Androgen Receptor In Prostate Cancer Cells Research Articles

Pleckstrin-2 Mediates the Activation of AKT in Prostate Cancer and Is Repressed by Androgen Receptor

PI3K-AKT and androgen receptor (AR) pathways are commonly activated in prostate cancers. Their reciprocal regulation makes it difficult to treat advanced prostate cancers. Here, we show that Pleckstrin-2 (PLEK2), a proto-oncoprotein involved in the activation and stabilization of AKT, connects these two pathways. We first provide genetic evidence that Plek2 deficiency largely reverted tumorigenesis in Pten prostate-specific knockout mice. Overexpression of PLEK2 promotes the proliferation and colony formation of prostate cancer cells in vitro. In addition, we found that PLEK2 is negatively regulated by AR. AR transcriptionally represses PLEK2 through binding to the PLEK2 promoter region. Overexpression of AR reduces PLEK2 expression, which inactivates AKT. Conversely, knockdown of AR in prostate cancer cells increases PLEK2 expression and activates the AKT pathway. This reciprocal inhibitory loop can be pharmacologically targeted using the PLEK2 inhibitor. We show that the PLEK2 inhibitor dose-dependently inhibits prostate cancer cell proliferation with the inactivation of AKT. Overall, our study uncovers the critical role of PLEK2 in prostate cancer proliferation and provides the rationale for targeting PLEK2 to treat prostate cancers.

RNA N6-methyladenosine-modified-binding protein YTHDF1 promotes prostate cancer progression by regulating androgen function-related gene TRIM68

PurposeThere is no report about the direct relationship between m6A modification and androgen receptor (AR)-related genes in prostate cancer (PC). We aimed to study the mechanisms of m6A methylation in regulating the pathogenesis of PC from the perspective of AR-related genes.MethodsqRT-PCR was applied to detect the expression of m6A-related genes in PC cell with or without AR inhibitor. The effects of YTHDF1 knockdown on PC cell viability, apoptosis, migration and invasion were investigated using flow cytometry, wound healing and transwell assays, respectively. The mechanism of YTHDF1 action was investigated using m6A RNA immunoprecipitation (MeRIP) sequencing. The biological functions of YTHDF1 were also explored through in vivo experiments.ResultsYTHDF1 was significantly down-regulated in AR inhibitor group. YTHDF1 knockdown significantly decreased AR level, viability and m6A methylation level of PC cells. TRIM68 was identified as a direct target of YTHDF1. Both YTHDF1 and TRIM68 knockdown increased apoptosis, and decreased cell viability, migration, and invasion of PC cells, while TRIM68 overexpression reversed the effects of YTHDF1 knockdown on PC cells. In addition, knockdown of YTHDF1 or TRIM68 significantly decreased the m6A methylation level, and mRNA and protein levels of YTHDF1, TRIM68 and AR in PC cells, while TRIM68 overexpression increased the expression levels above. Furthermore, subcutaneous xenografts of nude mice also revealed that TRIM68 could reverse the effects of YTHDF1 knockdown in PC in vivo.ConclusionThis study suggested the key role of YTHDF1-mediated m6A modification in PC progression by regulating androgen function-related gene TRIM68 in PC.

THU496 A Positive Relationship Exists Between CARF And AR In Prostate Cancer

Abstract Disclosure: K.M. Hasan: None. H. Khambati: None. R.R. Huerta: None. J. Vadgama: None. A.P. Sinha-Hikim: None. T.C. Friedman: None. Prostate cancer (PCa) is the second most prevalent cancer among men worldwide. Since PCa onset is androgen dependent, androgen deprivation therapy (ADT) is the first treatment choice for a primary tumor or metastatic PCa. However, ADT eventually leads to the development of castration resistant PCa (CRPC), which is the leading cause of higher mortality in PCa. Thus, there is a need to identify the underlying mechanisms of CRPC to manage this aggressive disease. Increased androgen receptor (AR) expression is one of the most important factors contributing to the development of CRPC. It has been shown that tumor suppressor p53 negatively regulates the expression of AR. So, loss of p53 functions has been associated with increased expression of AR in advanced PCa. However, the mechanism of p53-mediated inhibition of AR is not well understood. In this study, we demonstrated that CARF (CDKN2AIP), a p53 pathway regulatory protein, is highly expressed in PCa, but its significance in PCa development is unknown. Since, like AR, CARF is negatively regulated by p53, we hypothesize that a reciprocal relationship between CARF and p53 could regulate AR expression in PCa cells. Indeed, we found that activation of p53 by DNA damage stress inhibited both CARF and AR in LnCap and 22RV1, AR-positive, and wtp53 cell lines. Our result indicated that CARF and AR were decreased dose-dependent manner, and presumably, that could trigger cell death. Consistent with this speculation, we found that silencing CARF by ShRNA in LnCap triggered apoptosis and inhibition of cell proliferation. With great interest, we further demonstrated that silencing of CARF downregulated the AR level in LnCap and 22RV1 cells suggesting that CARF regulates the expression of AR in PCa. RT-PCR results showed that AR transcripts remained unchanged in CARF-depleted LnCap cells. This result indicated that CARF could regulate the AR stability but not the transcription. Intriguingly, we found that the expression of NEDD4, one of the AR-targeting E3 ligases, was increased in CARF-depleted cells. Therefore, by enhancing the stability, CARF could positively regulate the expression of AR in PCa cells. Further studies are in progress to know more details about the CARF-mediated AR regulation and its impact on androgen-independent PCa development and progression. Presentation: Thursday, June 15, 2023

