Patent Application
20140026234
The present invention relates to the biomarkers that are useful in the course of detection and/or treatment of cancer.
Cancer is one of the most significant diseases confronting mankind, and even though progress has been made in cancer treatment, particularly in the medical therapy of cancer, many challenges remain. In medical therapy of cancer, for example, the various anticancer agents for suppressing the growth of cancer cells that have been developed suppress the growth of not only cancer cells, but also normal cells, causing various side effects including nausea and vomiting, hair loss, myelosuppression, kidney damage, and nerve damage. Consequently, understanding the origins of these malignancies as well as developing models for the identification of new diagnostic arid therapeutic modalities is of significant interest to health care professionals.
Some research suggests that the environment around a cancer, for example, interstitial tissue which includes blood vessels, extracellular matrix (ECM), and fibroblasts, may play a role in the onset and progression of cancer. For example, Camps et al. (Proc. Natl. Acad. Sci. USA 1990, 87(1), 75-79) reported that when an athymic nude mouse was inoculated with tumor cells that do not form a tumor on their own or for which the tumor formation rate is low, together with tumorigenic fibroblasts, rapid and marked formation of a tumor was observed, and Olumi et al. (Cancer Res. 1999, 59(19), 5002-5011) reported that when peritumoral fibroblasts (i.e., cancer-associated fibroblasts or CAFs) from a prostate tumor patient were grafted on an athymic animal together with human prostate cells, neoplastic growth thereof was markedly accelerated. Furthermore, it has been clarified that a bioactive substance such as PDGF (platelet-derived growth factor), TGF-β (transforming growth factor-β), HGF (hepatocyte growth factor), or SDF-1 (stromal cell-derived factor-1) produced in the interstitium is involved in such growth of a tumor (Micke et al., Expert Opin Ther Targets. 2005, 9(6), 1217-1233).
Despite these findings, many needs remain unmet, including a better understanding of the environment around a cancer and effective models for the evaluation, diagnosis and generation of therapies for cancers, including metastatic cancers, in particular breast cancer. In this context, desirable models include those which provide insight into the processes underlying, for example, cancer onset and/or cancer progression, thereby facilitating diagnosis of cancer and/or generation of prophylactic and/or therapeutic treatments for cancer.
The present inventors have discovered a genetically tractable model system for identifying the genetic factors that govern the tumor promoting effects of cancer-associated fibroblasts. Accordingly, one aspect of the present invention provides a genetic model system for identifying the genetic factors that govern the tumor promoting effects of cancer-associated fibroblasts, the genetic model system comprising human Cav-1 deficient immortalized fibroblasts created using a targeted sh-RNA knock-down approach.
In certain embodiments, proteomics can be used to discover suitable biomarkers for use with the present invention. Proteomics is the study of proteome, the protein complement of the genome. The term proteome also used to refer to the totality of the proteins present in a sample (e.g. tissue, organism, or cell culture) at a certain point of time. Proteomics includes, among other things, study of the global changes of protein expression in a sample (also referred to as “expression proteomics”). Proteomics typically includes the following steps: (1) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (2) identification of the individual proteins recovered from the gel, e.g. by mass spectrometry and/or N-terminal sequencing, and (3) analysis of the data using bioinformatics. Proteomics methods are valuable supplements to other methods of gene expression profiling, and can be used, alone or in combination with other methods of the present invention, to detect the biomarkers of the present invention.
Accordingly, in another aspect, the present application concerns the identification, e.g., through proteomics, of one or more of a set of biomarkers (also referred to herein as “markers”) in tumor stroma that are predictive of the outcome of cancer in a cancer patient. These markers include ACO2, ALB, ANPEP, ANXA2, APEX1, ATP5A1, BAG2, CALR, CALU, CAPZB, CDC42, COL1A1, COL6A1, COL6A2, CRABP2, CRTAP, DMGDH, DNAJA3, DNM1L, ENO1, ETFB, FBN1, FKBP9, GAPDH, GDF2, GLUD1, HIST2H4B, HNRNPA2B1, HSPA8, HSPA9, HSPB1, HSPD1, IDH2, KIAA1409, LDHA, LDHAL6B, LGALS1, LGALS3, LMNA, MATR3, MT1M, MYL6, NDUFA5, NDUFS3, P4HA1, P4HA2, PITRM1, PKM2, PLOD1, PRDX1, PRDX4, PRDX6, PSME1, RAP1A, RCN1, RPLP2, S100A13, SCO2, SERPINH1, SHMT2, SOD2, SYNJ2BP, TPM1, TPM4, TRPC4AP, TXNDC5, UQCRFS1, VAT1, VIM, WDR78, XRCC6BP1, YWHAB and YWHAZ.
