprecipitation reactions, immunodiffusion assays, in situimmunoassays (e.g., using colloidal gold, enzyme or radioisotopelabels, for example), Western blots, precipitation reactions,agglutination assays (e.g., gel agglutination assays,hemagglutination assays, etc.), complement fixation assays,immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.

[0188] For example, antibody binding is detected by detecting alabel on the primary antibody. In another embodiment, the primaryantibody is detected by detecting binding of a secondary antibodyor reagent to the primary antibody. In a further embodiment, thesecondary antibody is labeled. Many methods are known in the artfor detecting binding in an immunoassay and are within the scope ofthe present invention.

[0189] In certain cases, an automated detection assay is utilized.Methods for the automation of immunoassays include those describedin U.S. Pat. Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691,each of which is herein incorporated by reference. In someembodiments, the analysis and presentation of results is alsoautomated. For example, in some embodiments, software thatgenerates a prognosis based on the presence or absence of a seriesof proteins corresponding to neoplastic disease markers isutilized.

[0190] Antibodies specific for the biomarkers of the invention aremade according to techniques and protocols well known in the art.The antibodies may be either polyclonal or monoclonal. Theantibodies are utilized in well-known immunoassay formats, such ascompetitive and non-competitive immunoassays, including ELISA,sandwich immunoassays and radioimmunoassays (RIAs), to determinethe presence or absence of the endothelial proliferation inhibitorsof the present invention in body fluids. Examples of body fluidsinclude but are not limited to blood, serum, peritoneal fluid,pleural fluid, cerebrospinal fluid, uterine fluid, saliva, andmucus.

[0191] The present invention provides isolated antibodies that canbe used in the diagnostic kits in the detection of the biomarkersof the invention. In preferred embodiments, the present inventionprovides monoclonal antibodies that specifically bind to thebiomarkers of the invention.

[0192] An antibody against the biomarkers of the invention in thepresent invention may be any monoclonal or polyclonal antibody, aslong as it can recognize the protein. Antibodies can be produced byusing the biomarkers of the invention or its analogues as theantigen using conventional antibody or antiserum preparationprocesses.

[0193] The present invention contemplates the use of bothmonoclonal and polyclonal antibodies. Any suitable method may beused to generate the antibodies used in the methods andcompositions of the present invention, including but not limitedto, those disclosed herein. For example, for preparation of amonoclonal antibody, protein, as such, or together with a suitablecarrier or diluent is administered to an animal (e.g., a mammal)under conditions that permit the production of antibodies. Forenhancing the antibody production capability, complete orincomplete Freund's adjuvant may be administered. Normally, theprotein is administered once every 2 weeks to 6 weeks, in total,about 2 times to about 10 times. Animals suitable for use in suchmethods include, but are not limited to, primates, rabbits, dogs,guinea pigs, mice, rats, sheep, goats, etc.

[0194] For preparing monoclonal antibody-producing cells, anindividual animal whose antibody titer has been confirmed (e.g., amouse) is selected, and 2 days to 5 days after the finalimmunization, its spleen or lymph node is harvested andantibody-producing cells contained therein are fused with myelomacells to prepare the desired monoclonal antibody producerhybridoma. Measurement of the antibody titer in antiserum can becarried out, for example, by reacting the labeled protein, asdescribed hereinafter with the antiserum and then measuring theactivity of the labeling agent bound to the antibody. The cellfusion can be carried out according to known methods, for example,the method described by Koehler and Milstein (Nature 256:495[1975]). As a fusion promoter, for example, Sendai virus (HVJ) or,preferably, polyethylene glycol (PEG), is used.

[0195] Polyclonal antibodies may be prepared by any known method ormodifications of these methods including obtaining antibodies frompatients. For example, a complex of an immunogen (an antigenagainst the protein) and a carrier protein is prepared and ananimal is immunized by the complex according to the same manner asthat described with respect to the above monoclonal antibodypreparation. A material containing the antibody against isrecovered from the immunized animal and the antibody is separatedand purified.

[0196] Methods of linking an antibody to a second agent such as acytotoxic agent in order to form a combination antibody, also knowas an immunotoxic, is well known in the art. Two major advances inthe immunotoxin field have been the use of the recombinant DNAtechnique to produce recombinant toxins with better clinicalproperties and the production of single-chain immunotoxins byfusing the DNA elements encoding combining regions of antibodies,growth factors, or cytokines to a toxin gene.

[0197] First-generation immunotoxins were constructed by couplingtoxins to MAb or antibody fragments using a heterobifunctionalcross-linking agent. It was also discovered that geneticengineering could be used to replace the cell-binding domains ofbacterial toxins with the Fv portions of antibodies or with growthfactors.

[0198] The present invention provides kits for the detection andcharacterization of the biomarkers of the invention in neoplasticdisease diagnostics. In some embodiments, the kits containantibodies specific for the biomarkers of the invention, inaddition to detection reagents and buffers. In other embodiments,the kits contain reagents specific for the detection of thebiomarkers of the invention. In preferred embodiments, the kitscontain all of the components necessary to perform a detectionassay, including all controls, directions for performing assays,and any necessary software for analysis and presentation ofresults.

[0199] Kits containing labels such as antibodies against thebiomarkers of the invention for measurement of the biomarkers ofthe invention are also contemplated as part of the presentinvention. Antibody solution is prepared such that it can detectthe presence of biomarkers peptides in extracts of plasma, urine,tissues, and in cell culture media are further examined toestablish easy to use kits for rapid, reliable, sensitive, andspecific measurement and localization of the biomarkers of theinvention. These assay kits include but are not limited to thefollowing techniques; competitive and non-competitive assays,radioimmunoassay, bioluminescence and chemiluminescence assays,fluorometric assays, sandwich assays, immunoradiometric assays, dotblots, enzyme linked assays including ELISA, microtiter plates,antibody coated strips or dipsticks for rapid monitoring of urineor blood, and immunocytochemistry. For each kit the range,sensitivity, precision, reliability, specificity andreproducibility of the assay are established according to industrypractices that are commonly known to and used by one with ordinaryskill in the art.

[0200] This immunohistochemistry kit provides instructions,biomarker molecules, preferably labeled and linked to a fluorescentmolecule such as fluorescein isothiocyanate, or to some otherreagent used to visualize the primary antiserum.Immunohistochemistry techniques are well known to those skilled inthe art. This immunohistochemistry kit permits localization of thebiomarkers of the invention in tissue sections and cultured cellsusing both light and electron microscopy. It is used for bothresearch and clinical purposes. For example, tumors are biopsied orcollected and tissue sections cut with a microtome to examine sitesof biomarker production. Such information is useful for diagnosticand possibly therapeutic purposes in the detection and treatment ofneoplastic disease.

[0201] Diagnostic Applications

[0202] The subject compositions may be used in a variety ofdiagnostic applications. Exemplary embodiments of such diagnosticapplications are described below.

[0203] As noted above, the present invention is based on thediscovery that expression of the biomarkers of the invention incells and/or fluids is increased in cells of high metastaticpotential relative to cells of low metastatic potential, cells ofnon-metastatic potential, and to normal cells. In general, theterms "high metastatic potential" and "low metastatic potential"are used to describe the relative ability of a cell to give rise tometastases in an animal model, with "high metastatic potential"cells giving rise to a larger number of metastases and/or largermetastases than "low metastatic potential" cells. Thus, a cell ofhigh metastatic potential poses a greater risk of metastases to thesubject than a cell of low metastatic potential. "Non-metastaticcells" are those cells that are cancerous, but that do not developdetectable metastases following injection in an animal model.

[0204] The invention thus features methods and compositions fordiagnosis and prognosis, as well as grading and staging of cancers,by detection of expression or activity of the biomarkers of theinvention in a biological test sample, e.g, cell sample or tissuesample. The methods of the invention can also be used to monitorpatients having a predisposition to develop a particular neoplasticdisease, e.g., through inheritance of an allele associated withsusceptibility to a neoplastic disease (e.g., BRCA1, BRCA2, TP53,ATM, or APC for breast cancer). Detection and monitoring ofexpression or activity levels the biomarkers of the invention canbe used to detect potentially malignant events at a molecular levelbefore they are detectable at a gross morphological level.

