Modulation Of Androgen Receptor Activity Research Articles

MED12 and CDK8/19 Modulate Androgen Receptor Activity and Enzalutamide Response in Prostate Cancer.

Prostate cancer progression is driven by androgen receptor (AR) activity, which is a target for therapeutic approaches. Enzalutamide is an AR inhibitor that prolongs the survival of patients with advanced prostate cancer. However, resistance mechanisms arise and impair its efficacy. One of these mechanisms is the expression of AR-V7, a constitutively active AR splice variant. The Mediator complex is a multisubunit protein that modulates gene expression on a genome-wide scale. MED12 and cyclin-dependent kinase (CDK)8, or its paralog CDK19, are components of the kinase module that regulates the proliferation of prostate cancer cells. In this study, we investigated how MED12 and CDK8/19 influence cancer-driven processes in prostate cancer cell lines, focusing on AR activity and the enzalutamide response. We inhibited MED12 expression and CDK8/19 activity in LNCaP (AR+, enzalutamide-sensitive), 22Rv1 (AR-V7+, enzalutamide-resistant), and PC3 (AR-, enzalutamide-insensitive) cells. Both MED12 and CDK8/19 inhibition reduced cell proliferation in all cell lines, and MED12 inhibition reduced proliferation in the respective 3D spheroids. MED12 knockdown significantly inhibited c-Myc protein expression and signaling pathways. In 22Rv1 cells, it consistently inhibited the AR response, prostate-specific antigen (PSA) secretion, AR target genes, and AR-V7 expression. Combined with enzalutamide, MED12 inhibition additively decreased the AR activity in both LNCaP and 22Rv1 cells. CDK8/19 inhibition significantly decreased PSA secretion in LNCaP and 22Rv1 cells and, when combined with enzalutamide, additively reduced proliferation in 22Rv1 cells. Our study revealed that MED12 and CDK8/19 regulate AR activity and that their inhibition may modulate response to enzalutamide in prostate cancer.

The Role of Androgen Receptor Signaling in Ovarian Cancer.

Emerging evidence has suggested that androgen receptor signaling plays an important role in ovarian cancer outgrowth. Specifically, androgen receptor activation appears to be associated with increased risks of developing ovarian cancer and inducing tumor progression. However, conflicting findings have also been reported. This review summarizes and discusses the available data indicating the involvement of androgens as well as androgen receptor and related signals in ovarian carcinogenesis and cancer growth. Although the underlying molecular mechanisms for androgen receptor functions in ovarian cancer remain far from being fully understood, current observations may offer effective chemopreventive and therapeutic approaches, via modulation of androgen receptor activity, against ovarian cancer. Indeed, several clinical trials have been conducted to determine the efficacy of androgen deprivation therapy in patients with ovarian cancer.

CD147 modulates androgen receptor activity through the Akt/Gsk-3β/β-catenin/AR pathway in prostate cancer cells.

The androgen signaling pathway serves an important role in the development of prostate cancer. β-Catenin is an androgen receptor (AR) cofactor and augments AR signaling. Glycogen synthase kinase-3β (GSK-3β), a target of phosphorylated serine/threonine protein kinase B (p-Akt), regulates β-catenin stability. In addition, β-catenin, a coregulator of AR, physically interacts with AR and enhances AR-mediated target gene transcription. The multifunctional glycoprotein cluster of differentiation (CD) 147 is highly expressed on the cell surface of the majority of cancer cells, and it promotes tumor invasion, metastasis and growth. In the present study, the molecular effects of CD147 on the Akt/GSK-3β/β-catenin/AR signaling network were investigated in LNCaP cells. Using short hairpin-mediated RNA knockdown of CD147 in LNCaP cells, it was demonstrated that downregulation of CD147 resulted in inhibitory phosphorylation of GSK-3β, and then promoted degeneration of β-catenin and reduced nuclear accumulation of β-catenin. In addition, immunoprecipitation studies demonstrated that CD147 downregulation decreased the formation of a complex between β-catenin and AR. It was shown that CD147 knockdown suppressed the expression of the AR target gene prostate-specific antigen and the growth of AR-positive LNCaP cells. Furthermore, inhibition of PI3K/Akt with LY294002 augmented CD147-mediated function. The present study indicates that the PI3K/Akt pathway may facilitate CD147-mediated activation of the AR pathway.

Persistence of androgen receptor (AR) expression in patients (pts) with small cell prostate cancer (SCPC): Preliminary results from the SU2C/PCF/AACR West Coast Prostate Cancer Dream Team (WCDT).

