Androgen Receptor In LNCaP Cells Research Articles

Astragalus membranaceus Extract Induces Apoptosis via Generation of Reactive Oxygen Species and Inhibition of Heat Shock Protein 27 and Androgen Receptor in Prostate Cancers.

Although Astragalus membranaceus is known to have anti-inflammatory, anti-obesity, and anti-oxidant properties, the underlying apoptotic mechanism of Astragalus membranaceus extract has never been elucidated in prostate cancer. In this paper, the apoptotic mechanism of a water extract from the dried root of Astragalus membranaceus (WAM) was investigated in prostate cancer cells in association with heat shock protein 27 (HSP27)/androgen receptor (AR) signaling. WAM increased cytotoxicity and the sub-G1 population, cleaved poly (ADP-ribose) polymerase (PARP) and cysteine aspartyl-specific protease 3 (caspase 3), and attenuated the expression of B-cell lymphoma 2 (Bcl-2) in LNCaP cells after 24 h of exposure. Consistently, WAM significantly increased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive LNCaP cells. WAM decreased the phosphorylation of HSP27 on Ser82 and inhibited the expression of the AR and prostate-specific antigen (PSA), along with reducing the nuclear translocation of p-HSP27 and the AR via the disturbed binding of p-HSP27 with the AR in LNCaP cells. WAM consistently inhibited the expression of the AR and PSA in dihydrotestosterone (DHT)-treated LNCaP cells. WAM also suppressed AR stability, both in the presence and absence of cycloheximide, in LNCaP cells. Taken together, these findings provide evidence that WAM induces apoptosis via the inhibition of HSP27/AR signaling in prostate cancer cells and is a potent anticancer candidate for prostate cancer treatment.

Antrodia salmonea Extracts Regulate p53-AR Signaling and Apoptosis in Human Prostate Cancer LNCaP Cells.

Antrodia salmonea (AS) is a genus of Antrodia, an epiphyte of Cunninghamia konishii in Taiwan. AS has been reported to have potential therapeutic effects on different diseases, including diarrhea, abdominal pain, and hypertension. AS has been reported to have anticancer effects on numerous cancer types, such as ovarian carcinoma and triple-negative breast cancer. Our previous studies demonstrated that antrocins and triterpenoids are possibly bioactive compositions. However, the effects of AS on prostate cancer remain unknown. Therefore, we investigated the role of AS in prostate cancer growth, apoptosis, and cell cycle regulation. The results showed that AS extracts significantly inhibited the proliferation of prostate cancer LNCaP cells in a dose-dependent manner and increased the levels of apoptotic markers (cleaved PARP and cleaved caspase 3/8/9). In addition, the cell cycle-related proteins CDK1, CDK2, CDK4, and their respective specific regulators Cyclin B1, Cyclin A, and Cyclin D were also affected. Besides, AS treatment increased p53 protein levels and slowed its degradation in LNCaP cells. Interestingly, we found that AS treatment reduced both total protein and Ser-81 phosphorylation levels of the androgen receptor (AR). Notably, the increase of nuclear p53 was accompanied by the down-regulation of AR, suggesting a reverse regulation between p53 and AR in LNCaP cells was triggered by AS treatment. These findings suggest that AS extracts trigger the apoptosis of prostate cancer cells through the reverse regulation of p53 and AR and elucidate that AS extracts might be a potential treatment for androgen-dependent prostate cancer in the near future.

Antitumor mechanism of combination of Angelica gigas and Torilis japonica in LNCaP prostate cancer cells via G1 arrest and inhibition of Wnt/β-catenin and androgen receptor signaling.

The goal of the current study is to assess the antitumor mechanism by the combination (7:3) of Angelica gigas and Torilis japonica (AT) that was found most effective through screening against prostate-specific antigen (PSA) in LNCaP prostate cancer cells. Here, AT reduced the viability and the number of colonies in androgen-dependent LNCaP cells more than in androgen independent PC3 and DU145 cells. Also, AT induced G1 phase arrest, cleaved PARP and caspase 3, activated p27 and decreased the expression of Cyclin D1, Cyclin E, cdk2 in LNCaP cells. Furthermore, AT decreased the expression of PSA and androgen receptor (AR) at mRNA and protein levels in LNCaP cells. Interestingly, AT attenuated the expression of AR, PSA and Wnt-3a and the stability of AR and PSA in LNCaP cells. Furthermore, AT reversed dihydrotestosterone (DHT)-induced upregulation of AR and PSA in LnCaP cells. Notably, AT disrupted the protein-protein interaction, nuclear translocation and fluorescent expression of β-catenin and AR in LNCaP cells. Consistently, β-catenin depletion enhanced the decreased expression of AR in AT treated LNCaP cells. Taken together, our findings highlight evidence that AT suppresses the proliferation of LNCaP cells via G1 arrest and inhibition of β-catenin and AR as a potential anticancer agent.