[0205] In general, diagnosis, prognosis, and grading and/or stagingof cancers may be performed by a number of methods to determine therelative level of expression of the differentially expressedbiomarker gene at the transcriptional level, and/or the absence orpresence or altered amounts of a normal or abnormal biomarkerpolypeptide in patient cells. As used herein, "differentiallyexpressed gene" is intended to refer to a gene having an expressionlevel (e.g., which in turn is associated with a level of biomarkerpolypeptide production and/or biomarker transcription) that isassociated with a decrease in expression level of at least about25%, usually at least about 50% to 75%, more usually at least about90% or more. In general, such a decrease in differentiallyexpressed biomarker is indicative of the onset or development ofthe metastatic phenotype

[0206] "Diagnosis" as used herein generally includes determinationof a subject's susceptibility to a disease or disorder,determination as to whether a subject is unaffected, susceptibleto, or presently affected by a disease or disorder, and/or toidentify a tumor as benign. non-cancerous, or cancerous (e.g.non-metastatic or metastatic, e.g., high metastatic potential orlow metastatic potential). "Prognosis" is used herein to generallymean a determination of the severity of disease (e.g.,identification or pre-metastatic or metastatic cancerous states,stages of cancer. etc.), which in turn can be correlated with thepotential outcome, response to therapy, etc. A complete diagnosisthus can include diagnosis as discussed above, as well asdetermination of prognosis, cancer staging, and tumor grading. Thepresent invention particularly encompasses diagnosis and prognosisof subjects in the context of cancers of various origins,particularly breast cancer (e.g., carcinoma in situ (e.g., ductalcarcinoma in situ), estrogen receptor (ER)-positive breast cancer,ER-negative breast cancer, or other forms and/or stages of breastcancer) and prostate cancer.

[0207] "Sample" or "biological sample" as used throughout here aregenerally meant to refer to samples of biological fluids ortissues, particularly samples obtained from tissues, especiallyfrom cells of the type associated with the disease for which thediagnostic application is designed (e.g., cells, and/or ductaladenocarcinoma), and the like. "Samples" is also meant to encompassderivatives and fractions of such samples (e.g., cell lysates).Where the sample is solid tissue, the cells of the tissue can bedissociated or tissue sections can be analyzed.

[0208] Methods of the subject invention useful in diagnosis orprognosis typically involve comparison of the amount of geneproduct of the biomarkers of the invention in a sample of interestwith that of a control to detect relative differences in theexpression of the gene product, where the difference can bemeasured qualitatively. and/or quantitatively. Quantitation can beaccomplished, for example, by comparing the level of expressionproduct detected in the sample with the amounts of product presentin a standard curve. A comparison can be made visually using ELISAto detect relative amounts of biomarker polypeptides in test andcontrol samples; by using a technique such as densitometry, with orwithout computerized assistance, to detect relative amounts ofdetectably labeled biomarker polypeptides; or by using an array todetect relative levels of anti-biomarker polypeptide antibodybinding, and comparing the pattern of antibody binding to that of acontrol.

[0209] In some embodiments of the methods of the invention it maybe particularly desirable to detect expression of a biomarker geneproduct as well as at least one gene product.

[0210] Other gene products that can serve as controls or increasethe sensitivity of classification of the metastatic phenotype of acell, as well as gene products that can serve as controls foridentification of normal cells (e.g., gene products that areexpressed in normal cells but not in cancerous cells, or expressedin normal cells, but not in metastatic cells, etc.) are known inthe art. In addition, the cells can be classified as normal orcancerous based on conventional methodologies such as generalmorphology as determined by light microscopy. For example,conventional techniques for classifying a cell as cancerous basedon morphology can be performed prior to or simultaneously withdetection of biomarker expression. Thus, a cell that exhibitsabnormal morphology associated with the neoplastic diseasephenotype, and that expresses a low level of biomarker relative toa normal cells or in which biomarker expression is not detectableis identified as a cell of high metastatic potential.

[0211] Methods for qualitative and quantitative detection ofbiomarker polypeptides in a sample, as well as methods forcomparing such to control samples are well known in the art. Thepatient from whom the sample is obtained can be apparently healthy,susceptible to disease (e.g., as determined by family history orexposure to certain environmental factors), or can already beidentified as having a condition in which altered expression of agene product of the invention is implicated.

[0212] In the assays of the invention, the diagnosis can bedetermined based on detected gene product expression levels of thebiomarkers of the invention, and may also include detection ofadditional diagnostic markers and/or reference sequences. Where thediagnostic method is designed to detect the presence orsusceptibility of a patient to metastatic cancer, the assaypreferably involves detection of a biomarker gene product andcomparing the detected gene product levels to a level associatedwith a normal sample, to levels associated with a low metastaticpotential sample, and/or to level associated with a high metastaticpotential sample. For example, detection of a higher level ofbiomarker expression relative to a normal level is indicative ofthe presence in the sample of a cell having high metastaticpotential. Given the disclosure provided herein, variations on thediagnostic and prognostic assays described herein will be readilyapparent to the ordinarily skilled artisan.

[0213] As used herein the term "stringency" is used in reference tothe conditions of temperature, ionic strength, and the presence ofother compounds such as organic solvents, under which nucleic acidhybridizations are conducted. Under "low stringency conditions" anucleic acid sequence of interest will hybridize to its exactcomplement, sequences with single base mismatches, closely relatedsequences (e.g., sequences with 90% or greater homology), andsequences having only partial homology (e.g., sequences with 50-90%homology). Under "medium stringency conditions," a nucleic acidsequence of interest will hybridize only to its exact complement,sequences with single base mismatches, and closely relationsequences (e. 90% or greater homology). Under "high stringencyconditions," a nucleic acid sequence of interest will hybridizeonly to its exact complement, and (depending on conditions such atemperature) sequences with single base mismatches. In other words,under conditions of high stringency the temperature can be raisedso as to exclude hybridization to sequences with single basemismatches.

[0214] "High stringency conditions" when used in reference tonucleic acid hybridization comprise conditions equivalent tobinding or hybridization at 42.degree. C. in a solution consistingof 5.times.SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH, PO.sub.4.H.sub.2O and1.85 g/1EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5.times.Denhardt's reagent and 100 microg/ml denatured salmon sperm DNAfollowed by washing in a solution comprising 0.1.times.SSPE, 1.0%SDS at 42.degree. C. when a probe of about 500 nucleotides inlength is employed.

[0215] "Medium stringency conditions" when used in reference tonucleic acid hybridization comprise conditions equivalent tobinding or hybridization at 42.degree. C. in a solution consistingof 5.times.SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH.sub.2PO.sub.4.H.sub.2Oand 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5.times. Denhardt's reagent and 100.mu.g/ml denatured salmon spermDNA followed by washing in a solution comprising 1.0.times.SSPE,1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides inlength is employed.

[0216] "Low stringency conditions" comprise conditions equivalentto binding or hybridization at 42.degree. C. in a solutionconsisting of 5.times.SSPE (43.8 g/1 NaCl, 6.9 g/1NaH.sub.2PO.sub.4H.sub.2O and 1.85 g/1 EDTA, pH adjusted to 7.4with NaOH), 0.1% SDS, 5.times. Denhardt's reagent (50.times.Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharamcia), 5g BSA (Fraction V; Sigma)) and 100 microg/ml denatured salmon spermDNA followed by washing in a solution comprising 5.times.SSPE, 0.1%SDS at 42.degree. C. when a probe of about 500 nucleotides inlength is employed.

[0217] The art knows well that numerous equivalent conditions maybe employed to comprise low stringency conditions; factors suchthat as the length and nature (DNA, RNA, base composition) of theprobe and nature of the target (DNA, RNA, base composition, presentin solution or immobilized, etc.) and the concentration of thesalts and other components (e.g., the presence or absence offormamide, dextran sulfate, polyethylene glycol) are considered andthe hybridization solution may be varied to generate conditions oflow stringency hybridization different from, but equivalent to, theabove listed conditions. In addition, the art knows conditions thatpromote hybridization under conditions of high stringency (e.g.,increasing the temperature of the hybridization and/or wash steps,the use of formamide in the hybridization solution, etc.) (seedefinition above for "stringency").

[0218] As used herein, the term "probe" refers to anoligonucleotide (i.e., a sequence of nucleotides), whetheroccurring naturally as in a purified restriction digest or producedsynthetically, recombinantly or by PC R amplification, that iscapable of hybridizing to another oligonucleotide of interest. Aprobe may be single-stranded or double-stranded. Probes are usefulin the detection, identification and isolation of particular genesequences. It is contemplated that any probe used in the presentinvention will be labeled with any "reporter molecule," so that isdetectable in any detection system, including, but not limited toenzyme (e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is notintended that the present invention be limited to any particulardetection system or label.

[0219] Any of a variety of detectable labels can be used inconnection with the various methods of the invention. Suitabledetectable levels include fluorochromes, radioactive labels, andthe like. Suitable labels include, but are not necessarily limitedto, fluorochromes, e.g. fluorescein isothiocyanate (FITC),rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX),5-carboxyfluorescein (5-FAM) orN,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. 32P, 35S, 3H; etc. The detectable label can involve atwo stage system (e.g., biotin-avidin, hapten-anti-hapten antibody,etc.).

[0220] Reagents specific for the polynucleotides and polypeptidesof the invention, such as detectably labeled antibodies ordetectably labeled nucleotide probes, can be supplied in a kit fordetecting the presence of an expression product in a biologicalsample. The kit can also contain buffers or labeling components, aswell as instructions for using the reagents to detect and quantifyexpression products in the biological sample. Exemplary embodimentsof the diagnostic methods of the invention are described below inmore detail.