288 Background: Historically, SCPC has been regarded as an AR-null subtype observed in ~ 1% of pts. In castration-resistant metastatic biopsies, we have observed a higher frequency of SCPC (~ 15%), associated with shortened survival. We sought to contrast AR signaling between adenocarcinoma and SCPC. Methods: Pts underwent a metastatic tumor biopsy of bone or soft tissue. Formalin fixed, paraffin-embedded tissue underwent pathologic review, fluorescence in situ hybridization (FISH) for assessment of AR amplification, and assessment of AR expression by immunohistochemistry (IHC). A novel AR transcriptional signature was applied to RNA sequencing data from frozen tissue. AR-V7 transcripts in biopsy tissue were quantified and compared to AR-full length (AR-FL) expression. Results: Overall, 70 of 114 biopsies were AR amplified by FISH (61%). AR amplification was seen in similar proportions of adenocarcinoma (53%) and small cell tumors (54%) (p = ns). AR expression by IHC was observed in 27/32 (84%) and 5/6 (83%) of evaluable adenocarcinoma and SCPC biopsies, respectively (p = ns). AR was localized to nucleus in 85% of biopsies and did not differ by histology. AR-V7 splice variants were detected in all evaluable biopsies. Neither the absolute AR-FL expression level nor the ratio of AR-V7/AR-FL differed by histology. AR transcriptional signature scores were lower in SCPC versus adenocarcinoma (p = 0.029); scores were not correlated with AR-V7/AR-FL transcript ratio (r = 0.16). Of the 5 pts with SCPC identified on biopsy subsequently treated with abiraterone or enzalutamide, 3 achieved PSA declines > 50%. Conclusions: Contrary to the classical description of SCPC as an AR-null phenotype, the majority of metastatic SCPC biopsies were positive for AR amplification and expression, reflecting their likely clonal origin from adenocarcinoma. AR transcriptional activity was lower in SCPC, potentially related to epigenetic or post-translational modulation of AR activity upon transdifferentiation from adenocarcinoma to SCPC. As novel therapeutic targets are identified in SCPC, continued co-targeting of the AR may be warranted.

Aberrant BAF57 Signaling Facilitates Prometastatic Phenotypes

BAF57, a component of the switching-defective and sucrose nonfermenting (SWI/SNF) chromatin-remodeling complex conglomerate, modulates androgen receptor activity to promote prostate cancer. However, the molecular consequences of tumor-associated BAF57 expression have remained undefined in advanced disease such as castration-resistant prostate cancer and/or metastasis. Clinical human specimens of primary and metastatic prostate cancer were immunohistochemically examined for tumor-grade association of BAF57 expression. Global gene expression analyses were conducted in models mimicking tumor-associated BAF57 expression. Aberrant BAF57-dependent gene expression changes, bypass of androgen-mediated signaling, and chromatin-specific SWI/SNF complex alterations with respect to cytoskeletal remodelers such as integrins were validated. Cell migration assays were used to profile the biologic phenotypes conferred under conditions simulating tumor-derived BAF57 expression. Immunohistochemical quantitation of primary human specimens revealed that BAF57 was significantly and aberrantly elevated as a function of tumor grade. Critically, gene expression analyses showed that BAF57 deregulation circumvented androgen-mediated signaling, elicited α2 integrin upregulation, and altered other SWI/SNF complex components at the α2 integrin locus. BAF57-dependent α2 integrin induction conferred a prometastatic migratory advantage, which was attenuated by anti-α2 integrin antibody blockade. Furthermore, BAF57 was found to be markedly upregulated in human prostate cancer metastases of the lung, lymph node, and dura. The findings herein, identifying tumor-associated BAF57 perturbation as a means to bypass androgen-signaling events that facilitate novel prometastatic phenotypes, link BAF57 upregulation to tumor dissemination. These data thereby establish BAF57 as a putative marker of metastatic potential that could be leveraged for therapeutic intervention.

ARF represses androgen receptor transactivation in prostate cancer.

