Treating & Diagnosing Cancer

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Abstract The present invention provides methods for increasing sensitivity of cancer cells to an antiestrogen agent, such as a selective estrogen receptor modulator (SERM). The methods include administering to the subject a polynucleotide in an amount effective to increase the antiestrogen agent sensitivity of the cancer cells. The cancer cells may be estrogen receptor positive, such as ER-α66 positive or ER-α36 positive, prior to the administering. Also provided are methods for decreasing the amount of estrogen receptor present in a cancer cell, methods for determining whether antiestrogen agent sensitivity of cancer cells in a subject can be increased, methods for diagnosing whether a subject has, or is at risk for developing, cancer, and methods for identifying an agent that increases the amount of let-7 miRNA in a cell.   Background Breast cancer is the second most common cause of death from cancer in women in Europe and North America. Annually, more than 1.3 million women are diagnosed with breast cancer worldwide, and approximately half a million die from the disease (Heneghan et al., 2009, J Oncol doi:10.1155/2010/950201). The involvement of estrogen in mammary carcinogenesis has been known for more than 100 years (Clemons and Goss, 2001, N. Engl. J. Med., 344:276-85). It is prevailingly known that estrogen signaling is mediated by two major estrogen receptors (ER), ER-α and ER-β (Weihua et al., 2003, FEBS Lett., 546:17-24), which share a common structural architecture. ER-α is often used to refer to a 66 kD protein that functions as a transcription factor and regulates the transcription of estrogen-responsive genes. ER-α is comprised of 6 domains, A-F (Evans, 1988, Science, 240:889-95). The A/B region contains a ligand-independent transactivation domain (AF-1). Regions C and E are responsible for DNA and ligand binding, respectively. A ligand-inducible transcription activating function (AF-2) is present in the ligand-binding domain D/E/F (Berry et al., 1990, EMBO J., 9:2811-8). Recent research revealed the existence of a truncated form of ER-α with a molecular weight of 46 kDa, which lacks the first 173 aa (AF-1 domain) of ER-α and is designated as ER-α46 (Flouriot et al., 2000, EMBO J., 19:4688-700). The full-length ER-α is therefore recognized as ER-α66. ER-α46 functions to inhibit the transcriptional activity mediated by the AF-1 domain of ER-α66 (Flouriot et al., 2000, EMBO J., 19:4688-700) and to signal a membrane-initiated estrogen pathway (Li et al., 2003, Proc. Natl. Acad. Sci. U.S.A., 100:4807-12). Previously, we identified and cloned a 36-kD novel isoform of ER-α66, ER-α36 (Wang et al., 2005, Biochem. Biophys. Res. Commun. 336:1023-7). ER-α36 is transcribed from a promoter located in the first intron of the ER-α66 gene and lacks both transcriptional activation domains (AF-1 and AF-2), but retains the DNA binding, dimerization, and partial ligand-binding domains. Additionally, it possesses an extra, unique 27-aa domain to replace the last 138 aa of the ER-α66. ER-α36 is predominantly localized on the plasma membranes and mediates membrane-initiated estrogen signal pathway (Wang et al., 2006, Proc. Natl. Acad. Sci. U.S. A., 103:9063-8) such as activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (the MAPK/ERK) signaling pathway (Segars and Driggers, 2002, Trends Endocrinol. Metab., 13:349-54). ER-α36 expression was detected in both ER-α66-positive and-negative breast cancer tumors (Lee et al., 2008, Anticancer Res. 28:479-83, Zhang et al., 2010, Oncogene, October 11, doi:10.1038/onc.2010.458). High levels of ER-α36 expression are also associated with tamoxifen resistance; breast cancer patients with tumors highly expressing ER-α36 benefit less from tamoxifen treatment (Shi et al., 2009, J. Clin. Oncol., 27:3423-9).   The discovery of microRNAs (miRNAs), which are ˜22 nt long, has brought new concepts to breast cancer research. A line of studies have shown that deregulation of mRNAs causes tumorigenesis and metastasis (Medina and Slack, 2008 Cell Cycle 7:2485-92). Further, growing evidence shows that miRNAs may be ‘master’ regulators that regulate cancer cell proliferation through different mechanisms. For example, miR-21 has been demonstrated to be an onco-microRNA (oncomiR) in breast tumorigenesis. Up-regulation of this miRNA causes aggressive malignant growth of breast cancer via regulation of the anti-apoptotic factors Bcl-2, TPM1, tumor suppressor PTEN, and PDCD4 (Ng et al., 2009, J Oncol, doi: 