In another aspect, the present invention provides a method for determining the prognosis of a cancer in a subject, the method comprising: (a) determining the expression level of at least one biomarker or a prognostic signature, said at least one biomarker or prognostic signature being associated with the prognosis of the cancer, wherein said at least one biomarker or prognostic signature comprises one or more biological molecules associated with the prognosis of the cancer, in a cancer sample obtained from the subject; (b) comparing the expression level of the at least one biomarker or the prognostic signature in the cancer sample with the expression level of the at least one biomarker or the prognostic signature expression in a control sample, wherein said prognosis is made when the expression level of the at least one biomarker or a prognostic signature in the sample of cancer is greater than the expression level of the at least one biomarker or the prognostic signature in the control sample.
It has been discovered that loss of stromal Cav-1 in human cancer associated fibroblasts dramatically promotes the growth of stromal cancers. In particular, loss of stromal Cav-1 in human cancer associated fibroblasts dramatically promotes the growth of triple negative breast cancer cells (MDA-MB-231), increasing both tumor mass and tumor volume by about 4-fold, without any increase in angiogenesis.
It has also been discovered that the phenotype of the Cav-1 knock-down fibroblasts can be significantly reverted by reducing oxidative stress in the tumor micro-environment. In particular, it was found that mitochondrial superoxide disumutase 2 (SOD2) significantly reverted the tumor promoting phenotype of Cav-1 deficient fibroblasts. Loss of Cav-1 is believed to increases reactive oxygen species (ROS) production in stromal fibroblasts. To combat the resulting oxidative stress, SOD2 was stably overexpressed in Cav-1 knock-down fibroblasts using a lenti-viral vector with puromycin resistance. Also, as a control, Cav-1 knock-down cells were transfected with the empty vector alone, in parallel. Then, these two fibroblast lines were co-injected with MDA-MB-231 cells into the flanks of nude mice. Relative to the control, Cav-1 knock-down fibroblasts with over expressed SOD2 reduced the tumor promoting effects of Cav-1 knock-down fibroblasts by nearly 2-fold. Therefore, SOD2 is useful as a genetic suppressor of Cav-1 deficient stromal cancers.
Accordingly, in another aspect, the present invention provides a method for identifying genetic suppressors and/or genes or screening for potential therapeutic agents that reduce oxidative stress associated stromal Cav-1 deficient cancers. The method comprising: (a) providing a wild-type mouse injected into its flanks with a cancer cell line, wherein the cancer has a stromal component, as a control mouse; (b) providing a Cav-1 deficient mouse injected with a cancer cell line in its flanks, as a test mouse; (c) providing a potential therapeutic agent or a potential genetic suppressor; (d) injecting a placebo into a test mouse; (e) injecting a placebo into a control mouse; (f) treating both a test mouse and a control mouse with the potential therapeutic agent or the potential genetic suppressor; (g) measuring the mass and/or the size of the resulting cancer tumor in the test mouse and the control mouse in the presence of placebo; (h) measuring the mass and/or the size of the resulting cancer tumor in the test mouse and the control mouse in the presence of the potential therapeutic agent or the genetic suppressor; and (i) comparing the mass and/or the size in the test mouse with the mass and/or the size in the control mouse, in the presence of either placebo or the potential therapeutic agent or the potential genetic suppressor, wherein a decrease in the mass and/or the size in the test mouse injected with the potential therapeutic agent or the potential genetic identifies a therapeutic agent or a genetic suppressor which treats stromal Cav-1 deficient cancer.
It has been discovered that overexpression of one or more biological molecules is associated with aggressive disease and poor prognosis in cancer. Accordingly, in an exemplary embodiment, the present invention provides a biomarker (or a prognostic signature) for determining the risk of recurrence or progression of a cancer, the biomarker or the prognostic signature comprising a biological molecule or a combination of biomarkers associated with prognosis of the cancer and is selected from the group consisting of ACO2, ALB, ANPEP, ANXA2, APEX1, ATP5A1, BAG2, CALR, CALU, CAPZB, CDC42, COL1A1, COL6A1, COL6A2, CRABP2, CRTAP, DMGDH, DNAJA3, DNM1L, ENO1, ETFB, FBN1, FKBP9, GAPDH, GDF2, GLUD1, HIST2H4B, HNRNPA2B1, HSPA8, HSPA9, HSPB1, HSPD1, IDH2, KIAA1409, LDHA, LDHAL6B, LGALS1, LGALS3, LMNA, MATR3, MT1M, MYL6, NDUFA5, NDUFS3, P4HA1, P4HA2, PITRM1, PKM2, PLOD1, PRDX1, PRDX4, PRDX6, PSME1, RAP1A, RCN1, RPLP2, S100A13, SCO2, SERPINH1, SHMT2, SOD2, SYNJ2BP, TPM1, TPM4, TRPC4AP, TXNDC5, UQCRFS1, VAT1, VIM, WDR78, XRCC6BP1, YWHAB, YWHAZ.
