Faculty Bibliography

Uterine carcinosarcoma (UCS) is a rare type of cancer and accounts for 5% of uterine malignancies. However, UCS patients suffer a high prevalence of chemo-resistance and a very poor prognosis compared to uterine cancer patients. URI is a chaperone with functions in transcription. We analyzed the somatic URI1 copy number variation in 57 post-menopausal non-metastatic UCS patients in comparison to 363 uterine corpus endometrial carcinomas. URI1 amplification was detected in 40% (23/57) of primary UCS and 5.5% (20/363) of uterine carcinomas. UCS patients with URI1 amplification exhibited 13% (3/23) tumor-free survival compared to 41% (14/34) in the absence of URI amplification (P=0.023). URI1 amplification (OR=6.54, P=0.027), weight (OR=1.068, P=0.024), hypertension (OR=3.35, P=0.044), and tumor stage (OR=2.358, P=0.018) associated with poor survival. Patients treated with hormone replacement therapy (OR=15.87, P=0.011) displayed enhanced overall survival. Combined radiation and chemotherapy improved patient survival (median survival=2043 days) compared to single (median survival=597 days) or no treatment (median survival=317 days, P=0.0016). Importantly, patients with URI1 amplification had poor response to adjuvant treatment compared to control group (P=0.013). Tumors with URI1 amplification displayed decreased transcription of genes encoding tumor suppressor and apoptotic regulators and increased expression of genes regulating oncogenesis, survival and metastasis. Overexpression of URI1 in a cultured cell model induced ATM expression and resistance to cisplatin. Our findings suggest that high prevalence in UCS may associate with poor prognosis and worse response to adjuvant treatment.

Despite decades of effort to develop effective therapy and to identify promising new drugs, prostate cancer is lethal once it progresses to castration-resistant disease. Studies show mis-regulation of multiple pathways in castration-resistant prostate cancer (CRPC), reflecting the heterogeneity of the tumors and also hinting that targeting androgen receptor (AR) pathway alone might not be sufficient to treat CRPC. In this study, we present evidence that the Wnt/beta-catenin pathway might be activated in prostate cancer cells after androgen-deprivation to promote androgen-independent growth, partly through enhanced interaction of beta-catenin with TCF4. Androgen-independent prostate cancer cells were more prone to activate a Wnt-reporter, and inhibition of the Wnt/beta-catenin pathway increased sensitivity of these cells to the second-generation antiandrogen, enzalutamide. Combined treatment of enzalutamide and Wnt/beta-catenin inhibitor showed increased growth repression in both androgen-dependent and -independent prostate cancer cells, suggesting therapeutic potential for this approach.

Androgen receptor (AR), a steroid hormone receptor, is critical for prostate cancer growth. However, activation of AR by androgens can also lead to growth suppression and differentiation. Transcriptional cofactors play an important role in this switch between proliferative and anti-proliferative AR target gene programs. Transducin beta-like-related protein 1 (TBLR1), a core component of the nuclear receptor corepressor complex, shows both corepressor and coactivator activities on nuclear receptors, but little is known about its effects on AR and prostate cancer. We characterized TBLR1 as a coactivator of AR in prostate cancer cells and determined that the activation is dependent on both phosphorylation and 19S proteosome. We showed that TBLR1 physically interacts with AR and directly occupies the androgen-response elements of the affected AR target genes in an androgen-dependent manner. TBLR1 is primarily localized in the nucleus in benign prostate cells and nuclear expression is significantly reduced in prostate cancer cells in culture. Similarly, in human tumor samples, the expression of TBLR1 in the nucleus is significantly reduced in the malignant glands compared with the surrounding benign prostatic glands (P<0.005). Stable ectopic expression of nuclear TBLR1 leads to androgen-dependent growth suppression of prostate cancer cells in vitro and in vivo by selective activation of androgen-regulated genes associated with differentiation (e.g. KRT18) and growth suppression (e.g. NKX3-1), but not cell proliferation of the prostate cancer. Understanding the molecular switches involved in the transition from AR-dependent growth promotion to AR-dependent growth suppression will lead to more successful treatments for prostate cancer.