Abstract B066: Genome-wide CRISPR screens identify PTGES3 as a druggable AR modulator

Abstract BACKGROUND The androgen receptor (AR) is the central therapeutic target in advanced prostate cancer (PCa). While treatments that directly target AR are effective in prolonging patient survival, aggressive prostate cancers invariably find a way to evade these therapies. Frequently, evasion mechanisms reestablish AR expression and therefore AR-driven oncogenic pathways are restored. Comprehensively understanding the protein regulation machinery of AR and identifying alternative therapeutic targets are crucial for effective treatment of this disease. METHODS To study regulators of AR, we utilized CRISPR-Cas9 technology to knock-in a split neon green fluorescent protein onto AR in prostate cancer cells. We evaluated the AR expression and activity in these AR reporter cell lines by multiple approaches including ARE-luciferase assay, ChIP, and RNA-seq. We further built a unique loss-of-function CRISPRi screening system in this AR reporter cell line where we performed a genome-wide fluorescence-activated cell sorting based screen and functionally interrogated hits that modulate AR protein levels. RESULTS We established the first prostate cancer cell line models harboring an endogenous AR reporter. Extensive genotypic and phenotypic characterization of this system demonstrated that the reporter is effective and does not alter AR activity. These models have enabled us to precisely measure the dynamic endogenous AR protein levels by its fluorescence signal. By utilizing a genome-wide CRISPRi screening method in this system, we identified classic AR regulators such as HOXB13, GATA2 and GRHL2, as well as many novel genes that have not been characterized in the context of prostate cancer. Among these novel hits, Prostaglandin E synthase 3 (PTGES3) is the highest ranked druggable target. We validated this finding by establishing PTGES3 knockdown cell lines and demonstrated that loss of PTGES3 downregulated AR and its downstream genes. Follow-up studies show that beyond the canonical chaperone function, PTGES3 physically interacts with AR in the nucleus, where it promotes AR activity. PTGES3 is required for cell proliferation in vitro and in vivo in multiple AR-driven PCa models but not in AR-independent PCa models, such as PC3 cells. From our preliminary data, this unique interaction further suggests that high tumor PTGES3 expression is associated with resistance to first-line androgen deprivation therapies in a retrospective clinical cohort. These results collectively confirm that PTGES3 regulates AR activity and is a potential therapeutic target. Lastly, using a disulfide tethering fragment screen, we developed a PTGES3 inhibitor that blocks the PTGES3/AR interaction and represses AR signaling in PCa cells. We aim to further optimize this PTGES3-specific inhibitor as a therapeutic alternative for patients with aggressive prostate cancer. CONCLUSIONS Together, these genetic, biochemical and chemical biology experiments point to a new class of AR-directed PTGES3 inhibitor therapies that may overcome known mechanisms of ARSI resistance in mCRPC. Citation Format: Haolong Li, James E. Melnyk, Becky Xu Hua Fu, Siyu Feng, Martin Sjöström, Raunak Shrestha, Meng Zhang, Lisa N. Chesner, Hyun Jin Shin, Marsha Calvert, Jonathan Chou, Rajdeep Das, Emily A. Egusa, Aidan Winters, Jun Zhu, Ashutosh Maheshwari, Junjie T. Hua, Mohammed Alshalalfa, William S. Chen, Bradley A. Stohr, Javed Siddiqui, Bo Huang, Eric J. Small, David A. Quigley, Kevan M. Shokat, Luke A. Gilbert, Felix Y. Feng. Genome-wide CRISPR screens identify PTGES3 as a druggable AR modulator [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B066.

