The development of intrinsic or acquired drug resistance by tumor cells significantly limits the efficacy of antineoplastic agents and is the major contributing factor to therapeutic failure of human malignancies. Drug resistance refers to progressive disease of the malignant tumors that occurs at doses associated with manageable toxicity of the drug. It is well known in the clinical practice that many malignant tumors are initially sensitive to chemotherapy, but the vast majority will eventually recur and develop broad resistance to conventional cytotoxic chemotherapeutic agents and radiotherapy (Nat. Rev. Cancer 3:502-516 (2003)).
Laboratory-based studies have identified a wide variety of genes and molecular pathways, such as MDR1 (P-glycoprotein) (Cancer Res. 53:747-754 (1993)) and p53 (Cell 74:957-967 (1993)), that can lead to increased resistance to treatments in malignant tumor cells. Nevertheless, significant discrepancies exist between drug resistance identified in experimental models and the multidrug resistance (MDR) phenotypes found in human malignant tumors (Br. J. Cancer 94:1087-1092 (2006)). For instance, acquisition of p53 mutations and gene amplification of MDR1 are rarely observed following chemotherapy in clinical human malignancies and so far there is little evidence demonstrating that single gene-mediated drug resistance individually correlated with treatment outcome of human malignancies (Nat. Rev. Cancer 3:502-516 (2003)).
The discrepancy between experimental and clinical drug resistance may partly stem from the distinct multicellular and spatial-dimensional contexts under which tumor drug resistance develops in vivo. Most of the current knowledge about the cellular responses to chemicals, toxins or radiation were gathered from studies using unicellular culture models, which lacked consideration of the heterotypic cell-cell interactions in the tumor-host interface (Nature 411:375-379 (2001)). Previously, it has been clearly shown that the drug resistance phenotype of tumor cells for alkylating agents only emerged in vivo mainly as a result of host-tumor interactions and could not be detected by unicellular culture models (Science 247:1457-1461 (1990)). The MDR phenotypes of cancers have only be recapitulated by culture models that incorporate elements mimicking in vivo tumor or tissue architectures instead of conventional monolayer cell cultures (Proc. Natl. Acad. Sci. U.S.A. 90, 3294-3298 (1993)). For instance, organization of tumor cells into three-dimensional (3D) multicellular spheroids endowed resistance to cytotoxic agents and radiation, which was reversible by disaggregation of the structures (Anticancer Drug Dev. 14:153-168 (1999); Crit. Rev. Oncol. Hematol. 36:193-207 (2000)).
A variety of mechanisms have been proposed for the development of MDR in tumor cells in 3D multicellular spheroids, including hypoxia, cellular attachment, cell cycle proteins such as KIP1, and cell surface signaling such as the phatidylinositol 3-kinase pathway (Nature Med. 2:1204-1210 (1996); Anticancer Drug Dev. 14:153-168 (1999); Anticancer Drug Dev. 14:169-177 (1999)). When maintained ex vivo with preserved tissue architecture, malignant tumors displayed differential sensitivities to cytotoxic drugs similar to those observed in vivo (Proc. Natl. Acad. Sci. U.S.A. 84:5029-5033 (1987)). The context-dependency of cell death sensitivity also holds true for non-neoplastic epithelial cells, as organization of mammary epithelial cells into 3D acinar architectures in response to reconstituted basement membrane (rBM) endowed them a MDR phenotype (Cancer Cell 2:205-216 (2002)).
Cell development and differentiation are governed by the hierarchical order of gene activation and repression controlled at the level of chromatin structures by epigenetic mechanisms. Epigenetic changes are heritable changes in gene expression that do not involve an alteration in the DNA sequence, which commonly involve changes in the patterns of modifications of DNA and histones, including methylation, acetylation, and phosphorylation, as well as in the architecture of the chromatin conformation (J. Cell Sci. 116:2117-2124 (2003)). Disruptions of the epigenetic regulation of chromatin structure, function, and gene expression therefore leads to the dysregulated of cell growth and differentiation, as well as cancer. Consistent with this view, there is now circumstantial evidence supporting the epigenetic progenitor model in favor of the classical clonal genetic model of cancer (Nat. Rev. Genet. 7:21-33 (2005)). Epigenetic alterations, such as global DNA hypomethylation and chromatin hyperacetylation, are found at very early stages of tumorigenesis. On the other hand, hypermethylation and chromatin hypoacetylation on selective promoters are common strategies which tumor use to silence selective tumor-suppressor genes, such as retinoblastoma 1 (RB1), p16 (CDKN2A), von Hippel-Lindau tumor suppressor (VHL), and MutL protein homologue 1 (MLH1).