The DNA/RNA helicase DHX9 contributes to the transcriptional program of the androgen receptor in prostate cancer

BackgroundProstate cancer (PC) is the most commonly diagnosed male malignancy and an important cause of mortality. Androgen deprivation therapy is the first line treatment but, unfortunately, a large part of patients evolves to a castration-resistant stage, for which no effective cure is currently available. The DNA/RNA helicase DHX9 is emerging as an important regulator of cellular processes that are often deregulated in cancer.MethodsTo investigate whether DHX9 modulates PC cell transcriptome we performed RNA-sequencing analyses upon DHX9 silencing in the androgen-responsive cell line LNCaP. Bioinformatics and functional analyses were carried out to elucidate the mechanism of gene expression regulation by DHX9. Data from The Cancer Genome Atlas were mined to evaluate the potential role of DHX9 in PC.ResultsWe found that up-regulation of DHX9 correlates with advanced stage and is associated with poor prognosis of PC patients. High-throughput RNA-sequencing analysis revealed that depletion of DHX9 in androgen-sensitive LNCaP cells affects expression of hundreds of genes, which significantly overlap with known targets of the Androgen Receptor (AR). Notably, AR binds to the DHX9 promoter and induces its expression, while Enzalutamide-mediated inhibition of AR activity represses DHX9 expression. Moreover, DHX9 interacts with AR in LNCaP cells and its depletion significantly reduced the recruitment of AR to the promoter region of target genes and the ability of AR to promote their expression in response to 5α-dihydrotestosterone. Consistently, silencing of DXH9 negatively affected androgen-induced PC cell proliferation and migration.ConclusionsCollectively, our data uncover a new role of DHX9 in the control of the AR transcriptional program and establish the existence of an oncogenic DHX9/AR axis, which may represent a new druggable target to counteract PC progression.

Antiandrogenic activity of Riboflavin 5′-phosphate (FMN) in 22Rv1 and LNCaP human prostate cancer cell lines

The androgen receptor is a hormone activated transcription factor that regulates the development and maintenance of male characteristics and represents one of the most well-established drug targets, being implicated not only in prostate cancer but also in many non-cancerous human diseases including androgenetic alopecia, acne vulgaris, and hirsutism. In this study, the antiandrogenic effects of FMN were investigated in 22Rv1 and LNCaP prostate cancer cells. FMN inhibited dihydrotestosterone (DHT)-induced protein expression of androgen receptor in 22Rv1cells. In another prostate cancer LNCaP cells, FMN decreased the protein level of DHT-induced prostate specific antigen (PSA). In addition, FMN downregulated DHT-induced mRNA expression of androgen regulated genes in both cell lines, showing less prominent inhibition in 22Rv1cells where androgen receptor had been significantly decreased by FMN. FMN was found to bind androgen receptor, demonstrating that it acted as a competitive androgen receptor antagonist. FMN increased the phosphorylation of Akt in 22Rv1 cells and this increment was abrogated by PI3K inhibitor wortmannin, resulting in a rescued androgen receptor protein level which was decreased by FMN. Additionally, FMN was found to increase the mRNA and protein level of E3 ligase mouse double minute 2. Our data suggest that the androgen receptor signaling is regulated through PI3K-Akt-MDM2 pathway in 22Rv1 cells. Together, our results indicate that FMN facilitated the degradation of androgen receptor in 22Rv1 cells and inhibited the expression of androgen regulated genes by competing the binding of DHT to androgen receptor in LNCaP cells, demonstrating its therapeutic potential as an antiandrogen.