A new xenograft system for modeling the lethality of a loss of stromal Cav-1 has been discovered. More specifically, it has been observed that a loss of stromal Cav-1 in human cancer associated fibroblasts dramatically promotes the growth of triple negative breast cancer cells (MDA-MB-231), increasing both tumor mass and tumor volume by about 4-fold, without any increase in angiogenesis. Furthermore, it has been shown that this phenotype can significantly reverted by reducing oxidative stress in the tumor micro-environment. The reduction of the oxidative stress was achieved via the recombinant overexpression of mitochondrially-targeted super-oxide dismutase (SOD2), in Cav-1 deficient cancer associated fibroblasts. As such, this new xenograft model provides a genetically tractable system for dissecting the key factors that govern the lethality of a Cav-1 deficient “pro-oxidative” tumor micro-environment.
According to a new paradigm for understanding tumor metabolism, called “The Autophagic Tumor Stroma Model of Cancer Metabolism” catabolism (autophagy) in the tumor stroma fuels the anabolic growth of aggressive cancer cells. It is believed that the tumor cells induce autophagy in adjacent cancer-associated fibroblasts via the loss of caveolin-1 (Cav-1), which is sufficient to promote oxidative stress in stromal fibroblasts. A human Cav-1 deficient immortalized fibroblasts created using a targeted sh-RNA knock-down approach have been used to demonstrate the role of Cav-1 deficient fibroblasts in promoting tumor growth. Relative to control fibroblasts, Cav-1 deficient fibroblasts dramatically promoted tumor growth in xenograft assays employing an aggressive human breast cancer cell line, namely MDA-MB-231 cells. Co-injection of Cav-1 deficient fibroblasts, with MDA-MB-231 cells, increased both tumor mass and tumor volume by about 4-fold.
Immuno-staining with CD31 indicated that this paracrine tumor promoting effect was independent of angiogenesis. Mechanistically, proteomic analysis of these human Cav-1 deficient fibroblasts identified >40 protein biomarkers that were upregulated, most of which were associated with (i) myofibroblast differentiation or (ii) oxidative stress/hypoxia.
In direct support of these findings, the tumor promoting effects of Cav-1 deficient fibroblasts could be functionally suppressed (nearly 2-fold) by the recombinant overexpression of SOD2 (superoxide dismutase 2), a known mitochondrial enzyme that de-activates superoxide, thereby reducing mitochondrial oxidative stress.
In contrast, cytoplasmic soluble SOD1 had no effect, further highlighting a specific role for mitochondrial oxidative stress in tumor promoting effect of Cav-1 deficient fibroblasts. The evidence directly support a key role for a loss of stromal Cav-1 expression and oxidative stress in cancer-associated fibroblasts, in promoting tumor growth, which is consistent with “The Autophagic Tumor Stroma Model of Cancer”. The human Cav-1 deficient fibroblasts described herein are a new genetically tractable model system for identifying suppressors of the cancer-associated fibroblast phenotype, via a genetic “complementation” approach.
The results disclosed herein elucidate the pathogenesis of triple negative and basal breasts cancers, as well as tamoxifen-resistance in ER-positive breast cancers, which are all associated with a Cav-1 deficient “lethal” tumor microenvironment, driving poor clinical outcome.
Accordingly, in some aspects, the present invention provides biomarkers and medical applications of the same, including methods of using the markers in diagnosis of cancer, determining prognosis of cancer, identifying potential cancer therapeutic agents, monitoring the progression of cancer in patients, and identifying genetic suppressors of cancer.
In one aspect the present invention provides a genetically tractable model system for identifying the genetic factors that govern the tumor promoting effects of cancer-associated fibroblasts. The genetic model system includes human fibroblast engineered to lack Cav-1, which in turn is co-injected with a human cancer cell line into immunodeficient mice.
In another aspect, the present invention provides a set of biomarkers or a prognostic signature associated with the prognosis of cancer, wherein in the set of biomarkers or the prognostic signature is selected from the group consisting of ACO2, ALB, ANPEP, ANXA2, APEX1, ATP5A1, BAG2, CALR, CALU, CAPZB, CDC42, COL1A1, COL6A1, COL6A2, CRABP2, CRTAP, DMGDH, DNAJA3, DNM1L, ENO1, ETFB, FBN1, FKBP9, GAPDH, GDF2, GLUD1, HIST2H4B, HNRNPA2B1, HSPA8, HSPA9, HSPB1, HSPD1, IDH2, KIAA1409, LDHA, LDHAL6B, LGALS1, LGALS3, LMNA, MATR3, MT1M, MYL6, NDUFA5, NDUFS3, P4HA1, P4HA2, PITRM1, PKM2, PLOD1, PRDX1, PRDX4, PRDX6, PSME1, RAP1A, RCN1, RPLP2, S100A13, SCO2, SERPINH1, SHMT2, SOD2, SYNJ2BP, TPM1, TPM4, TRPC4AP, TXNDC5, UQCRFS1, VAT1, VIM, WDR78, XRCC6BP1, YWHAB, YWHAZ and combinations thereof.