Androgen-Responsive Oncogenic lncRNA RP11-1023L17.1 Enhances c-Myc Protein Stability in Prostate Cancer

Long noncoding RNAs (lncRNAs) have been found as novel participants in the pathophysiology of prostate cancer (PCa), which is predominantly regulated by androgen and its receptor. The biological function of androgen-responsive lncRNAs remains poorly understood. Here, we identified that lncRNA RP11-1023L17.1, which is highly expressed in PCa. RP11-1023L17.1 expression, can be directly repressed by the androgen receptor in PCa cells. RP11-1023L17.1 depletion inhibited the proliferation, migration, and cell cycle progression, and promoted the apoptosis of PCa cells, indicating that RP11-1023L17.1 acts as an oncogene in PCa cells. Microarray results revealed that RP11-1023L17.1 depletion downregulated the c-Myc transcription signature in PCa cells. RP11-1023L17.1 depletion-induced cellular phenotypes can be overcome by ectopically overexpressed c-Myc. Mechanistically, RP11-1023L17.1 represses FBXO32 mRNA expression, thereby enhancing c-Myc protein stability by blocking FBXO32-mediated c-Myc degradation. Our findings reveal the previously unrecognized roles of RP11-1023L17.1 in c-Myc-dependent PCa tumorigenesis.

20-HETE/GPR75 pairing modulates the expression and transcriptional activity of the androgen receptor in androgen-sensitive prostate cancer cells

The androgen receptor (AR) and AR-driven genes are crucial in normal and neoplastic prostate tissue. Previous results showed a link between 20-hydroxyeicosatetraenoic acid (20-HETE) production and AR-driven prostate cancer (PCa) progression. This study aims to describe the contribution of GPR75, 20-HETE membrane receptor, in 20-HETE-mediated expression and transcriptional activity of AR in PCa.In LNCaP cells, 20-HETE increased AR expression, nuclear localization, and its transcriptional activity. Also, 20-HETE enhanced dihydrotestosterone (DHT) induced effects. All was abrogated by chemical antagonism of GPR75 (19-HEDE) or its transient knockdown. In human PCa, the expression of AR-driven genes correlated with GPR75. In LNCaP xenografts, tumors from castrated animals expressed higher levels of AR, this was impaired by inhibition of 20-HETE synthesis.These data suggest that 20-HETE, through the GPR75 receptor, regulates transcriptionally active AR in PCa cells, thus making 20-HETE/GRP75 potential targets to limit the expression of AR-driven phenotype in PCa cells.

Abstract 2353: Biphasic regulation of MYB by androgen signaling mediates its growth-promoting and repressive effects in prostate cancer cells

Abstract Prostate cancer is the most frequently diagnosed malignancy affecting millions of men worldwide. It is also the second leading cause of cancer-related death in American men. Androgen signaling plays a central role in prostate cancer development but results in growth inhibition at high doses. Mechanisms underlying such biphasic effect of androgens are not well understood. We recently identified MYB as an aberrantly expressed gene in prostate cancer cells, which promoted their growth and aggressive behavior. We also observed that MYB induced ligand-independent activation of androgen receptor (AR) and influenced its binding to selective target genes to promote castration-resistance. The goal of the present study was to examine the regulatory crosstalk between MYB and AR in prostate cancer cells. We observed that while MYB had no significant effect on AR expression, androgen (dihydrotestosterone, DHT) treatment resulted in a dose-dependent biphasic regulation of MYB at both transcript and protein levels correlating with its effect on the growth of prostate cancer cells. Interestingly, we observed that the treatment of prostate cancer cells with either low (1 nM) or high (100 nM) doses of DHT enhanced the transcriptional activity of MYB promoter. In silico analysis identified two potential androgen-responsive elements (AREs) in MYB promoter and AR bound more efficiently to the proximal sequence as determined by chromatin immunoprecipitation (ChIP). In a time-course experiment, we observed that both low and high doses of DHT increased MYB transcript and protein levels at early time points, which decreased sharply after 12 h in high dose DHT-treated prostate cancer cells. RNA stability assay following treatment with actinomycin D (inhibitor of transcription) showed decreased MYB mRNA half-life in prostate cancer cells at late time points when treated with high dose DHT. Profiling of MYB-targeted miRNAs showed significantly greater induction of miR-150 in high-dose DHT-treated prostate cancer cells than those treated with low-dose DHT. Two AREs were predicted in the 1000 bp upstream sequence of miR-150 transcription start site and AR binding was confirmed by ChIP assay. Functional inhibition of miR-150 using anti-miR relieved MYB suppression by high-dose DHT and promoted the growth of prostate cancer cells. Further, transfection of prostate cancer cells by miR-150 mimic reduced endogenous MYB levels and led to repression of growth of prostate cancer cells. Overall, our findings reveal a novel mechanism of MYB regulation that mediates, at least in part, the biphasic effect of androgens on the growth of prostate cancer cells. Citation Format: Srijan Acharya, Shashi Anand, Mohammad Aslam Khan, Haseeb Zubair, Sanjeev Kumar Srivastava, Seema Singh, Ajay Pratap Singh. Biphasic regulation of MYB by androgen signaling mediates its growth-promoting and repressive effects in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2353.