Androgen receptor (AR) signaling is essential for prostate cancer (PCa) development in humans. The initiation of prostate malignancy and progression to a castration-resistant stage are largely contributed by the modulation of AR activity through its coregulatory proteins. We and others previously reported that p14 alternative reading frame (ARF) expression is positively correlated with the disease progression and severity of PCa. Here, we provide evidence that p14ARF physically interacts with AR and functions as an AR corespressor in both an androgen-dependent and androgen-independent manner. Endogenous ARF (p14ARF in human and p19ARF in mouse) and AR colocalize in both human PCa cells in vitro and PCa tissues of mouse and human in vivo. Overexpression of p14ARF in PCa cells significantly attenuates the activities of androgen response region (ARR2)-probasin and prostate-specific antigen (PSA) promoters. The forced expression of p14ARF in cells resulted in a suppression of PSA and NK transcription factor locus 1 (NKX3.1) expression. Conversely, knockdown of endogenous p14ARF in human PCa cells with short hairpin RNA enhanced AR transactivation activities in a dose-dependent and p53-independent manner. Furthermore, we demonstrated that p14ARF binds to both the N-terminal domain and the ligand-binding domain of AR, and the human double minute 2 (HDM2)-binding motif of p14ARF is required for the interaction of p14ARF and AR proteins. p14ARF perturbs the androgen-induced interaction between the N terminus and C terminus of AR. Most importantly, we observed that the expression of PSA is reversely correlated with p14ARF in human prostate tissues. Taken together, our results reveal a novel function of ARF in modulation of AR transactivation in PCa.

Abstract 3927: Loss of bHLH transcription factor Id4 modulates androgen receptor activity in prostate cancer progression

Abstract Inhibitor of differentiation 4 (Id4), a crucial developmental gene also expressed in the normal prostate, has been shown to act as a tumor suppressor in prostate cancer. Promoter hypermethylation of Id4 is observed in several advanced stage cancers including prostate cancer. The loss of Id4 expression correlates with androgen independent prostate cancer progression. Our studies suggest that Id4 plays a crucial role in mediating the transition from androgen dependent to androgen independent prostate cancer by mediating the AR axis potential and reprogramming several cellular mechanisms including migration, senescence and proliferation. Stable transfection of a full length Id4 plasmid into the androgen-independent prostate cancer cell line DU145 resulted in re-expression of AR and downstream AR response gene PSA. Id4 transfected cells also observed an androgen regulated rate of proliferation in responsive to treatment with AR antagonist, Casodex. AR antagonism also significantly reduced the rate of migration as observed by a scratch wound assay. Under closer observation, AR antagonist treatment with Casodex also inhibited translocation of AR to the nucleus of DU145 cells as observed by immunofluorescence. Previous studies in our laboratory have demonstrated that DU145+Id4 transfected cells undergo a significant level of senescence (∼ 32%) compared to normal DU145 cells (∼ 4%). Treatment of DU145+Id4 cells with the AR antagonist Casodex demonstrate that the rate of senescence in DU145+Id4 transfected cells is closely related to inhibition of AR activity. Our results suggest that Id4 may play a crucial role in the loss of AR activity in advanced stage prostate cancer progression. These results also suggest that the antagonism of AR nuclear translocation post Id4 induction closely regulates the rate of proliferation and the induction of a senescent phenotype. These results provide a significant correlation between Id4 expression and AR activity during prostate cancer progression. Loss of AR activity through Id4 promoter hypermethylation may play a crucial role in understanding castration resistant prostate cancer. Therefore, induction of Id4 may provide a novel therapeutic target for advanced stage prostate cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3927. doi:1538-7445.AM2012-3927

Androgen receptor abnormalities in castration-recurrent prostate cancer

The androgen receptor (AR) plays a critical role in prostate cancer (PCa) development and progression. Despite the success of androgen-deprivation therapy, remission occurs in almost all cases. This stage of the disease is called castration-recurrent PCa (CRPC). CRPC cells adapt to low circulating levels of androgens, and active AR is maintained by numerous cellular mechanisms. Some mutations in the AR make it more responsive to lower androgen levels or other steroids. Furthermore, in this advance stage of the disease, PCa cells express the enzymes necessary for de novo synthesis of androgens. AR is also activated in a ligand-independent manner. Therefore, it is important to understand the mechanisms of AR activation and its deregulation during CRPC. The purpose of this article is to discuss mechanisms that are involved in modulation of AR activity and specificity.

Androgen Receptor Modulation Does Not Affect Longitudinal Growth of Cultured Fetal Rat Metatarsal Bones

Background: Systemic administration of the nonaromatizable androgen oxandrolone stimulates growth in girls with Turner syndrome and boys with a constitutional delay of growth and puberty. It is unknown if oxandrolone acts locally at the growth plate level to stimulate longitudinal bone growth. Methods: Metatarsal bones from female and male rat fetuses (day E20) were cultured for 14 days in the presence of oxandrolone, testosterone or the androgen receptor (AR) antagonist flutamide with/without insulin-like growth-factor-I (IGF-I) or charcoal-treated serum. Results: The AR was found to be expressed in both male and female fetal rat metatarsal bones. Neither oxandrolone nor testosterone had any effect on metatarsal bone growth when tested at a wide concentration range (1 nM to 10 μM), not even in the presence of IGF-I (100 ng/ml) or charcoal-treated serum (10%). Bone growth was also unaffected when the AR was blocked by flutamide. Control experiments confirmed that metatarsal bone growth was significantly stimulated by IGF-I (p < 0.001). Conclusion: Modulation of AR activity in the fetal rat growth plate does not affect linear bone growth. Extrapolating from these in vitro data, it could be speculated that oxandrolone stimulates longitudinal bone growth in treated children by acting indirectly rather than directly through AR activation in growth plate chondrocytes.