[0221] Polypeptide Detection in Diagnosis, Prognosis, CancerGrading and Cancer Staging

[0222] In one embodiment, the test sample is assayed for the levelof a polypeptide of the biomarkers of the invention. Diagnosis canbe accomplished using any of a number of methods to determine theabsence or presence or altered amounts of the differentiallyexpressed polypeptide in the test sample. For example, detectioncan utilize staining of cells or histological sections (e.g., froma biopsy sample) with labeled antibodies, performed in accordancewith conventional methods. Cells can be permeabilized to staincytoplasmic molecules. In general, antibodies that specificallybind a differentially expressed polypeptide of the invention areadded to a sample, and incubated for a period of time sufficient toallow binding to the epitope, usually at least about 10 minutes.The antibody can be detectably labeled for direct detection (e.g.,using radioisotopes, enzymes, fluorescers, chemiluminescers, andthe like), or can be used in conjunction with a second stageantibody or reagent to detect binding (e.g., biotin withhorseradish peroxidase-conjugated avidin, a secondary antibodyconjugated to a fluorescent compound, e.g. fluorescein, rhodamine,Texas red, etc.). The absence or presence of antibody binding canbe determined by various methods, including flow cytometry ofdissociated cells, microscopy, radiography, scintillation counting,etc. Any suitable alternative methods can of qualitative orquantitative detection of levels or amounts of differentiallyexpressed polypeptide can be used, for example ELISA, western blot,immunoprecipitation, radioimmunoassay, etc.

[0223] In general, the detected level of biomarker polypeptide inthe test sample is compared to a level of the differentiallyexpressed gene product in a reference or control sample, e.g., in anormal cell or in a cell having a known disease state (e.g., cellof high metastatic potential).

[0224] Immunological Methods

[0225] In the context of the present invention, "immunologicalmethods" are understood as meaning analytical methods based onimmunochemistry, in particular on an antigen-antibody reaction.Examples of immunological methods include immunoassays such asradioimmunoassay (RIA), enzyme immunoassay (ER, combined withsolid-phase technique: ELISA) or else immunofluorescence assays.The immunoassay is carried out by exposing the sample to beinvestigated to an SP-C-binding antibody and detecting andquantifying the amount of antibody which binds to SP-C. In theseassays. detection and quantification is carried out directly orindirectly in a known manner. Thus, detection and quantification ofthe antigen-antibody complexes is made possible by using suitablelabels which may be carried by the antibody directed against SP-Cand/or by a secondary antibody directed against the primaryantibody. Depending on the type of the abovementioned immunoassays,the labels are, for example, radioactive labels, fluorescent dyesor else enzymes. such as phosphatase or peroxidase, which can bedetected and quantified with the aid of a suitable substrate.

[0226] In one embodiment of the invention, the immunological methodis carried out with the aid of a suitable solid phase. Suitablesolid phases which may be mentioned include the customarycommercial microtiter plates made of polystyrene or membranes (forexample made of polyvinylidene difluoride, PVDF) which arecustomarily used for the ELISA technique. Surprisingly, it has beenfound that even chromatography plates are suitable for use as solidphase in the process according to the invention. The implementationof the process according to the invention using chromatographyplates is hereinbelow also referred to as immuno-TLC.

[0227] Screening for Targeted Drugs

[0228] In one embodiment, any of the biomarkers of the invention asdescribed herein are used in drug screening assays. The biomarkerproteins, antibodies, nucleic acids, modified proteins and cellscontaining the biomarkers of the invention are used in drugscreening assays or by evaluating the effect of drug candidates ona "gene expression profile" or expression profile of polypeptides.In one embodiment, the expression profiles are used, preferably inconjunction with high throughput screening techniques to allowmonitoring for expression profile genes after treatment with acandidate agent, Zlokamik, et al., Science 279, 84-8 (1998), Heid,et al., Genome Res., 6:986-994 (1996).

[0229] In another embodiment, the biomarker proteins, antibodies,nucleic acids, modified proteins and cells containing the native ormodified biomarker proteins are used in screening assays. That is,the present invention provides novel methods for screening forcompositions that modulate the cancer phenotype. This can be doneby screening for modulators of gene expression or for modulators ofprotein activity. Similarly, this may be done on an individual geneor protein level or by evaluating the effect of drug candidates ona "gene expression profile". In a preferred embodiment, theexpression profiles are used, preferably in conjunction with highthroughput screening techniques to allow monitoring for expressionprofile genes after treatment with a candidate agent, see Zlokamik,supra.

[0230] Having identified the biomarker genes herein, a variety ofassays to evaluate the effects of agents on gene expression may beexecuted. In a preferred embodiment, assays may be run on anindividual gene or protein level. That is, having identified aparticular gene as aberrantly regulated in neoplastic disease,candidate bioactive agents may be screened to modulate the gene'sregulation. "Modulation" thus includes both an increase and adecrease in gene expression or activity. The preferred amount ofmodulation will depend on the original change of the geneexpression in normal versus tumor tissue, with changes of at least10%. preferably 50%, more preferably 100-300%. and in someembodiments 300-1000% or greater. Thus, if a gene exhibits a 4 foldincrease in tumor compared to normal tissue, a decrease of aboutfour fold is desired; a 10 fold decrease in tumor compared tonormal tissue gives a 10 fold increase in expression for acandidate agent is desired, etc. Alternatively, where thebiomarkers of the invention has been altered but shows the sameexpression profile or an altered expression profile, the proteinwill be detected as outlined herein.

[0231] As will be appreciated by those in the art. this may be doneby evaluation at either the gene or the protein level; that is, theamount of gene expression may be monitored using nucleic acidprobes and the quantification of gene expression levels, or,alternatively, the level of the gene product itself can bemonitored, for example through the use of antibodies to thebiomarker protein and standard immunoassays. Alternatively, bindingand bioactivity assays with the protein may be done as outlinedbelow.

[0232] In a preferred embodiment, gene expression monitoring isdone and a number of genes, i.e. an expression profile, ismonitored simultaneously, although multiple protein expressionmonitoring can be done as well.

[0233] In this embodiment, the biomarker nucleic acid probes areattached to biochips as outlined herein for the detection andquantification of biomarker sequences in a particular cell. Theassays are further described below.

[0234] Generally, in a preferred embodiment, a candidate bioactiveagent is added to the cells prior to analysis. Moreover, screensare provided to identify a candidate bioactive agent that modulatesa particular type of cancer, modulates biomarker proteins, binds toa biomarker protein, or interferes between the binding of abiomarker protein and an antibody.

[0235] The term "potential therapeutic agent" "candidate bioactiveagent" or "drug candidate" or grammatical equivalents as usedherein describes any molecule, e.g., protein, oligopeptide, smallorganic or inorganic molecule, polysaccharide, polynucleotide,etc., to be tested for bioactive agents that are capable ofdirectly or indirectly altering either the cancer phenotype,binding to and/or modulating the bioactivity of a biomarkerprotein, or the expression of a biomarker sequence, including bothnucleic acid sequences and protein sequences. In a particularlypreferred embodiment, the candidate agent increases a biomarkerphenotype, for example to a normal tissue fingerprint. Generally aplurality of assay mixtures are run in parallel with differentagent concentrations to obtain a differential response to thevarious concentrations. Typically. one of these concentrationsserves as a negative control, i.e., at zero concentration or belowthe level of detection.

[0236] In one aspect, a candidate agent will neutralize the effectof a biomarker protein. By "neutralize" is meant that activity of aprotein is either inhibited or counter acted against so as to havesubstantially no effect on a cell.

[0237] Potential therapeutic agents encompass numerous chemicalclasses, though typically they are organic or inorganic molecules,preferably small organic compounds having a molecular weight ofmore than 100 and less than about 2,500 Daltons. Preferred smallmolecules are less than 2000, or less than 1500 or less than 1000or less than 500 D. Candidate agents comprise functional groupsnecessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine,carbonyl, hydroxyl or carboxyl group, preferably at least two ofthe functional chemical groups. The candidate agents often comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof. Particularly preferred are peptides.

[0238] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of awide variety of organic compounds and biomolecules, includingexpression of randomized oligonucleotides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant andanimal extracts are available or readily produced. Additionally,natural or synthetically produced libraries and compounds arereadily modified through conventional chemical, physical andbiochemical means. Known pharmacological agents may be subjected todirected or random chemical modifications, such as acylation,alkylation, esterification, or amidification to produce structuralanalogs.

[0239] In one embodiment, the candidate bioactive agents areproteins. By "protein" herein is meant at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The protein may be made up of naturallyoccurring amino acids and peptide bonds, or syntheticpeptidomimetic structures. Thus "amino acid", or "peptide residue",as used herein means both naturally occurring and synthetic aminoacids. For example, homo-phenylalanine, citrulline and norleucineare considered amino acids for the purposes of the invention."Amino acid" also includes imino acid residues such as proline andhydroxyproline. The side chains may be in either the (R) or the (S)configuration. In the preferred embodiment, the amino acids are inthe (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradations.