Histone hypoacetylation can be caused by inactivation of histone acetylase (HAT) activity due to gene mutations, inhibitory action of viral oncoproteins, and chromosomal translocations. For instance, mutations in CBP and P300 are associated with cancer predisposition (Trends Genet. 14:178-183 (1998), Nat. Genet. 24:300-303 (2000)). Fusion proteins involving MLL (mixed-lineage leukemia) or MORF (monocytic-leukemia-zinc-finger-protein related factor) and p300 or CBP have been associated with acute myelogenous leukemia (AML) (Blood 92:2118-2122 (1998), Hum. Mol. Genet. 10:395-404 (2001)). Histone hypoacetylation and tumorigenesis can also be caused by altered histone deacetylase (HDAC) activities. For instance, chromosomal translocation events in acute promyelocytic leukemia (APL) produce fusion proteins that contain retinoid acid receptor (RAR)α and PML (promyelocytic leukemia protein), and RARα and PLZF (promyelocytic zinc finger), which recruit HDACs with high affinity and result in constitutive repression of RAR-targeted genes (Oncogene 20:7204-7215 (2001)). Moreover, the fusions proteins AML1-ETO and TEL-AML1, expressed in AML and acute lymphoblastic leukemia, recruit HDACs and repress the AML1 transcriptional factor (Oncogene 20:5660-5679 (2001)). Inappropriate transcriptional repression mediated by HDACs may also operate in the tumorigenesis of solid tumors, although the precise mechanisms remain incompletely understood.
Epigenetic alterations not only play important roles in tumor initiation but may also contribute to malignant progression. Phenotypic plasticity mediated by epigenetic mechanisms has now been recognized as an important source of cancer-cell heterogeneity driving phenotypic evolution of tumors. For example, DNA hypomethylation can drive genomic instability as a result of decondensation of centromeric heterochromatin and the formation of new centromeres (Hum. Genet. 67:257-263 (1984)). A reduction in heterochromatin-associated protein 1 (HP1HSα), a nonhistone chromosomal protein that mediates transcriptional repression, is directly associated with breast tumor cell invasion and metastasis (Cancer Res. 60:3359-3363 (2000)). Recently, the polycomb group protein EZH2, a histone methyltransferase that causes gene silencing, was found to be overexpressed in metastatic prostate cancer and invasive breast cancer and promotes the proliferation and invasion of tumor cells through its interaction with HDAC2 (Nature 419:624-629 (2002), Proc. Natl. Acad. Sci. USA 100:11606-11611 (2003)). EZH2 was also found to be an independent predictor of prostate and breast cancer recurrence and death. Moreover, it was reported that the gene expression pathway associated with Bmi-1, a component of the chromatin remodeling complex PRC1 (polycomb repressive complex 1), which mediates ubiquitination of histone H2A, strongly predicts recurrence, metastasis, and death in various types of human cancers (J. Clin. Invest. 115:1503-1521 (2005)). If epigenetic plasticity is a common strategy used by tumor cells to evolve into more advanced malignant states, it's likely that more epigenetic regulators will be identified as contributors to tumor progression.
Epigenetic changes alter the expression of a large number of genes and may lead to a higher and faster phenotypic plasticity, through which tumor cells can adapt to new environments such as cytotoxic drug therapy, than genetic changes. Consistent with this possibility, there is now increasing evidence suggesting that epigenetic changes of malignant tumor cells may be a crucial driving force behind the acquisition of drug resistance (Br. J. Cancer 94:1087-1092 (2006)). For instance, methylation of CpG islands in genes involved in DNA repair, including BRCA1, GSTP1, and MGMT, was associated with increased response to chemotherapy in human ovarian cancers (Cancer Res. 65:8961-8967 (2005); N. Engl. J. Med. 343:1350-1354 (2000)). In contrast, methylation and epigenetic inactivation of the proapoptotic gene APAF1 is common in metastatic melanoma and confer resistance to conventional chemotherapy (Nature 409:207-211 (2001)). Similarly, a subset of patients with ovarian cancer acquired methylation of the DNA mismatch repair protein hMLH1 during chemotherapy, which was associated with poor overall survival (Clin. Cancer Res. 10:4420-4426 (2004)).