It has been discovered that levels of these biomarkers are significantly altered in human Cav-1 deficient fibroblasts, where Cav-1 deficient fibroblasts serve as a model for the “lethal tumor stroma” of human breast cancers having a poor clinical outcome. An increase in the expression level of any one or a panel of these biomarkers detected in a test biological sample compared to a normal control level indicates that the subject (from which/whom the sample was obtained) suffers from or is at risk of developing cancer.
In one embodiment, a deviation, increase or decrease in the expression level of any one or a panel of the above biomarkers detected in a test biological sample compared to a normal control level indicates that the subject (from which the sample was obtained) suffers from or is at risk of recurrence or progression cancer, such as breast cancer.
Alternatively, the expression level of any one or a panel of cancer-associated biomarkers in a biological sample may be compared to a cancer control level of the same biomarker or the same panel of biomarkers.
Thus, one aspect of the present invention is to provide a prognostic method for and/or treatment of cancer. In certain embodiments particular cancers having a stromal component/cells can be diagnosed and/or treated.
Stromal cells are connective tissue cells of an organ found in the loose connective tissue, including uterine mucosa (endometrium), prostate, bone marrow, bone marrow precursor cells, and the ovary and the hematopoietic system. The most common types of stromal cells include fibroblasts, immune cells, pericytes, endothelial cells, and inflammatory cells. Thus, cancers having a stromal component can occur in any organ or tissue with stromal component, including uterine mucosa (endometrium), prostate, bone marrow, bone marrow, the ovary and the hematopoietic system.
Accordingly, while any cancer can be considered within the scope of the present invention, particular cancers having a stromal component include leukemia, prostate cancer, ovarian sex cord-stromal cell cancers (e.g., Sertoli-Leydig cell tumor, granulosa-theca cell tumor, theca cell tumor, thecoma, fibroma, and gonadoblastoma), gastrointestinal stromal cancers (GIST), endometrial cancers, mesenchymal stromal cancers. In one embodiment, the cancer is breast cancer.
In one embodiment for diagnosing the presence or absence of a cancer, and in particular a cancer having a stromal component, includes the steps of: (a) providing a biological test sample from a subject afflicted with a cancer (e.g., breast cancer) or suspected of having cancer; (b) determining a level of at least one biomarker in the test sample that is associated with the prognosis of the cancer; (c) comparing the level of said at least one biomarker in the test sample to the level of the biomarker in a control sample, wherein an altered level, e.g., an increase or decrease in level, of the biomarker in said test sample relative to the level of the biomarker in said control sample is a prognostic indicator of the course of cancer disease in said subject.
In some embodiments the biomarker expression is increased or decreased 10%, 25%, 50% or more compared to the control level. Alternatively, biomarker expression is increased or decreased 1, 2, 3, 4, 5, 6, 7, or more, fold compared to the control level.
In some embodiments, the subject-derived biological sample may be any sample derived from a subject, e.g., a patient known to or suspected of having cancer. For example, the biological sample may be tissue containing sputum, blood, serum, plasma or cells from a breast tissue.
Another aspect of the present invention provides a method of monitoring the progression of breast cancer in a subject, the method comprising: (a) obtaining a first sample from a subject at a first time point and a second sample from said subject at a second time point; (b) determining the level of at least one biomarker in said first and second samples; (c) comparing the level of said at least one biomarker in said first sample to the level of said biomarker in said second sample, wherein an altered, e.g., elevated, level of the at least one biomarker in said second sample relative to the level in said first sample is an indication that the cancer has progressed in said subject.
One aspect of the present invention includes use of one or more isolated protein markers of the transformed fibroblasts, selected from a group consisting of ACO2, ALB, ANPEP, ANXA2, APEX1, ATP5A1, BAG2, CALR, CALU, CAPZB, CDC42, COL1A1, COL6A1, COL6A2, CRABP2, CRTAP, DMGDH, DNAJA3, DNM1L, ENO1, ETFB, FBN1, FKBP9, GAPDH, GDF2, GLUD1, HIST2H4B, HNRNPA2B1, HSPA8, HSPA9, HSPB1, HSPD1, IDH2, KIAA1409, LDHA, LDHAL6B, LGALS1, LGALS3, LMNA, MATR3, MT1M, MYL6, NDUFA5, NDUFS3, P4HA1, P4HA2, PITRM1, PKM2, PLOD1, PRDX1, PRDX4, PRDX6, PSME1, RAP1A, RCN1, RPLP2, S100A13, SCO2, SERPINH1, SHMT2, SOD2, SYNJ2BP, TPM1, TPM4, TRPC4AP, TXNDC5, UQCRFS1, VAT1, VIM, WDR78, XRCC6BP1, YWHAB, and YWHAZ for screening, detection, prognosis or determining therapeutic targets for cancer of the breast.