[0240] In a preferred embodiment, the candidate bioactive agentsare naturally occurring proteins or fragments of naturallyoccurring proteins. Thus, for example, cellular extracts containingproteins, or random or directed digests of proteinaceous cellularextracts, may be used. In this way libraries of prokaryotic andeukaryotic proteins may be made for screening in the methods of theinvention. Particularly preferred in this embodiment are librariesof bacterial, fungal, viral, and mammalian proteins, with thelatter being preferred, and human proteins being especiallypreferred.

[0241] In another preferred embodiment, the candidate bioactiveagents are peptides of from about 5 to about 30 amino acids, withfrom about 5 to about 20 amino acids being preferred, and fromabout 7 to about 15 being particularly preferred. The peptides maybe digests of naturally occurring proteins as is outlined above,random peptides, or "biased" random peptides. By "randomized" orgrammatical equivalents herein is meant that each nucleic acid andpeptide consists of essentially random nucleotides and amino acids,respectively. Since generally these random peptides (or nucleicacids, discussed below) are chemically synthesized, they mayincorporate any nucleotide or amino acid at any position. Thesynthetic process can be designed to generate randomized proteinsor nucleic acids, to allow the formation of all or most of thepossible combinations over the length of the sequence, thus forminga library of randomized candidate bioactive proteinaceousagents.

[0242] In one embodiment, the library is fully randomized, with nosequence preferences or constants at any position. In a preferredembodiment, the library is biased. That is, some positions withinthe sequence are either held constant, or are selected from alimited number of possibilities. For example, in a preferredembodiment, the nucleotides or amino acid residues are randomizedwithin a defined class, for example, of hydrophobic amino acids,hydrophilic residues, sterically biased (either small or large)residues, towards the creation of nucleic acid binding domains, thecreation of cysteines, for cross-linking, prolines for SH-3domains, serines, threonines, tyrosines or histidines forphosphorylation sites, etc., or to purines, etc.

[0243] In one embodiment, the candidate bioactive agents arenucleic acids. As described generally for proteins, nucleic acidcandidate bioactive agents may be naturally occurring nucleicacids, random nucleic acids, or "biased" random nucleic acids. Inanother embodiment, the candidate bioactive agents are organicchemical moieties, a wide variety of which are available in theliterature.

[0244] In assays for testing alteration of the expression profileof one or more biomarker genes, after the candidate agent has beenadded and the cells allowed to incubate for some period of time, anucleic acid sample containing the target sequences to be analyzedis prepared. The target sequence is prepared using known techniques(e.g., converted from RNA to labeled cDNA, as described above) andadded to a suitable microarray. For example, an in vitro reversetranscription with labels covalently attached to the nucleosides isperformed. Generally, the nucleic acids are labeled with a label asdefined herein, especially with biotin-FITC or PE, Cy3 and Cy5.

[0245] As will be appreciated by those in the art. these assays canbe direct hybridization assays or can comprise "sandwich assays",which include the use of multiple probes, as is generally outlinedin U.S. Pat. Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117,5,591,584, 5,571,670, 5,580,731, 5,571,670, 5,591,584, 5,624,802,5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681,697, all ofwhich are hereby incorporated by reference. In this embodiment, ingeneral, the target nucleic acid is prepared as outlined above, andthen added to the biochip comprising a plurality of nucleic acidprobes, under conditions that allow the formation of ahybridization complex.

[0246] A variety of hybridization conditions may be used in thepresent invention, including high, moderate and low stringencyconditions as outlined above. The assays are generally run understringency conditions that allow formation of the label probehybridization complex only in the presence of target. Stringencycan be controlled by altering a step parameter that is athermodynamic variable, including, but not limited to, temperature,formamide concentration, salt concentration, chaotropic saltconcentration, pH, organic solvent concentration, etc. Theseparameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus it may bedesirable to perform certain steps at higher stringency conditionsto reduce non-specific binding.

[0247] The reactions outlined herein may be accomplished in avariety of ways, as will be appreciated by those in the art.Components of the reaction may be added simultaneously, orsequentially, in any order, with preferred embodiments outlinedbelow. In addition, the reaction may include a variety of otherreagents in the assays. These include reagents like salts, buffers,neutral proteins, e.g. albumin, detergents, etc which may be usedto facilitate optimal hybridization and detection, and/or reducenon-specific or background interactions. Also reagents thatotherwise improve the efficiency of the assay, such as proteaseinhibitors, nuclease inhibitors, anti-microbial agents, etc., maybe used, depending on the sample preparation methods and purity ofthe target. In addition, either solid phase or solution based(i.e., kinetic PCR) assays may be used.

[0248] Once the assay is run, the data are analyzed to determinethe expression levels, and changes in expression levels as betweenstates, of individual genes, forming a gene expression profile.

[0249] In a preferred embodiment, as for the diagnosis andprognosis applications, having identified the differentiallyexpressed gene(s) or mutated gene(s) important in any one state,screens can be run to test for alteration of the expression of thebiomarker genes individually. That is, screening for modulation ofregulation of expression of a single gene can be done. Thus, forexample, in the case of target genes whose presence or absence isunique between two states, screening is done for modulators of thetarget gene expression.

[0250] In addition, screens can be done for novel genes that areinduced in response to a candidate agent. After identifying acandidate agent based upon its ability to modulate a biomarkerexpression pattern leading to a normal expression pattern, ormodulate a single biomarker gene expression profile so as to mimicthe expression of the gene from normal tissue, a screen asdescribed above can be performed to identify genes that arespecifically modulated in response to the agent. Comparingexpression profiles between normal tissue and agent treated tissuereveals genes that are not expressed in normal tissue, but areexpressed in agent treated tissue. These agent specific sequencescan be identified and used by any of the methods described hereinfor biomarker genes or proteins. In particular these sequences andthe proteins they encode find use in marking or identifyingagent-treated cells.

[0251] Thus, in one embodiment, a candidate agent is administeredto a population of cells, that thus has an associated expressionprofile. By "administration" or "contacting" herein is meant thatthe candidate agent is added to the cells in such a manner as toallow the agent to act upon the cell, whether by uptake andintracellular action, or by action at the cell surface. In someembodiments, nucleic acid encoding a proteinaceous candidate agent(i.e. a peptide) may be put into a viral construct such as aretroviral construct and added to the cell, such that expression ofthe peptide agent is accomplished; see PCT US97/01019, herebyexpressly incorporated by reference.

[0252] Once the candidate agent has been administered to the cells,the cells can be washed if desired and are allowed to incubateunder preferably physiological conditions for some period of time.The cells are then harvested and a new gene expression profile isgenerated, as outlined herein.

[0253] In a preferred embodiment, screening is done to alter thebiological function of the expression product of a biomarker gene.Again, having identified the importance of a gene in a particularstate, screening for agents that bind and/or modulate thebiological activity of the gene product can be run as is more fullyoutlined below.

[0254] In a preferred embodiment, screens are designed to firstfind candidate agents that can bind to biomarker proteins, and thenthese agents may be used in assays that evaluate the ability of thecandidate agent to modulate the biomarker activity and the cancerphenotype. Thus, as will be appreciated by those in the art, thereare a number of different assays that may be run; binding assaysand activity assays.

[0255] In a preferred embodiment, binding assays are done. Ingeneral, purified or isolated gene product is used; that is, thegene products of one or more biomarker nucleic acids are made. Ingeneral, this is done as is known in the art. For example,antibodies are generated to the protein gene products, and standardimmunoassays are run to determine the amount of protein present.Alternatively, cells comprising the biomarker proteins can be usedin the assays.

[0256] Thus, in a preferred embodiment, the methods comprisecombining a biomarker protein and a candidate bioactive agent, anddetermining the binding of the candidate agent to the biomarkerprotein. Preferred embodiments utilize the human or mouse biomarkerprotein, although other mammalian proteins may also be used, forexample for the development of animal models of human disease. Insome embodiments, as outlined herein, variant or derivativebiomarker proteins may be used.

[0257] Generally, in a preferred embodiment of the methods herein,the biomarker protein or the candidate agent is non-diffusablybound to an insoluble support having isolated sample receivingareas (e.g. a microtiter plate, an array, etc.). The insolublesupport may be made of any composition to which the compositionscan be bound, is readily separated from soluble material, and isotherwise compatible with the overall method of screening. Thesurface of such supports may be solid or porous and of anyconvenient shape. Examples of suitable insoluble supports includemicrotiter plates, arrays, membranes and beads. These are typicallymade of glass, plastic (e.g., polystyrene), polysaccharides, nylonor nitrocellulose, Teflon.RTM., etc. Microtiter plates and arraysare especially convenient because a large number of assays can becarried out simultaneously, using small amounts of reagents andsamples.

[0258] The particular manner of binding of the composition is notcrucial so long as it is compatible with the reagents and overallmethods of the invention, maintains the activity of the compositionand is nondiffusable. Preferred methods of binding include the useof antibodies (which do not sterically block either the ligandbinding site or activation sequence when the protein is bound tothe support), direct binding to "sticky" or ionic supports,chemical cross linking. the synthesis of the protein or agent onthe surface, etc. Following binding of the protein or agent, excessunbound material is removed by washing. The sample receiving areasmay then be blocked through incubation with bovine serum albumin(BSA), casein or other innocuous protein or other moiety.