Abstract LB-097: Linking prostate cancer cell AR heterogeneity to distinct castration and Enzalutamide responses

Abstract Expression of androgen receptor (AR) in prostate cancer (PCa) is heterogeneous but the functional significance of AR heterogeneity in regulating PCa biology and response to androgen/AR-targeted therapies remains unclear. Screening of ~200 castration-resistant PCa (CRPC) cores identified 3 AR expression patterns: nuclear (nuc-AR), mixed nuclear/cytoplasmic (nuc/cyto-AR), and low/no expression (AR-/lo). Xenograft CRPC modeling demonstrated that, although the AR+ CRPC responded to Enzalutamide, the AR-/lo CRPC was completely refractory to Enzalutamide. Genome editing-derived isogenic AR+ and AR-KO (knockout) PCa cells showed contrasting tumor-regenerating capacities and responses to castration and Enzalutamide. Whole-genome RNA-Seq and biochemical analyses coupled with experimental combinatorial therapy identified novel signaling pathways and BCL-2 as a critical therapeutic target in both AR+ and AR-/lo CRPC. Our study links AR heterogeneity to distinct castration/Enzalutamide responses and suggests the urgency in developing therapeutics to target AR-/lo PCa cells/clones. Citation Format: Qiuhui Li, Xin Chen, Qu Deng, Hsueh-Ping Chao, Amanda Tracz, Jason Kirk, Ruizhe Zhao, Kiera Rycaj, Dean G Tang. Linking prostate cancer cell AR heterogeneity to distinct castration and Enzalutamide responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-097.

Downregulation of androgen receptors by NaAsO2 via inhibition of AKT-NF-κB and HSP90 in castration resistant prostate cancer.

Androgen and androgen receptor (AR) play essential roles in the development and maintenance of prostate cancer. The recently identified AR splice variants (AR-Vs) have been considered as a plausible mechanism for the primary resistance against androgen deprivation therapy (ADT) in castration-resistant prostate cancer (CRPC). Sodium meta-arsenite (NaAsO2 ; KML001; Kominox), a trivalent arsenical, is an orally bioavailable and water soluble, which is currently in phase I/II clinical trials for the treatment of prostate cancer. It has a potent anti-cancer effect on prostate cancer cells and xenografts. The aim of this study was to examine the effect of NaAsO2 on AR signaling in LNCaP and 22Rv1 CRPC cells. We used hormone-sensitive LNCaP cells, hormone-insensitive 22Rv1 cells, and CRPC patient-derived primary cells. We analyzed anti-cancer effect of NaAsO2 using real-time quantitative reverse transcription-PCR, Western blotting, immunofluorescence staining and CellTiter Glo® luminescent assay. Statistical evaluation of the results was performed by one-way ANOVA. NaAsO2 significantly reduced the translocation of AR and AR-Vs to the nucleus as well as their level in LNCaP and 22Rv1 cells. Besides, the level of the prostate-specific antigen (PSA), downstream target gene of AR, was also decreased. This compound was also an effective modulator of AKT-dependent NF-κB activation which regulates AR. NaAsO2 significantly inhibited phosphorylation of AKT and expression and nuclear translocation of NF-κB. We then investigated the effect of NaAsO2 on AR stabilization. NaAsO2 promoted HSP90 acetylation by down-regulating HDAC6, which reduces the stability of AR in prostate cancer cells. Here, we show that NaAsO2 disrupts AR signaling at multiple levels by affecting AR expression, stability, and degradation in primary tumor cell cultures from prostate cancer patients as well as CRPC cell lines. These results suggest that NaAsO2 could be a novel therapeutics for prostate cancer.

Suppression of breast cancer cell growth by Her2-reduced AR serine 81 phosphorylation.