In another embodiment, the invention includes a method of screening, detecting, prognosticating cancer of the breast, comprising: isolating a set, i.e., two or more, of co-expressed differentiator marker proteins from the transformed fibroblast cells, wherein the co-expressed differentiator marker proteins being marker proteins selected from group consisting of ACO2, ALB, ANPEP, ANXA2, APEX1, ATP5A1, BAG2, CALR, CALU, CAPZB, CDC42, COL1A1, COL6A1, COL6A2, CRABP2, CRTAP, DMGDH, DNAJA3, DNM1L, ENO1, ETFB, FBN1, FKBP9, GAPDH, GDF2, GLUD1, HIST2H4B, HNRNPA2B1, HSPA8, HSPA9, HSPB1, HSPD1, IDH2, KIAA1409, LDHA, LDHAL6B, LGALS1, LGALS3, LMNA, MATR3, MT1M, MYL6, NDUFA5, NDUFS3, P4HA1, P4HA2, PITRM1, PKM2, PLOD1, PRDX1, PRDX4, PRDX6, PSME1, RAP1A, RCN1, RPLP2, S100A13, SCO2, SERPINH1, SHMT2, SOD2, SYNJ2BP, TPM1, TPM4, TRPC4AP, TXNDC5, UQCRFS1, VAT1, VIM, WDR78, XRCC6BP1, YWHAB, and YWHAZ.
In another embodiment, a method for treating a neoplastic disease in a patient is provided, comprising (a) obtaining a sample of stromal cells adjacent to the neoplasm from the neoplastic disease patient; (b) determining the level of caveolin-1 (Cav-1) protein expression in the stromal cells of the sample and comparing the level of Cav-1 protein expression in the stromal cells of the sample with the level of Cav-1 protein expression in a control; (c) predicting if the neoplasm will respond effectively to treatment with an anti-angiogenic agent, wherein said prediction is made when the level of Cav-1 protein expression in the stromal cells of the sample is lower than the level of Cav-1 protein expression in the control; and administering to said patient a therapeutically effective amount of an anti-angiogenic agent such as Avastin (bevacizumab).
In other embodiments, suitable anti-angiogenic agents include any agents that target the vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) pathways in patients with cancer. Examples the anti-angiogenic agents include selective inhibitors of the VEGF pathway (e.g., bevacizumab and VEGF Trap); VEGF/PDGF pathway inhibitors (e.g., sorafenib and sunitinib); and VEGF/PDGF/FGF pathway inhibitors (e.g., cediranib, pazopanib, and BIBF 1120)
In another embodiment, the invention provides a method of predicting whether a cancer in a cancer patient will respond effectively to treatment with an anti-angiogenic agent, comprising: (a) obtaining a sample of stromal cells adjacent to the cancer in a sample derived from the cancer patient; (b) determining the level of Cav-1 protein expression in the stromal cells of the sample; and (c) comparing the level of Cav-1 protein expression in the stromal cells of the sample with the level of Cav-1 protein expression in a control; (c) predicting if the cancer will respond effectively to treatment with an anti-angiogenic agent, wherein low expression levels of Cav-1 protein expression in the stromal layers relative to Cav-1 expression levels in the control correlate with a cancer that will not respond effectively to treatment with an angiogenic agent.
In another aspect, the present invention provides a method for identifying genetic suppressors that can block the tumor promoting properties of cancer-associated fibroblast cells specifically lacking Cav-1. The method comprises identifying genes which when expressed or undergo modulated expression reduce oxidative stress.
In another embodiment, the present invention includes a method of modulating one or more biomarker which are indicative of a disease state to thereby treat of cancer. In such an embodiment, one or more biomarkers can be modulated by the administration a compound which reduces oxidative stress. In one embodiment, acetylcysteine (commonly referred to as N-acetylcysteine) can be used.
It should be noted that the predictive value of stromal Cav-1 is independent of epithelial marker status, and is effective in all the major sub-types of breast cancer, including ER+, PR+, HER2+and triple negative (ER−, PR−, HER2−) breast cancer patients. In triple negative (TN) patients, which is one of the most lethal types of breast cancer, stromal Cav-1 effectively distinguished between low-risk and high-risk patients.