Evaluation of the effects of androgenic Chinese herbal medicines on androgen receptors and tumor growth in experimental prostate cancer models

Ethnopharmacological relevanceMany prostate cancer (PCa) patients in Mainland China and other Asian countries often use Chinese herbal medicines as an adjuvant treatment while receiving Western medicines. However, concerns have been raised about the potential herb-drug interaction when using herbal medicines containing phytoandrogens. Aim of the studyThis study aimed to investigate the effects of the selected 21 Chinese herbal medicines on the proliferation and tumor growth using the relevant in vitro and in vivo models of PCa. Materials and methodsAfter treatment of LNCaP and 22Rv1 cells with different concentrations of 70% ethanol extracts of the 21 selected herbal medicines for 48 h, the proliferative activity, the effects on androgen receptor (AR) and prostate specific antigen (PSA) were determined. The anti-tumor effects of the 21 herbs on PCa growth were also investigated on a subcutaneous mouse model of PCa. ResultsThe results showed that Epimedii Folium (EF) and Codonopsis Radix (CNR) could significantly increase the cell viability in LNCaP cells (p < 0.05 for both) and 22Rv1 cells (p < 0.05 for both), protein expressions of AR in LNCaP cells (p < 0.05 for both) and 22Rv1 cells (p < 0.05 for both), and PSA (p < 0.05 for both) in LNCaP cells. EF, CNR, and Cistanches Herba (CCH) markedly accentuated the tumor growth (p < 0.05 for three drugs) and AR expression (p < 0.05 for three herbs) in tumor tissues. On the other hand, treatment with Astragali Radix (AGR), Chuanxiong Rhizoma (CXR) and Bruceae Fructus (BF) significantly inhibited the cell viability in LNCaP cells (p < 0.05, p < 0.05 and p < 0.001, respectively) and in 22Rv1 cells (p < 0.05, p < 0.05 and p < 0.001, respectively), and the protein expression of AR in LNCaP cells (p < 0.05 for three herbs) and 22Rv1 cells (p < 0.05, p < 0.05 and p < 0.001, respectively), and the protein expression of PSA (p < 0.05 for three herbs) in LNCaP cells, as well as tumor growth (p < 0.05 for three herbs) and the AR expression (p < 0.05 for AGR and CXR, p < 0.001 for BF) in tumor tissues. ConclusionOur results revealed that AGR, CXR and BF suppressed the PCa development via inhibition of AR expression, while EF, CNR and CCH promoted the development and progression of PCa via enhancement of AR expression. The results strongly suggest that caution should be exercised when using androgenic Chinese herbal medicines in PCa patients.

Carbidopa suppresses prostate cancer via aryl hydrocarbon receptor-mediated ubiquitination and degradation of androgen receptor

Carbidopa, a peripheral decarboxylase inhibitor used with L-DOPA to treat Parkinson’s disease, has attracted significant interest in recent years for its anticancer effect. Increasing evidence reveals that Carbidopa can inhibit cancer cell growth and induce apoptosis through aryl hydrocarbon receptor (AHR) in some cancers. However, the antitumor effect of Carbidopa in prostate cancer (PCa) is not fully understood. Androgen receptor (AR) plays a central role in PCa, even in advanced “castrate-resistant” disease. In the present study, we report that Carbidopa suppresses the growth of PCa by downregulating the protein expression of AR. Carbidopa inhibits proliferation and migration of LNCaP cells and promotes apoptosis, but has no effect on the AR-independent prostate cell line DU145. Carbidopa increases ubiquitination of AR in LNCaP cells. Several studies have shown that AHR can act as an E3 ubiquitin ligase and promote the proteasomal degradation of AR. Quantitative RT-PCR, immunofluorescence staining and immunoblotting assay demonstrate that AHR is induced and activated by Carbidopa, and the co-immunoprecipitation assay shows that AR interacts with AHR, firmly confirming that Carbidopa decreases AR protein level though AHR-induced proteasomal degradation. In addition, Carbidopa suppresses PCa growth in vivo when xenografted into immunocompromised mice. Carbidopa treatment increases AHR protein level and decreases AR protein level in tumor tissues. Taken together, our study implicates Carbidopa for the first time in effective suppression of prostate cancer via a mechanism, involving AHR-mediated proteasomal degradation of AR.

Development of cell-penetrating bispecific antibodies targeting the N-terminal domain of androgen receptor for prostate cancer therapy.