[0259] In a preferred embodiment, the biomarker protein is bound tothe support, and a candidate bioactive agent is added to the assay.Alternatively, the candidate agent is bound to the support and thebiomarker protein is added. Novel binding agents include specificantibodies, non-natural binding agents identified in screens ofchemical libraries, peptide analogs, etc. Of particular interestare screening assays for agents that have a low toxicity for humancells. A wide variety of assays may be used for this purpose,including labeled in vitro protein-protein binding assays,electrophoretic mobility shift assays, immunoassays for proteinbinding, functional assays (phosphorylation assays, etc.) and thelike.

[0260] The determination of the binding of the candidate bioactiveagent to the biomarker protein may be done in a number of ways. Ina preferred embodiment, the candidate bioactive agent is labeled,and binding determined directly. For example, this may be done byattaching all or a portion of the biomarker protein to a solidsupport, adding a labeled candidate agent (for example afluorescent label), washing off excess reagent, and determiningwhether the label is present on the solid support. Various blockingand washing steps may be utilized as is known in the art.

[0261] By "labeled" herein is meant that the compound is eitherdirectly or indirectly labeled with a label which provides adetectable signal, e.g. radioisotope, fluorescers, enzyme,antibodies, particles such as magnetic particles, chemiluminescers,or specific binding molecules, etc. Specific binding moleculesinclude pairs, such as biotin and streptavidin, digoxin andantidigoxin etc. For the specific binding members, thecomplementary member would normally be labeled with a moleculewhich provides for detection, in accordance with known procedures,as outlined above. The label can directly or indirectly provide adetectable signal.

[0262] In some embodiments, only one of the components is labeled.For example, the proteins (or proteinaceous candidate agents) maybe labeled at tyrosine positions using .sup.125I, or withfluorophores. Alternatively, more than one component may be labeledwith different labels; using .sup.125I for the proteins, forexample, and a fluorophore for the candidate agents.

[0263] In a preferred embodiment, the binding of the candidatebioactive agent is determined through the use of competitivebinding assays. In this embodiment, the competitor is a bindingmoiety known to bind to the target molecule (i.e. the biomarkers ofthe invention), such as an antibody, peptide, binding partner,ligand, etc. Under certain circumstances, there may be competitivebinding as between the bioactive agent and the binding moiety, withthe binding moiety displacing the bioactive agent.

[0264] In one embodiment, the candidate bioactive agent is labeled.Either the candidate bioactive agent, or the competitor, or both,is added first to the protein for a time sufficient to allowbinding, if present. Incubations may be performed at anytemperature which facilitates optimal activity, typically between 4and 40.degree. C. Incubation periods are selected for optimumactivity, but may also be optimized to facilitate rapid highthroughput screening. Typically between 0.1 and 1 hour will besufficient. Excess reagent is generally removed or washed away. Thesecond component is then added, and the presence or absence of thelabeled component is followed, to indicate binding.

[0265] In a preferred embodiment, the competitor is added first,followed by the candidate bioactive agent. Displacement of thecompetitor is an indication that the candidate bioactive agent isbinding to the biomarkers of the invention and thus is capable ofbinding to, and potentially modulating, the activity of thebiomarkers of the invention. In this embodiment, either componentcan be labeled. Thus, for example, if the competitor is labeled,the presence of label in the wash solution indicates displacementby the agent. Alternatively, if the candidate bioactive agent islabeled, the presence of the label on the support indicatesdisplacement.

[0266] In an alternative embodiment, the candidate bioactive agentis added first, with incubation and washing, followed by thecompetitor. The absence of binding by the competitor may indicatethat the bioactive agent is bound to the biomarkers of theinvention with a higher affinity. Thus, if the candidate bioactiveagent is labeled, the presence of the label on the support, coupledwith a lack of competitor binding, may indicate that the candidateagent is capable of binding to the biomarkers of the invention.

[0267] In a preferred embodiment, the methods comprise differentialscreening to identity bioactive agents that are capable ofmodulating the activity of the biomarkers of the invention. In thisembodiment, the methods comprise combining a the biomarkers of theinvention and a competitor in a first sample. A second samplecomprises a candidate bioactive agent, a biomarkers of theinvention and a competitor. The binding of the competitor isdetermined for both samples, and a change, or difference in bindingbetween the two samples indicates the presence of an agent capableof binding to the biomarkers of the invention and potentiallymodulating its activity. That is, if the binding of the competitoris different in the second sample relative to the first sample, theagent is capable of binding to the biomarkers of the invention.

[0268] Positive controls and negative controls may be used in theassays. Preferably all control and test samples are performed in atleast triplicate to obtain statistically significant results.Incubation of all samples is for a time sufficient for the bindingof the agent to the protein. Following incubation, all samples arewashed free of non-specifically bound material and the amount ofbound, generally labeled agent determined. For example, where aradiolabel is employed, the samples may be counted in ascintillation counter to determine the amount of boundcompound.

[0269] A variety of other reagents may be included in the screeningassays. These include reagents like salts, neutral proteins, e.g.albumin, detergents, etc which may be used to facilitate optimalprotein-protein binding and/or reduce non-specific or backgroundinteractions. Also reagents that otherwise improve the efficiencyof the assay, such as protease inhibitors, nuclease inhibitors,anti-microbial agents, etc., may be used. The mixture of componentsmay be added in any order that provides for the requisitebinding.

[0270] Screening for agents that modulate the activity of thebiomarkers of the invention may also be done. In a preferredembodiment, methods for screening for a bioactive agent capable ofmodulating the activity of the biomarkers of the invention comprisethe steps of adding a candidate bioactive agent to a sample of thebiomarkers of the invention, as above, and determining analteration in the biological activity of proteins. "Modulating theactivity of a the biomarkers of the invention" includes an increasein activity, a decrease in activity, or a change in the type orkind of activity present. Thus, in this embodiment, the candidateagent should both bind to the biomarkers of the invention (althoughthis may not be necessary), and alter its biological or biochemicalactivity as defined herein. The methods include both in vitroscreening methods, as are generally outlined above, and in vivoscreening of cells for alterations in the presence, distribution,activity or amount of the biomarkers of the invention.

[0271] Thus, in this embodiment, the methods comprise combining asample and a candidate bioactive agent, and evaluating the effecton activity of the biomarkers of the invention. By "biomarkeractivity" or grammatical equivalents herein is meant one of thebiomarker protein's biological activities, including, but notlimited to, its role in tumorigenesis, including cell division,preferably in lymphatic tissue, cell proliferation, tumor growthand transformation of cells.

[0272] In a preferred embodiment, the activity of the biomarkers ofthe invention is increased; in another preferred embodiment, theactivity of the biomarkers of the invention is increased. Thus,bioactive agents that are antagonists are preferred in someembodiments, and bioactive agents that are agonists may bepreferred in other embodiments.

[0273] In a preferred embodiment, the invention provides methodsfor screening for bioactive agents capable of modulating theactivity of the biomarkers of the invention. The methods compriseadding a candidate bioactive agent, as defined above, to a cellcomprising biomarker proteins. Preferred cell types include almostany cell. The cells contain a recombinant nucleic acid that encodesa biomarker protein. In a preferred embodiment, a library ofcandidate agents is tested on a plurality of cells.

[0274] In one aspect, the assays are evaluated in the presence orabsence or previous or subsequent exposure of physiologicalsignals, for example hormones, antibodies, peptides, antigens,cytokines, growth factors, action potentials, pharmacologicalagents including chemotherapeutics, radiation, carcinogenics, orother cells (i.e. cell-cell contacts). In another example, thedeterminations are determined at different stages of the cell cycleprocess.

[0275] In this way, bioactive agents are identified. Compounds withpharmacological activity are able to enhance or interfere with theactivity of the biomarkers of the invention.

[0276] Animal Models and Transgenics

[0277] In another preferred embodiment the biomarkers of theinvention find use in generating animal models of cancers. As isappreciated by one of ordinary skill in the art, gene therapytechnology wherein antisense RNA directed to the biomarkers of theinvention will diminish or repress expression of the gene. Ananimal generated as such serves as an animal model of biomarkersthat finds use in screening bioactive drug candidates. Similarly,gene knockout technology, for example as a result of homologousrecombination with an appropriate gene targeting vector, willresult in the absence of the biomarker protein. When desired,tissue-specific expression or knockout of the biomarker protein maybe necessary.

[0278] It is also possible that the biomarkers of the invention isoverexpressed in neoplastic disease. As such, transgenic animalscan be generated that overexpress the biomarkers of the invention.Depending on the desired expression level, promoters of variousstrengths can be employed to express the transgene. Also, thenumber of copies of the integrated transgene can be determined andcompared for a deter ruination of the expression level of thetransgene. Animals generated by such methods find use as animalmodels of the biomarkers of the invention and are additionallyuseful in screening for bioactive molecules to treat neoplasticdisease.