An Adenosine Triphosphatase of the Sucrose Nonfermenting 2 Family, Androgen Receptor-Interacting Protein 4, Is Essential for Mouse Embryonic Development and Cell Proliferation

An adenosine triphosphatase of the sucrose nonfermenting 2 protein family, androgen receptor-interacting protein 4 (ARIP4), modulates androgen receptor activity. To elucidate receptor-dependent and -independent functions of ARIP4, we have analyzed Arip4 gene-targeted mice. Heterozygous Arip4 mutants were normal. Arip4 is expressed mainly in the neural tube and limb buds during early embryonic development. Arip4-/- embryos were abnormal already at embryonic d 9.5 (E9.5) and died by E11.5. At E9.5 and E10.5, almost all major tissues of Arip4-null embryos were proportionally smaller than those of wild-type embryos, and the neural tube was shrunk in some Arip4-/- embryos. Dramatically reduced cell proliferation and increased apoptosis were observed in E9.5 and E10.5 Arip4-null embryos. Mouse embryonic fibroblasts (MEFs) isolated from Arip4-/- embryos ceased to grow after two to three passages and exhibited increased apoptosis and decreased DNA synthesis compared with wild-type MEFs. Comparison of gene expression profiles of Arip4-/- and wild-type MEFs at E9.5 revealed that putative ARIP4 target genes are involved in cell growth and proliferation, apoptosis, cell death, DNA replication and repair, and development. Collectively, ARIP4 plays an essential role in mouse embryonic development and cell proliferation, and it appears to coordinate multiple essential biological processes, possibly through a complex chromatin remodeling system.

Androgen and anti-androgen treatment modulates androgen receptor activity and DJ-1 stability

Mechanisms regulating the transition from hormone responsive to hormone refractory prostate cancer (PCa) have remained unclear. We analyzed androgen and anti-androgen treatment on endogenous AR activity in primary human prostate epithelial (HPE) cells cultured directly from patient radical prostatectomy specimens utilizing a transiently infected gene reporter (TIGR) assay. Flutamide treatment exhibited agonist activities in HPE cells derived from tumor and non-tumor specimens which contained wild-type AR. After proteomic comparison of these cells to those where flutamide functioned normally as an antagonist, we identified DJ-1, a positive regulator of AR. DJ-1 expression increased in HPE and LNCaP cells during flutamide treatment as a result of DJ-1 protein stabilization. Stabilization of AR and its co-regulators in the absence of androgen may partially account for anti-androgen withdrawal syndrome and potentially contribute to the development of hormone refractory PCa.

Nuclear beta-catenin as a marker of outcome in localized prostate cancer

9570 Background: Beta-catenin, in its role as a nuclear signaling molecule, has been implicated in prostate carcinogenesis through modulation of androgen receptor activity. Mice in whom beta-catenin is overexpressed in the nuclei of prostatic epithelium develop hyperplastic and dysplastic lesions. Hence this study focused on nuclear beta-catenin expression and aimed to define the pattern of beta-catenin protein expression in the nuclei of normal, hyperplastic and malignant human prostate tissue and to determine whether changes in expression were associated with disease progression and/or prognosis. Methods: Five normal prostates, 26 benign prostatic hypertrophy specimens, 232 radical prostatectomy specimens from patients with clinically localized prostate cancer (PC), 186 specimens of hyperplasia adjacent to PC and 20 cases of advanced PC were assessed for beta-catenin expression using immunohistochemistry. Results: There were significantly lower levels of nuclear beta-catenin expression in localized PC compared to benign prostate tissue (p<0.001). Nuclear beta-catenin expression was also lower in advanced compared to localized PC (p<0.001). In addition, lower levels of nuclear beta-catenin expression (immunoreactivity in <10% of malignant cells) predicted for a shorter biochemical relapse-free survival in patients with localized PC (HR 1.9, 95%CI 1.2–3.0; p=0.008) and were inversely correlated with pre-operative prostate-specific antigen (PSA) levels (p=0.01). Analysis of a low-risk subgroup of patients with pre-operative PSA levels <10 ng/ml (n=114), demonstrated that lower levels of nuclear beta-catenin (<10% of malignant cells) again predicted for a poorer prognosis (HR 3.2, 95%CI 1.4–7.3; p=0.006). Conclusions: Lower levels of nuclear beta-catenin expression are found in malignant compared to benign prostate tissue. Lower nuclear beta-catenin expression is also associated with a poorer prognosis in localized PC, in particular in the low-risk subgroup of patients with pre-operative PSA levels <10 ng/ml. Consequently, nuclear beta-catenin expression may be of clinical utility as a pre-operative prognostic marker in patients with low-risk PC. No significant financial relationships to disclose.