Castration-resistant prostate cancer cells exhibit continued androgen receptor signaling in spite of low levels of ligand. Current therapies to block androgen receptor signaling act by inhibiting ligand production or binding. We developed bispecific antibodies capable of penetrating cells and binding androgen receptor outside of the ligand-binding domain. Half of the bispecific antibody molecule consists of a single-chain variable fragment of 3E10, an anti-DNA antibody that enters cells. The other half is a single-chain variable fragment version of AR441, an anti-AR antibody. The resulting 3E10-AR441 bispecific antibody enters human LNCaP prostate cells and accumulates in the nucleus. The antibody binds to wild-type, mutant and splice variant androgen receptor. Binding affinity of 3E10-AR441 to androgen receptor (284 nM) was lower than that of the parental AR441 mAb (4.6 nM), but could be improved (45 nM) through alternative placement of the affinity tags, and ordering of the VH and VK domains. The 3E10-AR441 bispecific antibody blocked genomic signaling by wild-type or splice variant androgen receptor in LNCaP cells. It also blocked non-genomic signaling by the wild-type receptor. Furthermore, bispecific antibody inhibited the growth of C4-2 prostate cancer cells under androgen-stimulated conditions. The 3E10-AR441 biAb can enter prostate cancer cells and inhibits androgen receptor function in a ligand-independent manner. It may be an attractive prototype agent for prostate cancer therapy.

Endogenous androgen receptor proteomic profiling reveals genomic subcomplex involved in prostate tumorigenesis.

Androgen receptor (AR) is a key player in prostate cancer development and progression. Here we applied immunoprecipitation mass spectrometry of endogenous AR in LNCaP cells to identify components of the AR transcriptional complex. In total, 66 known and novel AR interactors were identified in the presence of synthetic androgen, most of which were critical for AR-driven prostate cancer cell proliferation. A subset of AR interactors required for LNCaP proliferation were profiled using chromatin immunoprecipitation assays followed by sequencing, identifying distinct genomic subcomplexes of AR interaction partners. Interestingly, three major subgroups of genomic subcomplexes were identified, where selective gain of function for AR genomic action in tumorigenesis was found, dictated by FOXA1 and HOXB13. In summary, by combining proteomic and genomic approaches we reveal subclasses of AR transcriptional complexes, differentiating normal AR behavior from the oncogenic state. In this process, the expression of AR interactors has key roles by reprogramming the AR cistrome and interactome in a genomic location-specific manner.

Abstract P3-04-21: Androgen receptor in tamoxifen-resistant breast cancer is affected by SUMO

Abstract Resistance to tamoxifen is a major problem in treating women with estrogen receptor-positive (ERa+) breast cancer (BCa). Previous studies report elevated levels of the androgen receptor (AR) in tamoxifen-resistant (TamR) human mammary tumors. Inversely, in vitro overexpression of AR in ERa+-MCF-7 cells potentiates growth in the presence of tamoxifen. AR is a target for small ubiquitin like modifiers (SUMO) posttranslational modification (PTM). Prostate cancer (PCa) studies, show that SUMO-PTMs can have diverse effects on AR transcriptional activity, by modifying the AR itself or its co-regulators. SUMOylation regulates the AR's interaction with chromatin and modulates its function in a gene selective manner. Although SUMO-1 inhibited the AR transcriptional activity, SUMO-3 strongly enhanced the transactivation of AR in LNCaP cells but negatively affected the AR transcriptional activity in primary prostate epithelial cells. On the other hand, SUMO modifications and its subsequent effects on the AR in breast tumors and in particular TamR-BCa is unknown. Our objective is to understand how SUMO-PTMs affect AR activity in TamR-BCa. Others report that high Ubc9, a SUMO E2 conjugating enzyme, correlates with with aggressive phenotypes of BCa and that PIAS3, a SUMO E3 ligase, expression increases in breast tumors. Consistently, we now report that global SUMO2/3-conjugation is enhanced while the full-length SUMO protease SENP7 (SENP7L) is reduced in TamR-BCa cells. Collectively, these observations would suggest reduced SUMO dynamics in TamR-BCa as compared to tamoxifen-sensitive (TamS) BCa cells. Unlike PCa cells that favor unmodified over SUMOylated AR, we report that AR SUMOylation is potentiated in TamR-BCa cells. Functionally, SUMOylation of AR augments its chromatin binding and enhances its transcriptional activity. Our results suggest that the level of unmodified to SUMOylated AR is dramatically altered with onset of TamR and drives a functionally distinct population of AR in TamR versus TamS BCa. Consistently these biochemical reactions that define protein diversity could have a major impact on drug resistance in BCa. Citation Format: Bahnassy S, Kumar S, Ren J, Frutiz G, Karami S, Bawa-Khalfe T. Androgen receptor in tamoxifen-resistant breast cancer is affected by SUMO [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-21.