In an embodiment, the predictive value of stromal Cav-1 is independent of epithelial marker status, and is effective any cancer selected from the group consisting of basal cell carcinoma, glioma, breast cancer, chondrosarcoma, colon cancer, esophageal cancer, gastric cancer, gastrointestinal stromal tumor, hepatocellular cancer, lung cancer, medulloblastoma, melanoma, neuroectodermal tumors, osteogenic sarcoma, ovarian cancer, pancreatic cancer, prostate cancer, and testicular cancer.
In TN patients with high stromal Cav-1, their overall survival was >75% up to 12 years post-diagnosis. In contrast, in TN patients with absent stromal Cav-1, their overall survival was <10% at 5 years post-diagnosis. Thus, a loss of stromal Cav-1 is a key predictor of a “lethal” tumor micro-environment.
Accordingly, in an aspect, the present invention provides a method of identifying a potential therapeutic agent that treats stromal Cav-1 deficient cancer comprising: (a) providing a wild-type animal, e.g., a mouse, injected with mouse mammary cancer cells in the mammary fat pad as a control mouse; (b) providing a Cav-1 deficient mouse injected with mouse mammary cancer cells in the mammary fat pad as a test mouse; (c) providing a potential therapeutic agent; (d) injecting a placebo into a test mouse; (e) injecting a placebo into a control mouse; (f) treating both a test mouse and a control mouse with the potential therapeutic agent; (g) measuring the mass and/or the size of the resulting cancer tumor in the test mouse and the control mouse in the presence of placebo; (h) measuring the mass and/or the size of the resulting cancer tumor in the test mouse and the control mouse in the presence of the potential therapeutic agent; and (i) comparing the mass and/or the size in the test subject mouse with the mass and/or the size in the control mouse, in the presence of either placebo or the potential therapeutic agent, wherein a decrease in the mass and/or the size in the test mouse injected with the potential therapeutic agent identifies a therapeutic agent which treats stromal Cav-1 deficient cancer.
A “normal control level” indicates an expression level of a biomarker detected in a normal, healthy individual or in a population of individuals known not to be suffering from breast cancer. A control level is a single expression pattern derived from a single reference population or from a plurality of expression patterns. In contrast to a “normal control level”, the “control level” is an expression level of a biomarker detected in an individual or a population of individuals whose background of the disease state is known (i.e., cancerous or non-cancerous). Thus, the control level may be determined based on the expression level of a biomarker in a normal, healthy individual, in a population of individuals known not to be suffering from breast cancer, a patient suffering from breast cancer or a population of the patients. The control level corresponding to the expression level of a biomarker in a patient of breast cancer or a population of the patients are referred to as “breast cancer control level”. Furthermore, the control level can be a database of expression patterns from previously tested individuals.
The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They set forth for explanatory purposes only, and are not to be taken as limiting the invention.
General Techniques
Cell Cultures
Human immortalized fibroblasts (hTERT-BJ1) and human breast cancer cells (MDA-MB-231-GFP) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum in a 37° C., 5% CO2 incubator. hTERT-BJ1 fibroblasts stably-expressing eNOS, and the corresponding vector alone control, were prepared as previously described in Martinez-Outschoorn et al. Cell Cycle 2010, 9, 3256-3276.
sh-RNA silencing and retroviral infection. sh-RNA control and two pre-designed sh-RNAs targeting nucleotides 383-403 (5′-GCT GAG CGA GAA GCA AGT GTA-3′) or 660-680 (5′-TGG GCA GTT GTA CCA TGC ATT-3′) of the CAV1 mRNA (NM—001753.3) were obtained from Invitrogen and were subcloned into the pQCXIP-GFP retroviral vector (Clontech, Inc.). The sh-RNA negative control contains an insert that forms a hairpin structure that is predicted not to target any known vertebrate gene (Invitrogen, Inc.). For retroviral infection, vectors were transiently transfected into the amphotropic Phoenix packaging cell line, using a modified calcium phosphate method. Forty-eight hours post-transfection, the viral supernatant was collected, 0.45 μm sterile filtered, and added to the target cells. Two infection cycles were carried out (every 12 hours) with hTERT-BJ1 cells. Effective knockdown of CAV1 was determined by Western blot analysis of FACS-sorted GFP-positive cells.
sh-RNA Silencing and Retroviral Infection
sh-RNA control and two pre-designed sh-RNAs targeting nucleotides 383-403 (5′-GCT GAG CGA GAA GCA AGT GTA-3′) or 660-680 (5′-TGG GCA GTT GTA CCA TGC ATT-3′) of the CAV1 mRNA (NM—001753.3) were obtained from Invitrogen and were subcloned into the pQCXIP-GFP retroviral vector (Clontech, Inc.). The sh-RNA negative control contains an insert that forms a hairpin structure that is predicted not to target any known vertebrate gene (Invitrogen, Inc.). For retroviral infection, vectors were transiently transfected into the amphotropic Phoenix packaging cell line, using a modified calcium phosphate method. Forty-eight hours post-transfection, the viral supernatant was collected, 0.45 μm sterile filtered, and added to the target cells. Two infection cycles were carried out (every 12 hours) with hTERT-BJ1 cells. Effective knockdown of CAV1 was determined by Western blot analysis of FACS-sorted GFP-positive cells.