Breast cancer is a hormone-related carcinoma and the most commonly diagnosed malignancy in women. Although Her-2, estrogen receptor (ER), and progesterone receptor (PR) are the major diagnostic markers and therapeutic targets to breast cancer, searching for additional molecular targets remains an important issue and one of the candidates is androgen receptor (AR). AR has been shown expressed in 70% breast cancer patients and connects to low recurrence and high survival rate. Our previous study demonstrates that Ser81 phosphorylation of AR in prostate cancer cells is critical for its protein stability modulated by human epidermal growth factor receptor-2 (Her2). The aim of this study is to investigate the influence of Her2 and AR in proliferation of breast cancer cell line, MDA-MB-453. The data show that AR which was activated by synthetic androgen R1881 suppressed the proliferation of MDA-MB-453 cells. Notably, AR activation decreased the protein levels of cell growth-related proteins, including cyclin A, cyclin B, and early growth response protein 1 (Egr1), while cell-cycle inhibitor protein p27 was increased. Besides, Heregulin (HRG)-induced Her2 activation decreased the AR protein levels and its Ser81 phosphorylation. Her2 small molecular inhibitor, Lapatinib, dose-dependently suppressed cell proliferation while the levels of phospho-Ser81 AR and p27 protein were increased. Phospho-Ser81 AR was also increased after Her2 knockdown. Specifically, the influence of phospho-Ser81 AR by Lapatinib was primarily found in the nucleus of MDA-MD-453 cells, where the cell proliferation might directly be interfered. In conclusion, our findings indicate that Her2 might negatively regulate AR phosphorylation/activation and contribute to regulate the proliferation of MDA-MB 453 cells.

Targeting ACLY sensitizes castration-resistant prostate cancer cells to AR antagonism by impinging on an ACLY-AMPK-AR feedback mechanism.

The androgen receptor (AR) plays a central role in prostate tumor growth. Inappropriate reactivation of the AR after androgen deprivation therapy promotes development of incurable castration-resistant prostate cancer (CRPC). In this study, we provide evidence that metabolic features of prostate cancer cells can be exploited to sensitize CRPC cells to AR antagonism. We identify a feedback loop between ATP-citrate lyase (ACLY)-dependent fatty acid synthesis, AMPK, and the AR in prostate cancer cells that could contribute to therapeutic resistance by maintaining AR levels. When combined with an AR antagonist, ACLY inhibition in CRPC cells promotes energetic stress and AMPK activation, resulting in further suppression of AR levels and target gene expression, inhibition of proliferation, and apoptosis. Supplying exogenous fatty acids can restore energetic homeostasis; however, this rescue does not occur through increased β-oxidation to support mitochondrial ATP production. Instead, concurrent inhibition of ACLY and AR may drive excess ATP consumption as cells attempt to cope with endoplasmic reticulum (ER) stress, which is prevented by fatty acid supplementation. Thus, fatty acid metabolism plays a key role in coordinating ER and energetic homeostasis in CRPC cells, thereby sustaining AR action and promoting proliferation. Consistent with a role for fatty acid metabolism in sustaining AR levels in prostate cancer in vivo, AR mRNA levels in human prostate tumors correlate positively with expression of ACLY and other fatty acid synthesis genes. The ACLY-AMPK-AR network can be exploited to sensitize CRPC cells to AR antagonism, suggesting novel therapeutic opportunities for prostate cancer.

Corepressive function of nuclear receptor coactivator 2 in androgen receptor of prostate cancer cells treated with antiandrogen

BackgroundRecruitment of cofactors in the interaction of the androgen receptor (AR) and AR ligands plays a critical role in determining androgenic/antiandrogenic effects of the AR ligand on signaling, but the functions of key cofactors, including nuclear receptor coactivator (NCOA), remain poorly understood in prostate cancer cells treated with AR ligands.MethodsWe examined prostate cancer cell lines LNCaP and VCaP expressing mutated and wild-type ARs, respectively, to clarify the significance of NCOAs in the effect of antiandrogens. Hydroxyflutamide showed antagonistic activity against VCaP and an agonistic effect on LNCaP. Bicalutamide served as an antagonist for both. We analyzed mRNA transcription and protein expression of NCOAs in these cells pretreated with dihydrotestosterone and thereafter treated with the mentioned antiandrogens. Transcriptional silencing of candidate NCOAs and AR was performed using small interfering RNA (siRNA). Cell proliferation was evaluated with MTT assay.ResultsLNCaP treated with bicalutamide showed an about four-fold increase in the expression of NCOA2 mRNA compared to those pretreated with dihydrotestosterone alone (P <0.01). In VCaP pretreated with dihydrotestosterone, transcriptions of NCOA2 and NCOA7 were slightly increased with bicalutamide (1.96- and 2.42-fold, respectively) and hydroxyflutamide (1.33-fold in both). With Western blotting, the expression of NCOA2 protein also increased in LNCaP cells treated with bicalutamide compared with that in control cells pretreated with dihydrotestosterone alone. Following silencing with siRNA for NCOA2, PSA levels in media with LNCaP receiving bicalutamide were elevated compared with those in non-silencing controls (101.6 ± 4.2 vs. 87.8 ± 1.4 ng/mL, respectively, P =0.0495). In LNCaP cells treated with dihydrotestosterone and bicalutamide, NCOA2-silencing was associated with a higher proliferation activity compared with non-silencing control and AR-silencing.ConclusionNCOA2, which has been thought to be recruited as a coactivator, possibly plays a corepressive role in AR of prostate cancer cells when treated with antiandrogens, suggesting its potential as a therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2378-y) contains supplementary material, which is available to authorized users.