Abstract C132: Therapeutic reactivation of PP2A for prostate cancer treatment

Abstract Several new therapies have recently been approved for patients with castration-resistant prostate cancer (CRPC), however, none are curative and tumors ultimately develop resistance. Advances in the treatment of CRPC require novel approaches and therapies such as those outlined in this study. Most drug development efforts have focused on targeting single oncogenic proteins, an approach limited by the complexity of signaling networks and associated cross talk. Targeting phosphatases, the key negative regulators of signaling proteins, on the other hand, may overcome some of these limitations, particularly if these negative regulators themselves are altered.Through reverse engineering of tricyclic neuroleptic drugs, we have developed a series of small molecule activators of the serine/threonine phosphatase 2A (PP2A), a key negative regulator of numerous oncogenic signaling pathways. PP2A acts as a tumor suppressor and dephosphorylates several critical nodes in prostate cancer pathogenesis including the androgen receptor (AR). Decreased PP2A expression and/or activity have been correlated with castration-resistance in cell culture and human prostate cancer studies. These small molecule activators of PP2A (SMAPs), as represented by TRC-794, TRC-1154, and DT-061, directly bind and activate PP2A and have favorable pharmaceutical properties. In this study we sought to determine the activity of SMAPs in clinically relevant preclinical models of prostate cancer. Treatment of prostate cancer cell lines with SMAPs resulted in decreased cell viability and colony formation, cell cycle arrest, and an increase in apoptosis. Global Phosphoproteomic analysis of TRC-794 treated prostate cancer cells revealed that the AR and MYC were significantly perturbed in drug treated cells compared to controls which was subsequently confirmed by western blotting. Western blot analysis of prostate cancer cells demonstrated dose-dependent degradation of the AR resulting in PSA reduction and changes in canonical AR target gene expression. In order to investigate whether PP2A was mediating SMAP induced AR degradation, LNCAP cells were stably transduced with the SV40 small t antigen (ST), a potent oncoprotein that perturbs PP2A function. SMAPs were unable to degrade AR in LNCAP cells transduced with ST, suggesting that PP2A mediates SMAP induced AR degradation. SMAPs were evaluated in vivo in xenograft models representing prostate cancers that are sensitive to conventional therapy and resistant to enzalutamide, the current gold standard, due to overexpression of the AR or expression of androgen receptor splice variants (AR-SV). Single agent treatment with DT-1154 or DT-061 in vivo resulted in either significant tumor growth inhibition or tumor regression and induction of tumor cell apoptosis comparable to enzalutamide. Western blot analysis of the tumors demonstrated that the effects on tumor volume correlated strongly with target engagement as evidenced by significant decreases in PSA and AR expression in vivo. Additionally, these compounds demonstrated favorable pharmacokinetics and showed no overt toxicity. Combined these data highlight the potential for PP2A activation for both the treatment of CRPC and potentially for diverse PP2A inactivated tumor types and diseases. Citation Format: Kim McClinch, Rita Avelar, David Callejas, David Kastrinsky, Michael Ohlmeyer, Stephen Plymate, Matthew Galsky, Goutham Narla. Therapeutic reactivation of PP2A for prostate cancer treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C132.

Abstract 1849: Androgen receptor transcriptionally represses genes mediating DNA synthesis and repair in prostate cancer

Abstract In the U.S., prostate cancer (PCa) ranked second for cancer related deaths for men. It has been well established that androgen receptor (AR) plays an essential role in the development of primary PCa and its activity is restored in castration resistant prostate cancers (CRPC). While AR has been extensively characterized as a transcriptional activator, it can also function by direct or indirect mechanisms to suppress gene expression. Despite the critical role AR plays in PCa, mechanisms by which it functions as a transcriptional repressor are poorly understood. Using VCaP and VCaP derived CRPC cell lines, we have reported that androgen-repressed genes were functionally enriched for DNA synthesis and repair, while AR stimulated genes were associated with lipid/sterol synthesis and other aspects of cellular metabolism. Significantly, a subset of the genes that were androgen-repressed in VCaP cells were consistently increased in CRPC clinical samples, and this 53-gene subset was also highly enriched for genes mediating DNA synthesis and repair. Using AR ChIP-seq and transcriptome profiling in VCaP cells, we found that the majority of this AR-suppressed gene subset (39/53) have one or more AR binding sites within the gene locus. The expression level of these direct AR-suppressed genes is associated with poor survival of disease in clinical cohorts. Interestingly, although the suppression activity of AR on this subset of genes is direct, in other PCa cell lines such as LNCaP, there is a secondary mechanism that allows AR to indirectly stimulate the expression of these genes. Importantly, the direct AR mediated repression of these genes is enhanced in PCa cells expressing higher levels of AR, and overexpression of AR in LNCaP cells resulted in increased and more persistent AR binding to these genes, and also enhanced the direct AR repression of this gene subset. Mechanistically, we found that the indirect activation of these DNA synthesis genes by AR is mediated through transcriptionally stimulating lipid synthesis, which could subsequently drive the G1/S cell cycle progression and RB1 phosphorylation. Indeed, this indirect stimulatory effect of AR on these genes could be suppressed by cyclin dependent kinase inhibition or by preventing the AR stimulation of mTOR (rapamycin) or sterol synthesis (statins). Taken together, these findings indicate that the ability of AR to repress this gene set mediating DNA synthesis may persist in CRPC, and may be exploited therapeutically by combination therapies that stimulate AR while repressing its metabolic functions. Citation Format: Yanfei Gao, Shuai Gao, Housheng He, Xiaming Liu, Sen Chen, Fen Ma, X. Shirley Liu, Myles Brown, Steven P. Balk, Shaoyong Chen, Changmeng Cai. Androgen receptor transcriptionally represses genes mediating DNA synthesis and repair in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1849. doi:10.1158/1538-7445.AM2015-1849