It is well documented that androgen receptor (AR), a steroid hormone receptor, is important for prostate cancer (PCa) growth. Conversely, however, there is increasing evidence that activation of AR by androgens can also lead to growth suppression in prostate cells. AR mediated transcription is regulated by a number of different transcriptional coactivators. Changes in expression level or cellular localization of specific coactivators may play a crucial role in this switch between proliferative and anti- proliferative processes regulated by AR target gene programs. In this review, we discuss the expression and function of several AR coactivators exhibiting growth suppressive function in PCa, including ARA70/ELE1/NCOA4, androgen receptor coactivator p44/MEP50/WDR77, TBLR1, and ART-27. In luciferase reporter assays, they all have been shown to activate AR mediated transcriptional activation. ARA70 exists in two forms, the full length nuclear ARA70alpha and internally spliced cytoplasmic ARA70beta. For p44 and TBLR1, we identified nuclear and cytoplasmic forms with distinct expression and function. In comparison of their expression (ARA70alpha, p44, TBLR1 and ART-27) in prostate, these coactivators are expressed in the nucleus of benign prostate epithelial cells while they are more predominantly expressed in cytoplasmic form (ARA70beta, cytoplasmic p44 and TBLR1) in PCa. Consistent with their nuclear expression in benign prostate, the nuclear form of these coactivators inhibit PCa growth targeting a subset of AR target genes. In contrast, the cytoplasmic versions of these proteins enhance PCa growth and invasion. Interestingly, first characterized as an AR coactivator in luciferase assays, ART-27 functions as corepressor for endogenous AR target genes. Importantly, the growth inhibitions by these nuclear proteins are androgen-dependent processes and the regulation of invasion is androgen-independent. Understanding the molecular switches involved in the transition from AR dependent growth promotion to growth suppression and dysregulation of these coactivator proteins promoting androgen-independent invasion may lead to identification of novel therapeutic targets for PCa.

Prostate cancer (PCa) is the second leading cause of cancer-related death in American men and many prostate cancer patients develop skeletal metastasis. Current treatment modalities for metastatic prostate cancer are mostly palliative with poor prognosis. Epidemiological studies indicated that patients receiving the diabetic drug metformin have lower prostate cancer risk and better prognosis, suggesting that metformin may have anti-neoplastic effects. The mechanism by which metformin acts as chemopreventive agent to impede prostate cancer initiation and progression is unknown. The amplification of c-MYC oncogene plays a key role in early prostate epithelia cell transformation and prostate cancer growth. The purpose of this study is to investigate the effect of metformin on c-myc expression and prostate cancer progression. Our results demonstrated that: (1) In Hi-Myc mice murine prostate neoplasia and tumor model, metformin attenuated the development of prostate intraepithelial neoplasia (PIN, the pre-cancerous lesion of prostate) and PCa lesions. (2) Metformin reduced c-myc protein levels in vivo and in vitro. In Myc-CaP mouse prostate cancer cells, metformin decreased c-myc protein levels by 50% through protein degradation and inhibition of de novo protein synthesis. (3) Metformin selectively inhibited the growth of prostate cancer cells by stimulating cell cycle arrest and apoptosis without affecting the growth of normal prostatic epithelial cells (RWPE-1). (4) Metformin reduced androgen receptor and proliferation marker Ki-67 levels in Hi-Myc mouse prostate glands. Our novel findings suggest that by downregulating c-myc, metformin may act as a chemopreventive agent to restrict prostatic neoplasia initiation and transformation.