[0279] The invention will be illustrated in more detail withreference to the following Examples, but it should be understoodthat the present invention is not deemed to be limited thereto.

[0280] Microarrays

[0281] Microarrays have become well known and extensively used inthe art (See, e.g., Barinaga, Science 253: 1489 (1991); Bains,Bio/Technology 10: 757-758 (1992)). Guidance for the use ofmicroarrays is provided by Wang, E et al., Nature Biotechnology 18;457-459 (2000); Diehn M et al., Nature Genetics 25: 58-62(2000).

[0282] Polynucleotides, polypeptides, or analogues are attached toa solid support or substrate, which may be made from glass, plastic(e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, orother materials. "Substrate" refers to any suitable rigid orsemi-rigid support to which polynucleotides or polypeptides arebound and includes membranes, filters, chips, slides, wafers,fibers, magnetic or nonmagnetic beads, gels, capillaries or othertubing, plates, polymers, and microparticles with a variety ofsurface forms including wells, trenches, pins, channels and pores.Polynucleotides can be immobilized on a substrate by any methodknown in the art.

[0283] Among the vendors of microarrays and microarray technologyusage are Affymetrix, Inc. (USA), NimbleGen Systems, Inc. (Madison,Wis., USA), and Incyte Genomics (USA); Agilent Technologies (USA)and Grafinity Pharmaceutical Design, GmbH (Germany); and CLONTECHLaboratories (Becton Dickinson Bioscience) and BioRobotics, Ltd.(Great Britain) (See, e.g., Gwynne and Heebner G, Science(2001)).

[0284] In some embodiments, microarrays are utilized to monitor theexpression of genes from neoplastic disease (e.g., to compareexpression to normal HSCs). In some embodiments, microarrays areused to monitor the progression of disease. Differences in geneexpression between healthy (e.g., normal) HSCs and canceroustissues can be identified or monitored by analyzing changes inpatterns of gene expression compared with s (e.g., from a subjectwith neoplastic disease). In some embodiments, neoplastic diseasecan be diagnosed at earlier stages before the patient issymptomatic. The invention can also be used to monitor the efficacyof treatment. For example, when using a treatment with known sideeffects, a microarray can be employed to "fine tune" the treatmentregimen. A dosage is established that causes a change in geneticexpression patterns indicative of successful treatment. Expressionpatterns associated with undesirable side effects are avoided. Thisapproach may be more sensitive and rapid than waiting for thepatient to show inadequate improvement, or to manifest sideeffects, before altering the course of treatment.

[0285] Alternatively, animal models that mimic a disease, ratherthan patients, can be used to characterize expression profilesassociated with a particular disease or condition. This geneexpression data may be useful in diagnosing and monitoring thecourse of disease in a patient, in determining gene targets forintervention, and in testing novel treatment regimens.

[0286] Microarrays can be used to rapidly screen large numbers ofcandidate drug molecules, looking for ones that produce anexpression profile similar to those of known therapeutic drugs,with the expectation that molecules with the same expressionprofile will likely have similar therapeutic effects. Thus, in someembodiments, the invention provides the means to determine themolecular mode of action of a drug.

[0287] U.S. Pat. Nos. 6,218,122, 6,165,709, and 6,146,830, each ofwhich is herein incorporated by reference in their entireties,disclose methods for identifying targets of a drug in a cell bycomparing (i) the effects of the drug on a wild-type cell, (ii) theeffects on a wild-type cell of modifications to a putative targetof the drug, and (iii) the effects of the drug on a wild-type cellwhich has had the putative target modified of the drug. In variousembodiments, the effects on the cell can be determined by measuringgene expression, protein abundances, protein activities, or acombination of such measurements. In various embodiments,modifications to a putative target in the cell can be made bymodifications to the genes encoding the target, modification toabundances of RNAs encoding the target, modifications to abundancesof target proteins, or modifications to activities of the targetproteins. The present invention provides an improvement to thesemethods of drug discovery by providing s, for a more precise drugdiscovery program.

[0288] An "expression profile" comprises measurement of a pluralityof cellular constituents that indicate aspects of the biologicalstate of a cell. Such measurements may include, e.g., RNA orprotein abundances or activity levels. Aspects of the biologicalstate of a cell of a subject, for example, the transcriptionalstate, the translational state, or the activity state, aremeasured. The collection of these measurements, optionallygraphically represented, is called the "diagnostic profile".Aspects of the biological state of a cell which are similar tothose measured in the diagnostic profile (e.g., the transcriptionalstate) can be measured in an analogous subject or subjects inresponse to a known correlated disease state or, if therapeuticefficacy is being monitored, in response to a known, correlatedeffect of a therapy. The collection of these measurements,optionally graphically represented, is called herein the "responseprofile". The response profiles are interpolated to predictresponse profiles for all levels of protein activity within therange of protein activity measured. In cases where therapeuticefficacy is to be monitored, the response profile may be correlatedto a beneficial effect, an adverse effect, such as a toxic effect,or to both beneficial and adverse effects.

[0289] The invention will be illustrated in more detail withreference to the following Examples, but it should be understoodthat the present invention is not deemed to be limited thereto.

EXAMPLES

Example 1

Metabolomic Analysis of Cav-1 (-/-) Null Tissues: Evidence forOxidative Stress, Mitochondrial Dysfunction, and Autophagy

[0290] Mammary fat pads were harvested from age-matched female WTand Cav-1 (-/-) null mice (n=6 for each genotype) and subjected toan unbiased metabolomic analysis. Over 200 known compounds wereidentified by mass spectrometry analysis and were quantitated.Interestingly, there were a large number of compounds that weresignificantly changed in Cav-1 (-/-) mammary fat pads (n=103; 92UP; 11 DOWN), consistent with a severe metabolic phenotype (Table1, FIG. 21). Several observations are consistent with the presenceof oxidative stress. These include: 1) an increase in the amountsof several anti-oxidants, such as ascorbic acid (.about.11.2-fold),vitamin E (alpha-tocopherol; 2.7-fold), 5-hydroxyindoleacetate(2.7-fold), and hypotaurine (1.8-fold); 2) an increase in thenumber of amino-acid metabolites associated with the glutathionepathway, more specifically gamma-glutamyl amino acids andglutathione species (GSH, GSSH, 5-oxoproline,cys-glutathione-disulfide); 3) a shift towards gluconeogenesis, andthe pentose phosphate pathway, which is known to produce increasedamounts of NADPH, which can then be used as reducing equivalents tomaintain reduced glutathione; 4) the observed increase in riboseand nucleotides, which emanate from the pentose phosphate pathway;and 5) an increase in the amount of ADMA (asymmetric dimethylarginine; 3.3-fold), which is both a marker of protein catabolismand oxidative stress, and can also produce more oxidative stress.ADMA acts as an eNOS uncoupler, resulting in the production ofsuperoxide, instead of nitric oxide. Similarly, ADMA is also amarker of chronic hypoxia and mitochondrial dysfunction.

[0291] As oxidative stress also drives mitochondrial dysfunction,autophagy, and mitophagy, evidence of these catabolic biologicalprocesses was examined in the metabolic data set. Consistent with ageneralized catabolic phenotype, see: 1) higher levels of numerousamino acids and their catabolites; 2) elevation of 4 markers ofprotein or collagen degradation (assymetric dimethylarginine,trans-4-hydroxyproline, glycyl-proline, proline-hydroxy-proline);3) elevated levels of a marker of increased RNA turnover, namelypseudouridine (1.7-fold); 4) increased levels (4.3-fold) of3-hydroxybutryate (BHB), a ketone body, which is a well-acceptedmarker of mitochondrial dysfunction 22, 23; and 5) higher levels offree cholesterol (1.6-fold), which can also contribute tomitochondrial dysfunction 24. A decrease in mitochondrial functionis also consistent with the accumulation of certain metabolitesassociated with glycolysis (pyruvate; 1.4-fold), and the TCA cycle(fumarate and malate; both >1.4-fold). Interestingly, increasesin 5-hydroxyindole (2.7-fold). which is an anti-oxidant metaboliteof tryptophan, was observed which protects against oxidative damageand mitochondrial dysfunction, as it suppresses ROS generation,lipid peroxidation, peroxynitrite generation, and glutathionedepletion--thereby increasing mitochondrial membrane potential.

[0292] The results from the mammary fat pad were compared with lungtissue, as adipose tissue and lung tissue express the highestlevels of Cav-1. Only concordant changes were selected and areshown in Table 2, FIG. 22. Interestingly, ADMA, pyruvate, and3-hydroxybutyrate were significantly elevated in lung tissue,consistent with the idea that Cav-1 (-/-) null tissues areundergoing 1) oxidative stress and 2) mitochondrial dysfunction.Box plots for ADMA and BHB are shown in Figure ______, and for theantioxidant Vitamins C and E in Figure ______.