Mutation of androgen receptor N-terminal phosphorylation site Tyr-267 leads to inhibition of nuclear translocation and DNA binding.

Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.

Diabetes Protects from Prostate Cancer by Downregulating Androgen Receptor: New Insights from LNCaP Cells and PAC120 Mouse Model

Type 2 diabetes has been associated with decreased risk of prostate cancer in observational studies, and this inverse association has been recently confirmed in several large cohort studies. However the mechanisms involved in this protective effect remain to be elucidated. The aim of the present study was to explore whether different features of type 2 diabetes (hyperinsulinemia, hyperglycemia and tumor necrosis factor alpha [TNF-α]) protect against the development of prostate cancer. For this purpose LNCaP cells were used for in vitro experiments and nude mice in which PAC120 (hormone-dependent human prostate cancer) xenografts had been implanted were used for in vivo examinations. We provide evidence that increasing glucose concentrations downregulate androgen receptor (AR) mRNA and protein levels through NF-κB activation in LNCaP cells. Moreover, there was a synergic effect of glucose and TNFα in downregulating the AR in LNCaP cells. By contrast, insulin had no effect on AR regulation. In vivo experiments showed that streptozotocin-induced diabetes (STZ-DM) produces tumor growth retardation and a significant reduction in AR expression in PAC120 prostate cancer mice. In conclusion, our results suggest that hyperglycemia and TNF-α play an important role in protecting against prostate cancer by reducing androgen receptor levels via NF-κB.

Abstract 5455: Differential regulation of E2A (TCF3) by androgens in prostate cancer cells.

Abstract Introduction: E2A (TCF3) is a multifunctional basic helix loop helix (bHLH) transcription factor. E2A promotes cell differentiation, acts as a negative regulator of cell proliferation in normal cells and cancer cell lines and is required for normal B-cell development. Previous studies from our laboratory has shown that E2A expression is highly increased in prostate cancer as compared to normal prostate and that it acts as a tumor promoter in prostate cancer. Given the diverse biological pathways regulated/ influenced by E2A little is known about its regulation in prostate cancer. Experimental design: E2A expression in androgen sensitive LNCaP and insensitive C81 prostate cancer cell lines was determined by western blot after treatments with androgen receptor (AR) agonist R1881 and antagonist casodex. Putative Androgen Response Elements (ARE) were identified in the first intronic region of the E2A gene using some of the online bioinformatics tools and confirmed by Chromatin Immunoprecipitation (ChIP) with AR antibody and Luciferase reporter assays on the above mentioned cell lines after treatments with R1881 and casodex. Results: E2A expression was found to increase with the increasing aggrasiveness of prostate cancer cell lines as compared to normal prostate epithelial cell line RWPE1. When androgen responsive cell line LNCaP was treated with R1881 there was an increased expression of E2A which decreased upon treatment with antiandrogen casodex whereas the E2A expression remained unaltered upon similar treatments in androgen insensitive cell line C81. The first intronic region of the E2A gene was predicted to contain two putative ARE sites. ChIP after treatment of LNCaP and C81 cells with R1881 and casodex showed that the intronic region was bound by AR in LNCaP cells only in the presence of R1881, whereas C81 cells showed a pulldown with AR in presence as well as absence of R1881. Similar results were observed in luciferase reporter assays indicating that E2A is transactivated by AR in LNCaP cell lines whereas it is independent of androgens in C81 cell line. Furthermore, Luciferase reporter assays also confirmed that only one of the two predicted putative AREs was functionally active and responsible for the androgen mediated regulation of E2A. Conclusion: Our results indicate that E2A is differentially regulated in Prostate cancer cell lines. The increased expression of E2A and its role as a tumor promoter in prostate cancer cell lines may be contributed to its loss of androgen dependence as is evident in its progression from androgen dependent LNCaP to independent C81 cells. Acknowledgements: This study was supported by NIH/NCI RO1 CA128914 and NIH/NCRR/RCMI G12RR03062. Citation Format: Divya Patel, Jaideep Chaudhary. Differential regulation of E2A (TCF3) by androgens in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5455. doi:10.1158/1538-7445.AM2013-5455