10.1155/2009/305420). MiR-206 significantly down-regulated ER-α by binding to the 3′ UTR (Adams et al., 2007, Mol Endocrinol 21:1132-47, Kondo et al., 2008, Cancer Res 68:5004-8). MiR-17-5p regulates malignant growth of breast cancer by targeting a transcriptional factor, AIB1 (Hossain et al., 2006 Mol Cell Biol 26:8191-201), which is a coactivator for nuclear receptors, such as ER-α. Overexpression of miR-125a/b suppressed the activities of two important tyrosine kinase receptors, HER2 and HER3 (Scott 2007 J Biol Chem 282:1479-86), which are often deregulated in breast cancer. Recently, miR-10b has been shown to be associated with progression and metastasis in breast carcinoma (Ma et al., 2007 Nature 449:682-8). The same group also found that miR-31 is inversely correlated with metastasis in breast cancer (Valastyan et al., 2009, Cell 137:1032-46). Another interesting study shows that down-regulation of let-7 miRNAs was observed in breast tumor-initiation cells (BT-IC), and an increased level of let-7 was detected during BT-IC differentiation (Yu 2007, Cell 131:1109-23). Restoration of let-7 in BT-IC reduced proliferation and mammosphere formation in vitro, tumor formation, and metastasis in NOD/SCID mice (Yu 2007, Cell 131:1109-23). However, the detailed mechanisms underlying let-7 regulation in breast tumorigenesis are still unknown.   Previous studies have shown that let-7 sequences are highly conserved in vertebrates and invertebrates. Expression of let-7 miRNAs can be regulated temporally during cancer development (Yu 2007, Cell 131:1109-23) and embryonic development (Grosshans et al., 2005, Dev Cell 8:321-30). For example, expression of let-7 miRNAs increases during differentiation and in mature tissue, but is barely detectable in embryonic stage (Gunaratne 2009, Curr Stem Cell Res Ther 4:168-77). Let-7 is also considered as a tumor suppressor to inhibit malignant growth of cancer cells by targeting RAS (Johnson et al., 2005, Cell 120:635-47), HMGA2 (Lee and Dutta 2005, Genes Dev 21:1025-30, Mayr et al., 2007, Science 15:1576-9), and c-Myc (Kim et al., 2009, Genes Dev 23:1743-8). Reduced expression of let-7 miRNAs has been observed in colon cancer (Michael et al., 2003 Mol Cancer Res 1:882-91), lung cancer (Takamizawa et al., 2004 Cancer Res 64:3753-6), ovary cancer (Dahiya et al., 2008, PLoS One 3:e2436) and breast cancer (Yu 2007, Cell 131:1109-23).   Summary of the Invention The present invention provides a method for increasing sensitivity of cancer cells to an antiestrogen agent, such as a selective estrogen receptor modulator (SERM). The method may include identifying a subject that has cancer cells that are resistant to an antiestrogen agent. The antiestrogen agent may be a SERM, such as tamoxifen. The method may further include administering to the subject a polynucleotide in an amount effective to increase the antiestrogen agent sensitivity of the cancer cells compared to the cancer cells before the administering. The cancer cells may be breast cancer cells, ovarian cancer cells, pancreatic cancer cells, endometrial cancer cells, lung cancer cells, or colon cancer cells. The method may further include determining whether the cancer cells are ER-α66 negative or ER-α66 positive prior to the administering. The cancer cells may be ER-α66 positive prior to the administering, or ER-α66negative prior to the administering. The method may further include determining whether the cancer cells are ER-α36 negative or ER-α36 positive prior to the administering. The cancer cells may be ER-α36 positive prior to the administering, or ER-α36 negative prior to the administering. In one embodiment, the cancer cells are ER-α36 positive and ER-α66 negative prior to the administering. In one embodiment, the cancer cells are progesterone receptor negative and Human Epidermal growth factor Receptor 2 (HER2) negative prior to the administering. The method may further include administering an antiestrogen agent, such as a SERM.   The present invention also provides a method for decreasing the amount of estrogen receptor present in a cancer cell. The method may include administering to a subject in need thereof a polynucleotide in an amount effective to decrease the amount of estrogen receptor present in a cancer cell. The cancer cell may be a breast cancer cell, an ovarian cancer cell, a pancreatic cancer cell, an endometrial cancer cell, a lung cancer cell, or a colon cancer cell. The method may further include identifying whether the subject has a cancer cell that is ER-α66 positive or ER-α66 negative. The method may further include identifying whether the subject has a cancer cell that is ER-α36 positive or ER-α36 negative. In one embodiment, the subject as a cancer cell is ER-α66 negative and ER-α36 positive. In one embodiment, the cancer cells are progesterone receptor negative and Human Epidermal growth factor Receptor 2 (HER2) negative prior to the administering. The method may further include administering an antiestrogen agent, such as a SERM.   