As mentioned, inappropriate transcriptional repression by altered HDAC activities is a common epigenetic mechanism used by oncoproteins and plays a significant role in tumorigenesis (Nat. Rev. Drug Disc. 1:287-299 (2002)). Currently, compounds that bind and inhibit a broad genus of HDACs are in phase I and II clinical trials for their potentials as anti-tumor agents (Nat. Rev. Cancer 6:38-51 (2006)). These HDAC inhibitors induce histone hyperacetylation, reactivate suppressed genes, and have pleiotropic cellular effects. Most promisingly, HDAC inhibitors has been shown to induce apoptosis in MDR tumor cells and to sensitize them to chemotherapeutic agents or ionizing radiation through activation of both the death-receptor and intrinsic apoptotic pathways (Int. J. Cancer 104:579-586 (2003); Cancer Res. 63:4460-4471 (2003); Oncogene 24:4609-4623 (2005); Nat. Rev. Drug Disc. 1:287-299 (2002)).
HDACs alone or in combination with DNA-demethylating agents have been shown to increase sensitivity to chemotherapeutic agents in cell line models (Anticancer Drugs 13:869-874 (2002)) and are currently being assessed for their potentials as chemosensitizers in clinical trials. However, the key cellular targets of HDAC inhibitors, as well as patients and tumor types that most likely respond to HDAC inhibitors, remain unknown. Moreover, the inhibitors currently in clinical trials do not demonstrate specificity for individual HDACs. This is a significant problem, as individual HDACs have differential substrate specificities and functions. Determining which of these activities most readily effects tumorogenesis is critical for the efficient targeting of individual molecules. Thus, the use of HDAC inhibitors in clinical studies has very limited success to date (Nat. Rev. Drug Disc. 1:287-299 (2002)).
Given the pleiotropic effects of HDAC inhibitors on a wide variety of histone and non-histone substrates (Nat. Rev. Cancer 6:38-51 (2006)), it is unlikely that a single surrogate marker, such as the genomic level of histone acetylation, can serve as a predictor for drug efficacy. As HDAC inhibition can induce alterations in the transcription of a large number (up to 20% of known genes) of genes (Mol. Cancer. Ther. 2:151-163 (2003); Proc. Natl. Acad. Sci. USA 101:540-545 (2004); Proc. Natl. Acad. Sci. USA 102:3697-3702 (2005)), transcriptional profiles associated with HDAC mutation or inhibition may show particular promise in the prediction of response to HDAC inhibitors.
The Nuclear Corepressor 2 (N-CoR2) (gene symbol: NCOR2; NCBI RefSeq #NM—006312; UniGene ID Hs.137510) and its paralog N-CoR (gene symbol: NCOR1; NCBI RefSeg #NM—006311; UniGene ID Hs.462323) are epigenetic regulators that mediate transcriptional repression by recruiting and activating various histone deacetylases (HDACs) (Annu. Rev. Physiol. 66:315-360 (2004)). N-CoR2 and N-CoR were originally identified as transcriptional corepressors of unliganded nuclear receptors, such as reteinoic acid and thyroid hormone receptors (Nature 377:454-457 (1995)). It has become increasingly evident that N-CoR2 and N-CoR also mediate repression of a wide array of non-receptor transcriptional factors, including the myogenic specific bHLH protein MyoD (Mol. Endocrinol. 13:1155-1168 (1999)), B-Myb (Mol. Cell. Biol. 22, 3663-3673 (2002)), the Pbx family of homeobox genes (Mol. Cell. Biol. 19:8219-8225 (1999)), the signal transducers and activators of transcription-5 (STAT5) (EMBO J. 20:6836-6844 (2001)), the oncoproteins PLZF-RAR (Nature 391:811-814 (1998)) and LAZ3/BCL6 (Proc. Natl. Acad. Sci. U.S.A. 94:10762-10767 (1997)), serum response factor (SRF), activating protein-1 (AP-1), and nuclear factor-KB (NFκB) (J. Biol. Chem. 275:12470-12474 (2000)).
Biochemical purification of the N-CoR2/N-CoR complexes demonstrated that both N-CoR2 and N-CoR exist in large protein complexes comprising GPS2 (G-protein pathway suppressor 2), which mediates inhibition of the JNK pathway (Cell 9:611-623 (2002)), TBL-1 (transducin β-like protein 1) and TBL-R1, which serve as E3 ligases that recruit the ubiquitin conjugating/19S proteosome complex and thereby degrades the N-COR2/N-CoR complex (Gene Dev. 14:1048-1057 (2000), Cell 116:511-526 (2004)), and HDAC3, which exhibits histone deacetylase activities. Interestingly, the purified N-CoR2-HDAC3 complex possesses deacetylase activity, whereas HDAC3 alone does not function as a HDAC, suggesting that N-CoR2 or N-CoR not only serves as the adaptor but also the activator of the HDAC3 enzymatic activity (Mol. Cell. Biol. 21:6091-6101 (2001)). Biochemistry studies have further shown that a particular deacetylase activation domain of N-CoR2 is required for the activation of the otherwise inert HDAC3 (Proc. Natl. Acad. Sci. USA 102:6009-6014 (2005)).