The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer.

Patterns of glycosylation are important in cancer, but the molecular mechanisms that drive changes are often poorly understood. The androgen receptor drives prostate cancer (PCa) development and progression to lethal metastatic castration-resistant disease. Here we used RNA-Seq coupled with bioinformatic analyses of androgen-receptor (AR) binding sites and clinical PCa expression array data to identify ST6GalNAc1 as a direct and rapidly activated target gene of the AR in PCa cells. ST6GalNAc1 encodes a sialytransferase that catalyses formation of the cancer-associated sialyl-Tn antigen (sTn), which we find is also induced by androgen exposure. Androgens induce expression of a novel splice variant of the ST6GalNAc1 protein in PCa cells. This splice variant encodes a shorter protein isoform that is still fully functional as a sialyltransferase and able to induce expression of the sTn-antigen. Surprisingly, given its high expression in tumours, stable expression of ST6GalNAc1 in PCa cells reduced formation of stable tumours in mice, reduced cell adhesion and induced a switch towards a more mesenchymal-like cell phenotype in vitro. ST6GalNAc1 has a dynamic expression pattern in clinical datasets, beingsignificantly up-regulated in primary prostate carcinoma but relatively down-regulated in established metastatic tissue. ST6GalNAc1 is frequently upregulated concurrently with another important glycosylation enzyme GCNT1 previously associated with prostate cancer progression and implicated in Sialyl Lewis X antigen synthesis. Together our data establishes an androgen-dependent mechanism for sTn antigen expression in PCa, and are consistent with a general role for the androgen receptor in driving important coordinate changes to the glycoproteome during PCa progression.

CSL regulates AKT to mediate androgen independence in prostate cancer progression

Aberrant signaling pathways leads to cancer initiation and progression. Both Notch and PI3K/AKT signaling pathways are believed to be involved in prostate cancer. How the interaction between the two pathways contributes to prostate cancer progression to androgen independence is still elusive. Prostate cancer cells were grown in RPMI 1,640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) or 10% charcoal-stripped heat-inactivated fetal bovine serum (FCS), 1% penicillin-streptomycin in 75 cm2 polystyrene flasks, and maintained at 37 °C in a humidified atmosphere with 5% CO2 . Cell proliferation, invasion were performed with cell counting, matrigel assay in vitro. Dual luciferase assays were performed using reporter plasmids with ARE (Androgen Response Element, ARE). RNA interference was applied to gene silence. Tumorigenicity of cancer cells was evaluated by mouse xenograft in vivo. A subpopulation of casodex resistant prostate cancer cells were identified with an overexpressed androgen receptor (AR) and aggressive phenotypes, characterized with high proliferation, invasion in vitro and enhanced tumorigenesis in vivo. Gene profiling for androgen-dependent LNCaP and androgen-independent LNCaP-CR revealed that both CSL and AKT gave the similar expressional pattern upon casodex treatment. Immunoblot demonstrated that CSL and AKT were dramatically suppressed in androgen dependent LNCaP cells, but slightly inhibited in LNCaP-CR cells as well as other androgen independent prostate cancer cells. Further studies indicated that CSL regulates AKT, and subsequently AR in prostate cancer cells. AKT mediates casodex resistance and androgen independence through regulation of cyclin D1. CSL-AKT-AR axis might play an important role in prostate cancer progression. Targeting CSL depleted the casodex resistant population through inhibition of the AKT, suggesting a more effective therapeutic strategy for abrogating casodex resistance in advanced prostate cancer.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Identification of novel genes that regulate androgen receptor signaling and growth of androgen-deprived prostate cancer cells.

Prostate cancer progression to castration refractory disease is associated with anomalous transcriptional activity of the androgen receptor (AR) in an androgen-depleted milieu. To identify novel gene products whose downregulation transactivates AR in prostate cancer cells, we performed a screen of enzymatically-generated shRNA lenti-libraries selecting for transduced LNCaP cells with elevated expression of a fluorescent reporter gene under the control of an AR-responsive promoter. The shRNAs present in selected populations were analyzed using high-throughput sequencing to identify target genes. Highly enriched gene targets were then validated with siRNAs against selected genes, testing first for increased expression of luciferase from an AR-responsive promoter and then for altered expression of endogenous androgen-regulated genes in LNCaP cells. We identified 20 human genes whose silencing affected the expression of exogenous and endogenous androgen-responsive genes in prostate cancer cells grown in androgen-depleted medium. Knockdown of four of these genes upregulated the expression of endogenous AR targets and siRNAs targeting two of these genes (IGSF8 and RTN1) enabled androgen-independent proliferation of androgen-dependent cells. The effects of IGSF8 appear to be mediated through its interaction with a tetraspanin protein, CD9, previously implicated in prostate cancer progression. Remarkably, homozygous deletions of IGSF8 are found almost exclusively in prostate cancers but not in other cancer types. Our study shows that androgen independence can be achieved through the inhibition of specific genes and reveals a novel set of genes that regulate AR signaling in prostate cancers.