A polynucleotide used in methods described herein may be substantially complementary to 5′-AACACCAGGAAGGCCUACCUCA (SEQ ID NO:16). For instance, in one embodiment, between 12 and 17 nucleotides of the polynucleotide are complementary to 5′-AACACCAGGAAGGCCUACCUCA (SEQ ID NO:16). In another embodiment, the polynucleotide may be substantially complementary to 5′-UUUCUAAGUAAUUGCUGCCUCU (SEQ ID NO:6). For instance, in one embodiment, between 15 and 17 nucleotides of the polynucleotide are complementary to 5′-UUUCUAAGUAAUUGCUGCCUCU (SEQ ID NO:6). In one embodiment the polynucleotide may be between 17 and 26 nucleotides. For instance, the polynucleotide may be chosen from SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24. The polynucleotide may be double stranded or single stranded, and may include ribonucleotides, deoxynucleotides, or be a combination thereof. The polynucleotide may be present in a vector, and the polynucleotide may include one or more modifications. The modifications may be selected from a modified nucleic acid sugar, a modified base, a modified backbone, or a combination thereof. In one embodiment two or more polynucleotides are administered.   The present invention further provides a method for determining whether antiestrogen agent sensitivity of cancer cells in a subject can be increased. Such a method may also be used to evaluate treatment options for a subject having a cancer. The antiestrogen agent may be a SERM, such as tamoxifen. The method may include determining the estrogen receptor status of cancer cells from the subject. For instance, the method may include determining the expression of ER-α66, ER-α36, or the combination thereof. The presence of an antiestrogen agent resistant breast cancer cell that is positive for either ER-α36 or ER-α66 indicates the sensitivity of cancer cells to an antiestrogen agent in a subject can be increased. The method may further include obtaining a biological sample from the subject, wherein the biological sample includes antiestrogen agent resistant cancer cells. The biological sample may be, for instance, blood, plasma, serum, or urine. The cancer cells may be breast cancer cells, ovarian cancer cells, pancreatic cancer cells, endometrial cancer cells, lung cancer cells, or colon cancer cells.   Further provided by the present invention is a method for diagnosing whether a subject has, or is at risk for developing, cancer. The method may include measuring the level of a let-7 miRNA in the biological sample from a subject, wherein an increase in the level of let-7 miRNA in the biological sample relative to the level of the let-7 miRNA in a control sample, indicates the subject has, or is at risk for developing, cancer. The biological sample may be blood, plasma, serum, or urine. The cancer cell may be breast cancer, ovarian cancer, pancreatic cancer, endometrial cancer, lung cancer, or colon cancer. In one embodiment, the cancer is an early stage breast cancer. The cancer may be an estrogen receptor positive cancer, such as ER-α36 positive or ER-α66 positive. The subject may have one or more signs, symptoms, or a combination thereof, of a cancer. The method may further include treating the subject with a polynucleotide described herein in an amount effective to increase the level of the polynucleotide in a cancer cell of the subject.   Also provided by the present invention is a method for identifying an agent that increases the amount of let-7 miRNA in a cell. The method may include exposing a cell to an agent, and measuring the amount of let-7 in the cell, wherein an increase in the amount of let-7 miRNA in the cell compared to the cell not exposed to the agent indicates the agent increases the amount of let-7 miRNA. The cell may be a cancer cell, and may be positive or negative for ER-α36 or ER-α66. In one embodiment the cancer cell is ER-α36 positive, ER-α66 negative, progesterone receptor negative, and HER2 negative. The cancer cell may be a breast cancer cell, an ovarian cancer cell, a pancreatic cancer cell, an endometrial cancer cell, a lung cancer cell, or a colon cancer cell.