To date, most of the studies on N-CoR or N-CoR2 have been focused on protein biochemistry and their role in hormone receptor signaling and much less was known about their other biological functions. Recently, aside form its nuclear receptor corepressor functions, N-CoR has been found to play important roles in differentiation (Mol. Endocrinol. 13:1155-1168 (1999)) and stem cell maintenance (Nature 419:934-939 (2002)). Similarly, N-CoR2 was also found to be involved in forebrain development and in maintenance of the neural stem cell state in mice (Nature 450:415-420 (2007)).
Recent advances in high-throughput analytical tools that can measure the expression of a large number of genes have enabled molecular profiling of human malignant tumors. This has greatly enhanced tumor classification and allows for prediction of disease progression and clinical outcome. For instance, unsupervised hierarchical clustering on gene expression data allowed the classification of breast cancers into several distinct subgroups or molecular subtypes (Proc. Natl. Acad. Sci. USA 100:8418-8423 (2003)). In a second study, a 32-gene molecular classifier was used to place human bladder cancers into subclasses with prognostic significance (Nat. Genet. 33:90-96 (2003)). Gene expression profiling in another study allowed the classification of high-grade gliomas with higher accuracy and reproducibility (Cancer Res. 63:1602-1607 (2003)). Molecular profiling of childhood medulloblastomas demonstrated their distinct molecular and clinical features from other types of brain tumors (Nature 415:436-442 (2002)). A 133-gene signature accurately predicted survival among patients with acute myeloid leukemia (N. Engl. J. Med. 350:1605-1616 (2004)). Furthermore, a 70-gene or 76-gene prognostic signature has been developed which successfully predicts survival in patients with breast cancer (N. Engl. J. Med. 347:1999-2009 (2002); Lancet 365:671-679 (2005)). Gene expression signatures have also successfully predicted clinical outcome of prostate cancers (J. Clin. Invest. 113:913-923 (2004)). A more “universal” signature comprising 128 genes have been developed, which could distinguish primary and metastatic adenocarcinomas of diverse origin and primary tumors carrying the signature were associated with metastasis and poor clinical outcome (Nat. Genet. 33:49-54 (2003)).
Aside from the predictive value for long-term disease outcome, it is increasingly recognized that molecular characteristics, such gene expression profiles, of malignant tumors also affect their sensitivity to adjuvant (post-operative) or neo-adjuvant (pre-operative) chemotherapy (Nat. Clin. Pract. Oncol. 3:621-632 (2006)). To this end, several multigene signatures have been developed to predict patient response to preoperative chemotherapy in breast cancers based on the gene expression profiles of tumor biopsies (J. Clin. Oncol. 24:4236-4244 (2006); J. Clin. Oncol. 22:2284-2293 (2004); J. Clin. Oncol. 23:7265-7277 (2005); J. Translantional Med. 3:32 (2005)). Of note, these signatures were extracted by combining mathematical and statistical methods and none of them were directly related to a cellular pathway that is involved in the process of cell death, stress response, or drug metabolism. As such, a rationale approach to treat resistant malignant tumors based on the gene expression signatures has been hampered by the lack of biological relevance thereof. Recently, Nevins et al. have developed gene expression signatures that reflect the patterns of oncogenic pathway deregulation, which can be used to predict the sensitivity to therapeutic agents that target the deregulated pathway identified (Nature 439:353-357 (2006); Nat. Med. 12:1294-1300 (2006)). An experimentally derived gene-expression signature of the interferon (INF)-related DNA damage signaling pathway was found to be a therapy-predictive marker of adjuvant chemotherapy or radiation in breast cancer (Proc. Natl. Acad. Sci. U.S.A. 105:18490-18495 (2008)). It is conceivable that gene expression signatures associated with particular cellular pathways like these examples can offer a better opportunity to guide the use of pathway-specific drugs and is of considerable value in a more rationalized design of chemotherapies for human malignancies.
The current invention satisfies a need in the art for such a gene expression signature associated with multidrug resistance and HDAC activity in tumor cells.