Inhibition of Stat5a/b Enhances Proteasomal Degradation of Androgen Receptor Liganded by Antiandrogens in Prostate Cancer.

Although poorly understood, androgen receptor (AR) signaling is sustained despite treatment of prostate cancer with antiandrogens and potentially underlies development of incurable castrate-resistant prostate cancer. However, therapies targeting the AR signaling axis eventually fail when prostate cancer progresses to the castrate-resistant stage. Stat5a/b, a candidate therapeutic target protein in prostate cancer, synergizes with AR to reciprocally enhance the signaling of both proteins. In this work, we demonstrate that Stat5a/b sequesters antiandrogen-liganded (MDV3100, bicalutamide, flutamide) AR in prostate cancer cells and protects it against proteasomal degradation in prostate cancer. Active Stat5a/b increased nuclear levels of both unliganded and antiandrogen-liganded AR, as demonstrated in prostate cancer cell lines, xenograft tumors, and clinical patient-derived prostate cancer samples. Physical interaction between Stat5a/b and AR in prostate cancer cells was mediated by the DNA-binding domain of Stat5a/b and the N-terminal domain of AR. Moreover, active Stat5a/b increased AR occupancy of the prostate-specific antigen promoter and AR-regulated gene expression in prostate cancer cells. Mechanistically, both Stat5a/b genetic knockdown and antiandrogen treatment induced proteasomal degradation of AR in prostate cancer cells, with combined inhibition of Stat5a/b and AR leading to maximal loss of AR protein and prostate cancer cell viability. Our results indicate that therapeutic targeting of AR in prostate cancer using antiandrogens may be substantially improved by targeting of Stat5a/b.

Abstract 2120: Cdk5 involves in interleukin-6 induces AR activation through phosphorylation of serine 727 Stat3 and serine 81 AR in prostate cancer cells

Abstract Signal transducer and activator of transcription 3 (Stat3), a common transcription factor, has been reported to regulate cancer development, including prostate cancer. Stat3 has also been suggested that its transcriptional activity is initiated by phosphorylation. The tyrosine 705 (Y705) and serine 727 (S727) phosphorylation of Stat3 were documented to Stat3 activation. Androgen receptor (AR) is a ligand-activated transcription factor that mediates the biological responses of androgens. Cyclin-dependent kinase 5 (Cdk5), which belongs to Cdk family, is a proline-directed serine/threonine kinase. Our previous results indicate that Cdk5 may phosphorylate AR at serine 81 site (S81) and increase AR stabilization and transactivation. On the other hand, previous studies have shown that Interleukin-6 (IL-6), which is a cytokine, activates androgen receptor (AR) via MAPK and Stat3-related pathway. Therefore, the aim of this study is to investigate if Cdk5 regulates S81 phosphorylation-related AR activation through S727 Stat3 phosphorylation under IL-6 treatment. First we observed IL-6 enhanced S727 phosphorylation of Stat3 and S81 phosphorylation of AR as well as AR protein level. Next, IL-6 increased AR biochemical interaction with Stat3 and AR stabilization, nuclear localization, and transactivation, which is correlated to S727 phosphorylation of Stat3. Subsequently, we found that IL-6 induced tyrosine 15 (Y15) phosphorylation of Cdk5, which is an indicator of Cdk5 activation, and this phosphorylation was inhibited by AG490 (JAK inhibitor). We also found that Y15-Cdk5 mutant (Y15F) decreased IL-6-induced phosphorylation of AR S81 and Stat3 S727 and the biochemical interaction between AR and Stat3 was also affected. In summary, these results suggest that IL-6 activated AR by S81 phosphorylation via S727 phosphorylation of Stat3 and Cdk5 might be an upstream regulator in the pathway. Citation Format: Jo-Hsin Wang, Pei-Chi Li, Li-Chiung Lin, Fu-Ning Hsu, Mei-Chih Chen, Ho Lin. Cdk5 involves in interleukin-6 induces AR activation through phosphorylation of serine 727 Stat3 and serine 81 AR in prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2120. doi:10.1158/1538-7445.AM2014-2120