Increased Akt Signaling Resulting from the Loss of Androgen Responsiveness in Prostate Cancer

The mechanisms responsible for the switch of prostate cancer from androgen-sensitive (AS) to androgen-insensitive (AI) form are not well understood. Regulation of androgen receptor (AR), through which androgens control the expression of genes involved in prostate cells proliferation, migration and death also involves its cross-talk with the other signaling pathways, transcription factors and coregulatory proteins, such as β-catenin. With the aim to determine their possible contribution in triggering the switch from AS to AI form, which occurs upon androgen deprivation therapy - AR, Akt and β-catenin expression were knocked-down with respective siRNAs. Treatment of LNCaP prostate cells with siRNA for AR significantly reduced their proliferation (45-70%), expression of nuclear β- catenin, cyclin-D1, cyclin-G1, c-Myc as well as activity of metalloproteinases (MMPs) -2,-7,-9 and cell migration. Surprisingly, after longer (over 72 hrs) silencing of AR in LNCaP cells, elevated levels of p-Akt were detected and enhanced proliferation as well as expression of nuclear β-catenin, cyclin-D1, c-Myc and activity of MMPs were observed. Such effects were not observed in either PC-3 or DU145 AI cells. However, silencing of Akt and /or β-catenin in those as well as in LNCaP cells led to their decreased proliferation and migration. Our findings suggest that in prostate cancer cells, either AR or Akt signaling prevails, depending on their initial androgen sensitivity and its availability. In AI prostate cancer cells, Akt takes over the role of AR and more effectively contributes through the same signaling molecule, β-catenin, to AI cancer progression.

Activation of p53 Signaling and Inhibition of Androgen Receptor Mediate Tanshinone IIA Induced G1 Arrest in LNCaP Prostate Cancer Cells

Our group previously reported that tanshinone IIA induced apoptosis via a mitochondria dependent pathway in LNCaP prostate cancer cells. In the present study, the roles of androgen receptor (AR) and p53 signaling pathways were investigated in tanshinone IIA-induced G1 arrest in LNCaP cells. Tanshinone IIA significantly inhibited the growth and proliferation of LNCaP cells by colony formation and BrdU incorporation assays, respectively. Tanshinone IIA induced cell cycle arrest at G1 phase and down-regulated cyclin D1, CDK2 and CDK4. Furthermore, tanshinone IIA activated the phosphorylation of p53 at Ser 15 residue and its downstream p21 and p27. Additionally, tanshinone IIA suppressed the expression of AR and prostate specific antigen (PSA). Conversely, silencing p53 using its specific siRNA reversed cyclin D1 expression inhibited by tanshinone IIA. However, knockdown of AR had no effect on the p53/p21/p27 signaling pathway activated by tanshinone IIA in LNCaP cells. In AR siRNA-transfected cells, tanshinone IIA did not cause cell cycle arrest and reduce cyclin D1, implying that AR is essential to induce G1 arrest by tanshinone IIA in LNCaP cells. Taken together, the findings suggest that tanshinone IIA induces G1 arrest via activation of p53 signaling and inhibition of AR in LNCaP cells.