Recombinant Expression of SOD2
hTERT-BJ1 Cav-1 knock-down cells were transduced with a lenti-viral vector encoding SOD2 (Human superoxide dismutase 2, mitochondrial; Accession #'sY00985.1/NM—000636.2), with puromycin resistance (pReceiver-I0569-Lv105) or with the vector alone (pReceiver-Lv105), as a critical negative control (GeneCopoeia, Inc.). The same strategy was also used to overexpress SOD1 (Human superoxide dismutase 1, soluble; Accession #X02317), with the construct pReceiver-K2710-Lv105 (GeneCopoeia, Inc.), Athymic Ncr-nu/nu mice (7-to-9 weeks old) were purchased from Taconic. For each injection, 106 MDA-MB-231 cells and 300,000 hTERT-BJ1 fibroblasts in 100 μl of sterile PBS were injected subcutaneously into the flanks of the nude mice. Two flank injections were performed per mouse. Tumors weights and volumes were then measured at 4,5 weeks post-injection (Chiavarina et al., Cell Cycle 2010, 9, 3534-3551; Bonuccelli et al. Cell Cycle 2010, 9, 1960-1971; Migneco et al. Cell Cycle 2010, 9, 2412-2422; and Bonuccelli et al., Cell Cycle 2010, 9, 3506-3514).
Quantitation of Tumor Angiogenesis
Immunohistochemical staining for CD31 was performed on frozen tumor sections using a 3-step biotin-streptavidin-horseradish peroxidase method. Frozen tissue sections (6 μm) were fixed in 2% paraformaldehyde in PBS for 10 min and washed with PBS. After blocking with 10% rabbit serum the sections were incubated overnight at 4° C. with rat antimouse CD31 antibody (BS Biosciences) at a dilution of 1:200, followed by biotinylated rabbit anti-rat IgG (1:200) antibody and streptavidin-HRP. Immunoreactivity was revealed with 3,3′-diaminobenzidine. For quantitation of vessels, CD31-positive vessels were enumerated in 4-6 fields within the central area of each tumor using a 20× objective lens and an ocular grid (0.25 mm2 per field). The total number of vessels per unit area was calculated and the data was represented graphically.
Protcomic Analysis
2-D DICE (two-dimensional difference gel electrophoresis) and mass spectrometry protein identification were run by Applied Biomics (Hayward, Calif.). Image scans were carried out immediately following the SDS-PAGE using Typhoon TRIO (Amersham BioSciences) following the protocols provided. The scanned images were then analyzed by Image QuantTL software (GE-Healthcare), and then subjected to in-gel analysis and cross-gel analysis using DeCyder software version 6.5 (GE-Healthcare). The ratio of protein differential expression was obtained from in-gel DeCyder software analysis. The selected spots were picked by an Ettan Spot Picker (GE-Healthcare) following the DeCyder software analysis and spot picking design. The selected protein spots were subjected to in-gel trypsin digestion, peptides extraction, desalting and followed by MALDI-TOF/TOF (Applied Biosystems) analysis to determine the protein identity.
Proteomic analysis of Cav-1 deficient fibroblasts provides evidence for the onset of a myofibroblast phenotype and mitochondrial oxidative stress. In order to mechanistically dissect the tumor promoting activity associated with human Cav-1 knockdown fibroblasts, they were subjected to an extensive unbiased proteomic analysis, as detailed above. Cav-1 deficient fibroblasts showed the upregulation of 15 gene products associated with the myo-fibroblast phenotype, including numerous proteins associated with collagen synthesis and processing (COL1A1; COL6A1/2; P4HA1/2; HSP47; PLOD1), muscle-specific proteins (VIM; MYL6; TPM1/4), and other components of the extracellular matrix (LGALS1/3; CRTAP) (see Table 1).
While not wishing to be bound by any particular mechanism of action or theory, the observed upregulation of factors associated with the autophagic destruction of mitochondria (DNM1L), and lipid/protein catabolism (DMGDH; PSME1) is in accordance with the idea that oxidative stress induces autophagy/mitophagy. The upregulation of CDC42 is consistent with the activation of ROS production via NADPH oxidase and the overexpression of DMGDH (dimethylglycine dehydrogenase) is also consistent with oxidative stress. DMGDH functions in the catabolism of choline, catalyzing the oxidative demethylation of dimethylglycine to form sarcosine. Increase in sarcosine directly parallels a loss of reduced glutathione, indicative of an oxidative tumor micro-environment. Thus, increased sarcosine is a biomarker for oxidative stress, which is associated with advanced prostate cancer and the development of metastatic disease. Furthermore, observed upregulation of 4 calcium-binding proteins (CALU; CALR; RCN1; S100A13), suggests that there may be dys-regulation of calcium homeostasis, in Cav-1 deficient fibroblasts (see Table 1).