[0293] It is also known that oxidative stress is indeed sufficientto induce ketone production in an animal model of AmyotrophicLateral Sclerosis (ALS). These mice express a mutant form of SOD1(G86R) and show progressively increased serum levels of ketonebodies. Furthermore, ALS patients show increase serum levels ofketone bodies, both 3-hydroxybutyrate and acetone, as documented byNMR spectroscopy. Finally, autophagy has also been implicated inthe pathogenesis of ALS, both using transgenic SOD1-mutant mousemodels and human patient samples. Thus, oxidative stress,mitochondrial dysfunction, and autophagy/mitophagy are allclustered together in various neurodegenerative disorders, such asALS and Alzheimer's disease.

[0294] Other noteworthy metabolites that were increased includehistamine (2.5-fold) and arachidonic acid (1.5-fold), which maydirectly or indirectly contribute towards an inflammatorymicro-environment. As arachidonic acid is the precursor of bothprostaglandins and leukotrienes, increased free arachidonic acidcould drive the generation of increased inflammatory mediators.Histamine also increases the differentiation of stromal cellstowards a more myo-fibroblastic phenotype, consistent with thebehavior of cancer-associated fibroblasts.

Example 2

Transcriptional mRNA Profiling of Cav-1 (-/-) Stromal CellsProvides Validating Evidence for a Stromal Catabolic State

[0295] The inventors have previously traced the lethality of aCav-1 negative tumor micro-environment to the stromal fibroblast orcancer-associated fibroblast compartment. Thus, to garnervalidating evidence for our metabolic profiling studies, theinventors re-interrogated the transcriptional profiling dataobtained via the analysis of WT and Cav-1 (-/-) stromal cells. Themetabolic features observed could be explained by oxidative stressinduced autophagy and mitophagy. In direct support of this notion,Table 3, FIG. 23 shows that many the genes that are involved inmediating autophagy and mitophagy are indeed upregulated in Cav-1(-/-) null stromal cells. Since autophagy and mitophagy aredependent on increased lysosomal degradation activity. thetranscriptional profiles of lysosomal proteases (the cathepsins)and other lysosomal associated proteins was assessed (Table 4, FIG.24). Interestingly, numerous cathepsin genes and lysomal associatedproteins were transcriptionally upregulated in Cav-1 (-/-) stromalcells.

[0296] In further support of increased oxidative stress, thetranscriptional overexpression of numerous genes associated withglutathione metabolism, genes responsive to oxidative stress) andhypoxia, as well as numerous anti-oxidant proteins (Table 5, FIG.25). Thus, the transcriptional mRNA profile of Cav-1 (-/-) stromalcells is consistent with oxidative stress induced autophagy andmitophagy, and lysosome-related and peroxisome-related genetranscripts.

Example 3

Transcriptional mRNA Profiling of Human Breast Cancer StromaProvides Evidence for Stromal Autophagy and Mitophagy In Vivo

[0297] To further test the possible clinical relevance of ourobservations regarding autophagy and mitophagy, the transcriptionalprofiles of human tumor stroma that was isolated by laser-capturemicro-dissection of breast cancer tumor tissue was analyzed. Themethods and origins of these samples have been previously describedin detail. Using these raw transcriptional profiling data, threerelated gene lists were created: 1) tumor stroma, 2) recurrencestroma, and 3) metastasis stroma. The tumor stroma list containsgenes that were upregulated in the stroma of primary tumors, ascompared with normal mammary gland stroma. The recurrence stromalist contains stromal genes, from the primary tumor, that areupregulated in patients that underwent tumor recurrence, ascompared with patients that did not recur. The metastasis stromallist contains stromal genes, from the primary tumor, that areupregulated in patients that underwent lymph node metastasis atdiagnosis, as compared with patients that did not show lymph nodemetastasis. Thus, these 3 complementary gene lists were analyzedfor evidence of autophagy and mitophagy. Table 6 (FIG. 26) showsthat many of the genes that are associated with autophagy andmitophagy are transcriptionally upregulated in the tumor stroma ofhuman breast cancer patients. In further support of this idea,upregulation of lysosomal proteases (the cathepsins and) legumain),as well as other lysosomal associated proteins was observed (Table7, FIG. 27).

[0298] Finally, genes associated with glutathione metabolism,oxidative and hypoxic stress, as well as anti-oxidants are alltranscriptionally upregulated in the tumor stroma obtained fromhuman breast cancer patients (Table 8, FIG. 28). Perhaps. mostinterestingly, many of these gene transcripts are also associatedwith tumor recurrence and metastasis (See Tables 6-8, FIG. 28). Itis important to note that telomerase-related genes are alsotranscriptionally upregulated in both Cav-1 (-/-) stromal cells andthe tumor stroma of human breast cancers (See Tables 5 and 8, FIGS.25 and 28). Thus, over-expression of telomerase activity couldprovide an escape mechanism to keep stromal cell cells alive formuch longer periods of time under conditions of oxidative stress,autophagy, and mitophagy.

[0299] To independently assess the statistical association ofautophagy, lysosomes, peroxisomes, and telomere-related genetranscripts with the human tumor stroma of breast cancer patients,the more comprehensive gene ontology lists were used to intersectwith the tumor stroma, recurrence stroma, and metastasis stromagene lists. The results of this more detailed analysis arepresented in FIGS. 3, 4, and 5, and are represented as Venndiagrams. More specifically, FIG. 3 shows that autophagy-relatedgenes are significantly associated with tumor stroma,recurrence-stroma, and metastasis-stroma. Similarly,lysosome-related genes were significantly associated with tumorstroma, and recurrencestroma, while telomere-related genes wereonly associated with metastasis-stroma FIG. 4, panels A and B).Finally, peroxisome-related genes were significantly associatedwith both tumor stroma and recurrence-stroma (FIG. 5). Thus, all ofthese inter-related biological processes (oxidative stress,autophagy/lysosomal degradation, and telomere-maintenance) may playa significant pathogenic role in generating an activated lethaltumor stroma.

Example 4

ADMA and Ketone Metabolism in Cav-1 (-/-) Stromal Cells and HumanTumor Stroma

[0300] Here, ADMA and 3-hydroxybutyrate (BHB) were identified asthe two major metabolites, which increased in Cav-1 (-/-) nullmammary fat pads and lung tissue, along with pyruvate to a lesserextent. These 2 metabolites (ADMA and BHB) are reflective ofoxidative stress and mitochondrial dysfunction in Cav-1 (-/-)stromal cells.

[0301] To further validate these observations, transcriptionalprofiling data for the expression of the relevant enzymes that areinvolved in ADMA and ketone metabolism were analyzed. Bothtranscriptional profiles from Cav-1 (-/-) null stromal cells andhuman breast cancer tumor stroma were analyzed in parallel and arepresented in Table 9, FIG. 29. For this purpose, the mRNAexpression of the genes involved in ADMA production (PRMT genefamily members) and degradation (DDAH1/2), as well as nitric oxide(NO) related genes, as ADMA drives NOS uncoupling and theproduction of superoxide, instead of NO 19 was analyzed.Interestingly, using this approach, the genes involved in both ADMAproduction (PRMT2/7/8) and degradation (DDAH1/2), as well as nitricoxide production (NOS1/2/3 or NOS trafficking), are alltranscriptionally over-expressed. both in human tumor stroma and inCav-1 (-/-) stromal cells.

[0302] Next, ketone metabolism was assessed (Table 9, FIG. 29). Forthis purpose the transcriptional profiles of the genes associatedwith both ketone production (ACYL, HMGCS1/2, HMGCL, HMGCLL1, andBDH1/2) and ketone re-utilization (ACAT1/2 and) OXCT1/2) were,analyzed. Interestingly, only the genes associated with ketoneproduction, but not ketone re-utilization were associated withhuman tumor stroma. This is exactly as would be predicted, as theepithelial cancer cells should express the genes associated withketone re-utilization, so that they can re-use 3-hydroxybutyrate asan energy source for mitochondrial oxidative metabolism. Also, manyof the stromal genes involved in ketone production are specificallyassociated with tumor recurrence (ACLY, HMGCS2, HMGCLL1, and BDH1)and/or metastasis (BDH2). Many of these ketone production genes arealso transcriptionally over-expressed in Cav-1 (-/-) stromal cells,consistent with our current metabolic analysis.

Example 5

Micro-RNA (miR) Profiling Provides New Mechanistic Insight into howLoss of Stromal Cav-1 Drives Oxidative Stress, Autophagy, andMitochondrial Dysfunction

[0303] Since miRs have recently taken center stage in the molecularanalysis of tumor progression and metastasis, Cav-1 (-/-) stromalcells were subjected to miR transcriptional profiling. Using thisapproach, a select subset of miRs that could explain the oxidativeand catabolic phenotypes we observe in metabolically in Cav-1 (-/-)mammary fat pads could be identified. Table 10 shows that only aselect number of miRs were transcriptionally upregulated in Cav-1(-/-) stromal cells. For this analysis, a cut-off of 1.5-foldincreased (KO/WT) was chosen. P-values are as shown. Note that top2 miRs showed the most significant pvalues. Notably, miR-3) andmiR-34c were increased 4.2-fold and nearly 3-fold,respectively.