Nuclear beta-catenin as a marker of outcome in localized prostate cancer

9570 Background: Beta-catenin, in its role as a nuclear signaling molecule, has been implicated in prostate carcinogenesis through modulation of androgen receptor activity. Mice in whom beta-catenin is overexpressed in the nuclei of prostatic epithelium develop hyperplastic and dysplastic lesions. Hence this study focused on nuclear beta-catenin expression and aimed to define the pattern of beta-catenin protein expression in the nuclei of normal, hyperplastic and malignant human prostate tissue and to determine whether changes in expression were associated with disease progression and/or prognosis. Methods: Five normal prostates, 26 benign prostatic hypertrophy specimens, 232 radical prostatectomy specimens from patients with clinically localized prostate cancer (PC), 186 specimens of hyperplasia adjacent to PC and 20 cases of advanced PC were assessed for beta-catenin expression using immunohistochemistry. Results: There were significantly lower levels of nuclear beta-catenin expression in localized PC compared to benign prostate tissue (p<0.001). Nuclear beta-catenin expression was also lower in advanced compared to localized PC (p<0.001). In addition, lower levels of nuclear beta-catenin expression (immunoreactivity in <10% of malignant cells) predicted for a shorter biochemical relapse-free survival in patients with localized PC (HR 1.9, 95%CI 1.2–3.0; p=0.008) and were inversely correlated with pre-operative prostate-specific antigen (PSA) levels (p=0.01). Analysis of a low-risk subgroup of patients with pre-operative PSA levels <10 ng/ml (n=114), demonstrated that lower levels of nuclear beta-catenin (<10% of malignant cells) again predicted for a poorer prognosis (HR 3.2, 95%CI 1.4–7.3; p=0.006). Conclusions: Lower levels of nuclear beta-catenin expression are found in malignant compared to benign prostate tissue. Lower nuclear beta-catenin expression is also associated with a poorer prognosis in localized PC, in particular in the low-risk subgroup of patients with pre-operative PSA levels <10 ng/ml. Consequently, nuclear beta-catenin expression may be of clinical utility as a pre-operative prognostic marker in patients with low-risk PC. No significant financial relationships to disclose.

Heat shock protein 90.

Heat shock protein 90 (Hsp90) is a molecular chaperone required for the stability and function of a number of conditionally activated and/or expressed signaling proteins, as well as multiple mutated, chimeric, or overexpressed signaling proteins, which promote cancer cell growth or survival or both. Hsp90 inhibitors, by interacting specifically with a single molecular target, cause the inactivation, destabilization, and eventual degradation of Hsp90 client proteins, and they have shown promising antitumor activity in preclinical model systems. One Hsp90 inhibitor, 17-AAG, has completed Phase I clinical trial, and several Phase II trials are in progress. Hsp90 inhibitors are unique in that, although they are directed towards a specific molecular target, they simultaneously inhibit multiple signaling pathways that frequently interact to promote cancer cell survival. Recently identified clients of Hsp90 participate, frequently in overlapping pathways, in mediating cancer cell survival. These include Akt, Her2, and HIF-1 alpha. Thus, by inhibiting multiple survival pathways used by cancer cells, combination of an Hsp90 inhibitor with standard chemotherapeutic agents may dramatically increase the in vivo efficacy of the standard agent. Furthermore, Hsp90 modulates androgen receptor activity and the activity of several mutated kinases characteristic of several leukemias and lymphomas, making Hsp90 inhibition an attractive modality in these cases. Hsp90 inhibitors may circumvent the characteristic genetic plasticity that has allowed cancer cells to eventually evade the toxic effects of most molecularly targeted agents. The mechanism-based use of Hsp90 inhibitors, both alone and in combination with other drugs, should augment the treatment of multiple forms of cancer.