Since oxidative stress is known to be associated with the disruption of calcium homeostasis, the upregulation of both myofibroblast and oxidative stress markers accords with oxidative stress as being sufficient and/or required for the induction of the myofibroblast phenotype. Also, many of the proteins induced by Cav-1 knock-down in fibroblasts are highly expressed in the tumor stroma of human breast cancer patients, and are associated with tumor recurrence or metastasis (See Table 2).
), mitochondrial (IDH2) (induced by oxidative
indicates data missing or illegible when filed
CALR
CALU
CDC42
COL1A1
COL6A1
CRTAP
DMGDH
DNM1L
FBN1
FKBP9
HSPB1
LDHAL6B
LGAL53
MYL6
P4HA2
S100A13
SERPINH1
TPM1
TPM4
XRCC6BP1
YWHAB
indicates data missing or illegible when filed
Proteomic Analysis of hTERT eNOS Fibroblasts
Proteomic analysis of eNOS transfected fibroblasts identified SOD2 as a potential stromal tumor suppressor (
The eNOS proteomic data is also consistent with analyses of several other Cav-1 deficient fibroblastic cell lines, including murine Cav-1 (−/−) knock-out fibroblasts, murine mesenchymal stem cells, HIF-alpha and IKBKE-transfected hTERT-BJ1 human fibroblasts, which also lack Cav-1. Thus, the induction of mitochondrial oxidative stress with eNOS is indeed sufficient to induce a proteomic profile similar to the one we observed due to a loss of Cav-1.
This observation supports the idea that a loss of Cav-1 leads to increased NO production. However, unlike Cav-1 deficient fibroblasts (
Thus, it would appear that eNOS overexpressing fibroblasts may have upregulated a “tumor suppressor protein” to allow them to adjust or adapt to a very high-level of mitochondrial oxidative stress, thereby “repressing” their tumor promoting activity. The sequence listings associated with the accession numbers set forth in Table 3 are hereby incorporated by reference as if set forth fully herein.
superoxide dismutase 2, mitochondrial (SOD2)
1.44
gi|30841309
48
transporting, mitochondrial F1 complex, alpha subunit 1, cardiac
indicates data missing or illegible when filed
The proteomic profiles obtained from Cav-1 deficient and eNOS transfected fibroblasts were found to overlap with the transcriptional stromal profiles obtained from human breast caners. Transcriptional profiles of a large data set of human breast cancer patients whose tumors were subjected to laser-capture micro-dissection in classified based on stroma into three subgroups: Tumor stroma vs. normal stroma list; Recurrence stroma list; and Lymph-node (LN) metastasis stroma list. The tumor stroma vs. normal stroma list compared the transcriptional profiles of tumor stroma obtained from 53 patients to normal stroma obtained from 38 patients. Gene transcripts that were consistently upregulated in tumor stroma were selected and assigned a p-value, with a cut-off of p<0.05 (contains 6,777 genes). The recurrence stroma list compared the transcriptional profiles of tumor stroma obtained from 11 patients with tumor recurrence to the tumor stroma of 42 patients without tumor recurrence. Gene transcripts that were consistently upregulated in the tumor stroma of patients with recurrence were selected and assigned a p-value, with a cut-off of p<0.05 (contains 3,354 genes). And the lymph-node (LN) metastasis stroma list compared the transcriptional profiles of tumor stroma obtained from 25 patients with LN metastasis to the tumor stroma of 25 patients without LN metastasis. Gene transcripts that were consistently upregulated in the tumor stroma of patients with LN metastasis were selected and assigned a p-value, with a cut-off of p<0.05 (contains 1,182 genes). All three gene lists were then individually intersected with the proteomic profiles of Cav-1 deficient and eNOS-transfected fibroblasts. The results are shown in Table 4.
ACO2
ALB
APEX1
BAG2
COL6A2
DNAJA3
GDF2
PKM2
PRDX6
SHMT2
SOD2
VAT1
WDR78
Proteins that were transcriptionally upregulated in laser-capture microdissected human breast cancer tumor stroma are shown in BOLD. Those gene products that are associated with tumor recurrence or metastasis are shown in BOLD and are underlined. P-values are as shown.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US12/22933 | 1/27/2012 | WO | 00 | 10/1/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61437108 | Jan 2011 | US | |
| 61437120 | Jan 2011 | US |