Androgen receptor (AR) is the major therapeutic target in aggressive prostate cancer. However, targeting AR alone can result in drug resistance and disease recurrence. Therefore, simultaneous targeting of multiple pathways could in principle be an effective approach to treating prostate cancer. Here we provide proof-of-concept that a small-molecule inhibitor of nuclear beta-catenin activity (called C3) can inhibit both the AR and beta-catenin-signaling pathways that are often misregulated in prostate cancer. Treatment with C3 ablated prostate cancer cell growth by disruption of both beta-catenin/T-cell factor and beta-catenin/AR protein interaction, reflecting the fact that T-cell factor and AR have overlapping binding sites on beta-catenin. Given that AR interacts with, and is transcriptionally regulated by beta-catenin, C3 treatment also resulted in decreased occupancy of beta-catenin on the AR promoter and diminished AR and AR/beta-catenin target gene expression. Interestingly, C3 treatment resulted in decreased AR binding to target genes accompanied by decreased recruitment of an AR and beta-catenin cofactor, coactivator-associated arginine methyltransferase 1 (CARM1), providing insight into the unrecognized function of beta-catenin in prostate cancer. Importantly, C3 inhibited tumor growth in an in vivo xenograft model and blocked renewal of bicalutamide-resistant sphere-forming cells, indicating the therapeutic potential of this approach.

Integration of cellular signaling pathways with androgen receptor (AR) signaling can be achieved through phosphorylation of AR by cellular kinases. However, the kinases responsible for phosphorylating the AR at numerous sites and the functional consequences of AR phosphorylation are only partially understood. Bioinformatic analysis revealed AR serine 213 (S213) as a putative substrate for PIM1, a kinase overexpressed in prostate cancer. Therefore, phosphorylation of AR serine 213 by PIM1 was examined using a phosphorylation site-specific antibody. Wild-type PIM1, but not catalytically inactive PIM1, specifically phosphorylated AR but not an AR serine-to-alanine mutant (S213A). In vitro kinase assays confirmed that PIM1 can phosphorylate AR S213 in a ligand-independent manner and cell type-specific phosphorylation was observed in prostate cancer cell lines. Upon PIM1 overexpression, AR phosphorylation was observed in the absence of hormone and was further increased in the presence of hormone in LNCaP, LNCaP-abl and VCaP cells. Moreover, phosphorylation of AR was reduced in the presence of PIM kinase inhibitors. An examination of AR-mediated transcription showed that reporter gene activity was reduced in the presence of PIM1 and wild-type AR, but not S213A mutant AR. Androgen-mediated transcription of endogenous PSA, Nkx3.1 and IGFBP5 was also decreased in the presence of PIM1, whereas IL6, cyclin A1 and caveolin 2 were increased. Immunohistochemical analysis of prostate cancer tissue microarrays showed significant P-AR S213 expression that was associated with hormone refractory prostate cancers, likely identifying cells with catalytically active PIM1. In addition, prostate cancers expressing a high level of P-AR S213 were twice as likely to be from biochemically recurrent cancers. Thus, AR phosphorylation by PIM1 at S213 impacts gene transcription and is highly prevalent in aggressive prostate cancer.Oncogene advance online publication, 17 September 2012; doi:10.1038/onc.2012.412.

BACKGROUND: Androgen receptor (AR) expression in breast cancers may serve as a prognostic and predictive marker. We examined the expression pattern of AR and its phosphorylated forms, Ser-213 (AR-Ser[P]-213) and Ser-650 (AR-Ser[P]-650), in breast cancer and evaluated their association with clinicopathological parameters. METHODS: Immunohistochemistry was performed on primary and distant metastatic breast cancers and benign breast tissue using antibodies against AR, AR-Ser(P)-213, and AR-Ser(P)-650. The levels of cytoplasmic and nuclear expression were scored semiquantitatively using a histoscore. RESULTS: Nuclear staining of AR was observed in all benign breast tissue and 67% of cancer cases. Nuclear and cytoplasmic AR-Ser(P)-213 was increased in breast cancers 2-fold (P = .0014) and 1.7-fold (P = .05), respectively, compared with benign controls, whereas nuclear and cytoplasmic AR-Ser(P)-650 expression was decreased in tumors by 1.9-fold and 1.7-fold (both P < .0001), respectively. Increased expression of nuclear or cytoplasmic AR-Ser(P)-213 was observed in metastatic breast cancers (1.3-fold, P = .05), ER-negative (2.6-fold, P = .001), and invasive ductal carcinoma (6.8-fold, P = .04). AR-Ser(P)-650 expression was downregulated in lymph node-positive breast cancers (1.4-fold, P = .02) but was upregulated in invasive ductal carcinomas (3.2-fold, P < .0001) and metastases (1.5-fold, P = .003). Moreover, in ER-negative breast cancers, nuclear AR-Ser(P)-650 was decreased (1.4-fold, P = .005), and cytoplasmic AR-Ser(P)-650 was increased (1.4-fold, P = .003). CONCLUSIONS: AR and its phosphorylation at serines 213 and 650 are differentially expressed in breast cancer tumorigenesis and progression. Phosphorylation of AR at serines 213 and 650 is increased in ER-negative breast cancers, ductal carcinomas, and metastases and may have predictive value in breast cancer prognosis. Cancer 2013;000:000-000. (c) 2013 American Cancer Society.