[0304] A large body of evidence suggests that both miR-31 andmiR-34c play prominent roles in both tumorigenesis and metastasis.miR-34c is normally induced under conditions of oxidative stress,DNA damage, and cellular senescence, consistent with our metabolicand mRNA transcriptional profiling data related to oxidativestress. miR-31, only the other hand, targets FIR (factor inhibitingHIF) 18. This, in turn, leads to the loss of FIH proteinexpression, driving HIF1-alpha transcriptional activation 18. Bothhypoxia and HIF1-alpha activation itself are known to be sufficientto induce autophagy and mitophagy. Thus, loss of Cav-1 expression,driving miR-31 over-expression, and HIF1-alpha transcriptionalactivation, may be sufficient to explain our current findingsrelated to oxidative stress, autophagy, and mitophagy. Inaccordance of role for Cav-1 in hypoxia regulation and HIF1-alphatranscriptional activation, an increase in miR-210 was observed,which is known to mediate many of the effects attributed to thehypoxia genetic transcriptional program 38. However, althoughmiR-210 was increased nearly 2-fold, it did not reach statisticalsignificance (p=0.07). miR-31 has recently been shown to beincreased in the serum of patients with oral squamous cell cancers,and is dramatically reduced upon tumor resection, indicating thatit can function as a marker for monitoring the course of cancertherapy. miR-31 is also upregulated in human colon cancers.Similarly, miR-210 is increased in the serum of pancreatic cancerpatients.

Example 6

Over-Expression of Autophagy and Mitophagy Markers in Cav-1 (-/-)Null Mammary Fat Pads: Cathepsin B and BNIP3

[0305] To further validate that a loss of Cav-1 drives the onset ofoxidative-stress induced autophagy, the expression of establishedautophagy markers, namely cathepsin B 42 and BNIP3, in Cav-1 (-/-)mammary fat pads was assessed. Cathepsin B is a well-knownlysosomal cysteine protease that is up-regulated in the tumorstroma of human breast cancers, and its expression is alsoassociated with tumor recurrence and metastasis (Table 7, FIG. 27).BNIP3 is a marker of autophagy which mediates the autophagicdestruction of mitochondria, by a process called mitophagy. BNIP3is also up-regulated by oxidative stress and/or hypoxia, and isunder the direct transcriptional control of HIF1a. The stromalexpression of BNIP3 is also associated with breast cancer tumorrecurrence (Table 6, FIG. 26). Importantly, FIG. 18 directly showsthat both cathepsin B (the pro-enzyme and activated form) and BNIP3are significantly over-expressed in Cav-1 (-/-) null mammary fatpads, relative wild-type controls processed in parallel.Immunoblotting with Cav-1 and beta-actin are shown for comparison.These results are consistent with the idea that a loss of Cav-1expression promotes the onset of autophagy in the tumor stromalcompartment.

Example 7

Ketones Promote Tumor Growth, without any Increase inAngiogenesis

[0306] To evaluate the potential tumor promoting properties of theproducts of aerobic glycolysis (such as 3-hydroxy-butyrate andL-lacate), a xenograft model employing MDA-MB-231 breast cancercells injected into the flanks of athymic nude mice was used. Tumorgrowth was assessed by measuring tumor volume, at 3-weeks posttumor cell injection. During this time period, mice wereadministered either PBS alone, or PBS containing 3-hydroxy-butyrate(500 mg/kg) or L-lactate (500 mg/kg), via daily intra-peritoneal(i.p.) injections.

[0307] Interestingly, FIG. 10 shows that 3-hydroxy-butyrate issufficient to promote an .about.2.5-fold increase in tumor growth,relative to the PBS-alone control. Under these conditions,L-lactate had no significant effect on tumor growth.

[0308] One mechanism that could account for the tumor-promotingproperties of 3-hydroxy-butyrate is increased tumor angiogenesis.Thus, the status of tumor vascularity using antibodies directedagainst CD31 was evaluated. Interestingly, FIG. 11 shows that thevascular density (number of vessels per field) was not increased bythe administration of either 3-hydroxy-butyrate or L-lactate. Thus,other mechanisms, such as the "Reverse Warburg Effect" may beoperating to increase tumor growth.

Example 8

Ketones and Lactate Function as Chemo-Attractants, StimulatingCancer Cell Migration

[0309] Next, whether 3-hydroxy-butyrate or L-lactate can functionas chemo-attractants was assessed, using a modified "BoydenChamber" assay, employing Transwell cell culture inserts.MDA-MB-231 cells were placed in the upper chambers, and3-hydroxy-butyrate (10 mM) or L-lactate (10 mM) were introducedinto the lower chambers. Interestingly, using this assay system,both 3-hydroxy-butyrate and L-lactate promoted cancer cellmigration by nearly 2-fold (FIG. 13). Thus, the metabolic productsof aerobic glycolysis can also function as chemo-attractants forcancer cells, probably via a form of nutrient sensing.

Example 9

Lactate Fuels Lung Metastasis

[0310] The effect of 3-hydroxy-butyrate and L-lactate on cancercell metastasis were measured. For this purpose, a well-establishedlung colonization assay was used, where MDA-MB-231 cells areinjected into the tail vein of athymic nude mice. After 7 weekspost-injection, the lungs were harvested and the metastases werevisualized with India ink staining. In this method, the lungparenchyma stains black, while the tumor metastatic foci remainunstained, and appear white. For quantitation purposes. the numberof metastases per lung lobe was scored.

[0311] FIG. 14 shows that relative to PBS-alone, the administrationof L-lacate stimulated the formation of metastatic foci by.about.10-fold. Under these conditions, 3-hydroxy-butryate had noeffect on metastasis formation. Representative examples of lungmetastasis in PBS-alone controls and L-lactate-treated animals areshown in FIG. 15. Note that the metastatic foci formed in L-lactatetreated animals are more numerous, and are also larger in size.

[0312] Thus, 3-hydroxy-butryate fuels tumor growth, while L-lactatestimulates lung metastasis. As such, the "tumor-promoting" effectsof 3-hydroxy-butyrate and L-lactate are remarkably specific to agiven phase of tumor progression.

Example 10

Human Breast Cancer Epithelial Cells In Vivo Show the Induction ofMitochondrial Oxidative Phosphorylation, Relative to Adjacent TumorStromal Cells

[0313] To further validate the model that human breast cancer cellsshow a shift towards mitochondrial oxidative metabolism, rawtranscriptional profiling data obtained by laser-capturemicro-dissection of human breast cancer samples was assessed. Inthis data set, breast cancer epithelial cells and adjacent tumorstroma were isolated from the same tumors, allowing their directcomparison by transcriptional profiling.

[0314] For this purpose, we generated a list of genes that weretranscriptionally up-regulated in human breast cancer epithelialcells, relative to the adjacent stromal cells. Then, this list ofgenes was intersected with existing databases to determine thecellular processes that were up-regulated in epithelial cancercells, relative to adjacent stromal cells. Interestingly, usingthis approach, shows that numerous gene sets associated withoxidative mitochondrial metabolism are indeed increased or"enriched" in human breast cancer cells, relative to adjacentstromal cells (Table 1, FIG. 21). Conversely, this means thatoxidative mitochondrial metabolism in down-regulated in the tumorstromal compartment, relative to the cancer cells, consistent withthe "Reverse Warburg Effect".

[0315] Moreover, the genes that were up-regulated in epithelialcancer cells were also down-regulated in response to starvation,hypoxia, and Alzheimer's disease/aging (associated with oxidativestress) (Table 1, FIG. 21). This is a strong indication theseepithelial cancer cells are not experiencing starvation, hypoxia,or oxidative stress, as they are presumably being "fed" byglycolysis in adjacent stromal cells.

[0316] Interestingly, the number one gene up-regulated in cancercells, relative to stromal cells, is a subunit of the mitochondrialenzyme isocitrate dehydrogenase (IDH3B; .about.7.5-fold increased;p=3.2.times.10-10) which catalyzes the oxidative de-carboxylationof isocitrate to 2-oxoglutarate, in the TCA cycle. During hypoxia,2-oxoglutarate would accumulate, leading to HIF-stabilization viainhibition of the prolyl-hydroxylases. However, this appears not tobe the case in the epithelial cancer cells, as the genes that theyup-regulate are down-regulated in response to hypoxia and/orHIF1-alpha activation (Table 1, FIG. 21). This is a furtherindication that the epithelial cancer cells are indeed usingoxidative mitochondrial metabolism.

[0317] These results provide independent clinically-relevantevidence that human epithelial breast cancer cells in vivo useoxidative mitochondrial metabolism in patients.

[0318] While the invention has been described in detail and withreference to specific examples thereof, it will be apparent to oneskilled in the art that various changes and modifications can bemade therein without departing from the spirit and scopethereof.

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