Abstract 4033: Epigenetic alterations in the multifaceted inhibitory effects of metformin on castration resistant prostate cancer

Abstract We recently demonstrated that metformin reduced MYC oncogene protein levels both in vitro and in vivo to inhibit prostate cancer (PCa) growth. MYC oncogene, known for its critical role in prostate carcinogenesis, interacts and recruits protein complexes with histone acetylases activities on the bound targets indicating epigenetic alterations are associated with MYC function. The importance of epigenetic events in PCa growth include facilitation of androgen receptor (AR) signaling and stabilizing AR in PCa cells by histone-modifying enzymes. The conversion of early stage tumors into invasive malignancies with an aggressive phenotype associated with the irreversible loss of epithelial markers from hyper-methylation. We hypothesize that metformin reduces the growth and migration of castration resistant prostate cancer (CRPC) via epigenetic alterations. We demonstrated that metformin inhibits cellular proliferation of the castration resistant cell lines, human C4-2b PCa and Myc-CaP mouse PCa cells. In vivo, metformin reduced the incidence of prostatic intraepithelial neoplasia lesion development in Hi-Myc mice after 4-week-treatment and further reduced PCa incidence after 24-week-treatment. Metformin also reduced tumor growth in subcutaneous xenograft model with C4-2b cells in athymic mice after 4-week treatment. The reduction of CRPC cell growth was resulted from a combined force from metformin induced cell cycle arrest at G1 phase, stimulation of apoptosis and autophagy. Western blots and IHC analysis were performed to demonstrate that metformin inhibition of PCa growth is associated with reduced AR expression in vitro and in vivo. The expression of a methyltransferase Suv39H1, which was recently identified to regulated PCa growth, migration and AR activation, was significantly suppressed by metformin at mRNA and protein levels in CRPC cells. Metformin also reduced the interaction between Suv39H1 and class III deacetylase Sirt1 in CRPC cells. Suv39H1 and Sirt1 could regulate the levels of the H3K9 methylation of the target genes, which is strongly associated with regional variation in chromatin organization and mutation rate variation in somatic cells. As expected with the reduction of Suv39H1 expression and activity to stimulate H3K9 methylation, cell migration and mobility was suppressed in metformin treated CRPC cells. Our data indicate that metformin altered epigenetic enzymes, Suv39H1 and Sirt1, to down-regulate AR expression and activity, cell growth and migration. Taken together, this study revealed a novel molecular mechanism through Suv39H1 and Sirt1 to mediate the multifaceted inhibitory effects of metformin against CRPC including the stimulation of cell cycle arrest, apoptosis, autophagy and migration. Citation Format: Xin Li, Juncheng Wei, Satoko Matsumura, Yuqi Guo, Huawei Yuan. Epigenetic alterations in the multifaceted inhibitory effects of metformin on castration resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4033. doi:10.1158/1538-7445.AM2014-4033

Determinants of Gli2 co-activation of wildtype and naturally truncated androgen receptors.

Gli2, a transcription factor in the Hedgehog pathway, is overexpressed in castrate-resistant prostate cancer (PCa). Previously we showed that Gli2 overexpression increased transcriptional activity of androgen receptor (AR) and conferred androgen growth-independence to normally growth-dependent PCa cells. Here we localized the regions of AR-Gli2 protein interaction and determined the domains within Gli2 needed for AR co-activation. Co-immunoprecipitation and GST-pulldown assays were used to define AR-Gli binding domains. Co-activation assays using androgen-responsive promoter reporters were used to define Gli2 regions needed for AR co-activation. Chromatin immunoprecipitation (ChIP) assays were used to confirm nuclear interactions of Gli2 with AR in PCa cells. The Gli2 C-terminal domain (CTD) is sufficient for AR co-activation. Two elements within the CTD were required: (1) an AR binding domain within aa628-897; and (2) at least part of the Gli2 transactivation domain within aa1252-1586. In turn, Gli2 binds the tau5/AF5 ligand-independent activation domain in the AR N-terminus. Mutations in the WxxLF motif in tau5/AF5 greatly diminished binding to Gli2-CTD. Gli2 interaction with AR tau5/AF5 was further substantiated by the ability of Gli2/Gli2-CTD to co-activate truncated AR splice variants (AR-V7/ARV567es). ChIP assays confirmed that Gli2 associates with chromatin at androgen response elements found near androgen-responsive genes in LNCaP cells. These assays also showed that AR associates with chromatin containing a Gli-response element near a Gli-responsive gene. Our findings indicate that Gli2 overexpression in PCa cells might support development of castration resistant PCa through AR co-activation and suggests that AR might modulate transcription from Gli2.