Background: Chronic inflammation has been linked to cancer initiation and progression in a variety of tissues. The impact of acute and chronic inflammatory signaling on androgen receptor (AR) function has not been widely studied. We employed IL-1beta, one of important cytokines responsible for inflammation to treat LNCaP, which is both androgen- and bicalutamide-sensitive prostate cells. Methods: To mimic the effects of inflammation on AR function, we treated the LNCaP prostate cancer cell line either acutely or chronically with IL-1beta. AR protein and mRNA expression levels were examined over time course of IL-1beta stimulation in either the presence or absence of the synthetic androgen, R1881. The population of cells chronically treated with IL-1beta are called LNCaP-CI. Chromatin immunoprecipitation was used to measure the active epigenetic marks H3K4 dimethylation on the enhancer of M phase genes such as CDC20, CDK1 and UBE2C. mRNA level of AR, CDC20, CDK1 and UBE2C was done with quantitative PCR. Results: Acute IL-1beta treatment inhibits the transcription of the AR gene itself, resulting in the reduction of AR protein levels. In cells subjected to chronic IL-1beta stimulation, the transcription of the AR gene is restored within a few cell passages, and the cells acquire the ability to grow in the presence of the anti-androgen, bicalutamide. Using chromatin immunoprecipitation, we found that acquired bicalutamide resistance in chronic IL-1beta treatment correlates with increased selective active epigenetic mark H3K4me2 occupancy on the M-phase gene enhancer of UBE2C, CDC20 and CDK1 through AR recruitment and subsequent up-regulation of the M-phase genesin LNCaP-CI cells. Conclusions: Bicalutamide resistance induced by IL-1beta recapitulates the biological properties of LNCaP-abl (CRPC cells) and CRPC clinical cases through upregulation of M-phase genes, suggesting that chronic inflammation, induced by IL-1beta characterizes the development of CRPC from androgen-dependent prostate cancer t!

The androgen receptor (AR) is a mediator of both androgen-dependent and castration-resistant prostate cancers. Identification of cellular factors affecting AR transcriptional activity could in principle yield new targets that reduce AR activity and combat prostate cancer, yet a comprehensive analysis of the genes required for AR-dependent transcriptional activity has not been determined. Using an unbiased genetic approach that takes advantage of the evolutionary conservation of AR signaling, we have conducted a genome-wide RNAi screen in Drosophila cells for genes required for AR transcriptional activity and applied the results to human prostate cancer cells. We identified 45 AR-regulators, which include known pathway components and genes with functions not previously linked to AR regulation, such as HIPK2 (a protein kinase) and MED19 (a subunit of the Mediator complex). Depletion of HIPK2 and MED19 in human prostate cancer cells decreased AR target gene expression and, importantly, reduced the proliferation of androgen-dependent and castration-resistant prostate cancer cells. We also systematically analyzed additional Mediator subunits and uncovered a small subset of Mediator subunits that interpret AR signaling and affect AR-dependent transcription and prostate cancer cell proliferation. Importantly, targeting of HIPK2 by an FDA-approved kinase inhibitor phenocopied the effect of depletion by RNAi and reduced the growth of AR-positive, but not AR-negative, treatment-resistant prostate cancer cells. Thus, our screen has yielded new AR regulators including drugable targets that reduce the proliferation of castration-resistant prostate cancer cells.