Y-box binding protein-1 promotes castration-resistant prostate cancer growth via androgen receptor expression

The androgen receptor (AR) is well known to play a central role in the pathogenesis of prostate cancer (PCa). In several studies, AR was overexpressed in castration-resistant PCa (CRPC). However, the mechanism of AR overexpression in CRPC is not fully elucidated. Y-box binding protein-1 (YB-1) is a pleiotropic transcription factor that is upregulated in CPRC. We aimed to elucidate the role of YB-1 in castration resistance of PCa and identify therapeutic potential of targeting YB-1. Using immunohistochemistry, we found that nuclear YB-1 expression significantly correlated with the Gleason score and AR expression in PCa tissues. In PCa cells, YB-1 regulated AR expression at the transcriptional level. Furthermore, YB-1 expression and nuclear localization were upregulated in CRPC cells. Overexpression of AR, as well as YB-1, conferred castration-resistant growth in LNCaP and 22Rv1 cells. Conversely, knocking down YB-1 resulted in suppressed cell growth and induced apoptosis, which was more efficient than knocking down AR in LNCaP cells. In other types of PCa cells, such as CRPC cells, knocking down YB-1 resulted in a significant reduction of cell growth. In conclusion, these findings suggested that YB-1 induces castration resistance in androgen-dependent PCa cells via AR expression. Thus, YB-1 may be a promising therapeutic target for PCa, as well as CRPC.

Abstract B45: The short-term effects of arsenic on Cdk5-dependent apoptosis of prostate cancer cells

Abstract Arsenic compounds have been used widely in medicine to treat a variety of diseases. However, it has also been believed to involve the carcinogenesis of prostate cancer and the hormone disruption might be one of the causes. Our previous results indicate that digoxin treatment leads to apoptosis of prostate cancer cells through Cdk5 overactivation, in which the regular activator of Cdk5, p35, is cleaved into more potent one, p25. Here, our latest data showed that arsenic also caused p35 cleavage and subsequently Cdk5 overactivation in prostate cancer cells. Similarly, this arsenic- induced stimulatory effect on Cdk5 resulted in apoptosis of prostate cancer cells accompanying with the degradation of androgen receptor in LNCaP cells. This result implies that androgen is no longer a trophic factor for prostate cancer cells after arsenic treatment. Although STAT3 was identified by us as a Cdk5 substrate responding to tumor growth, arsenic-triggered Cdk5 overactivation did not increase STAT3 phosphorylation. In conclusion, although long-term exposure of arsenic possibly causes prostate cancer, our present results provide evidence indicating that in vitro and short-term treatments of arsenic might lead to apoptosis of prostate cancer cell lines through Cdk5 overactivation pathway. This study may arouse the new indication for the old chemical and also shed light on the application on cancer therapy. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B45.

Abstract 1709: Activation of androgen signaling axis by NF-kappaB2/p52

Abstract INTRODUCTION: Castration-resistant progression of prostate cancer occurs in &amp;gt;80% of patients after failure of hormonal therapy but the underlying mechanisms are incompletely understood. Emerging evidence implies that ligand-independent activation of the androgen receptor (AR) is of critical importance during this process. Our previous studies show that the levels of NF-κB2/p52 are elevated in prostate cancer cells and that active NF-κB2/p52 promotes prostate cancer cell growth in vitro and in vivo. In this study, we examined the role of NF-κB2/p52 in the aberrant activation of the AR during castration resistant progression, in contrast to the interaction between the AR and RelA/p65. METHODS: The effects of NF-κB2/p52 on cell survival, growth and androgen responsiveness were examined in prostate cancer cells. Modulation of LNCaP tumor growth by p52 was examined in intact and castrated male nude mice. Effect of p52 or p65 on AR activation was examined using luciferase assays, EMSA, ChIP assays and ELISA. RESULTS: Expression of NF-κB2/p52 enhanced androgen-sensitive LNCaP human CaP cell growth and clonogenic ability in androgen-deprived conditions in vitro. LNCaP cells expressing p52 exhibited protection from apoptotic cell death and cell cycle arrest induced by androgen-deprivation. Adenoviral mediated p52 expression in LNCaP cells induced tumor growth in castrated male nude mice. NF-κB/p52 induced the activation of the AR resulting in increased transactivation of AR-responsive genes such as PSA and NKX3.1 in a ligand-independent manner. NF-κB2/p52 enhanced nuclear translocation and transactivation of AR by interacting with its N-terminal domain and enhanced the recruitment of co-activators like p300 to the promoters of AR-dependent genes. These results were confirmed in 3 different prostate cancer cell lines: LAPC-4 (wild type AR), LNCaP (mutant AR) and C4-2 (androgen-insensitive). Transfection of p52 into LAPC-4 and LNCaP cells (which express low levels of p52) showed increased activation of the endogenous AR. Downregulation of endogenous p52 in C4-2 cells resulted in abrogation of AR constitutive activation. In contrast, we observed that p65 exhibited transcriptional repression of the AR, and did not induce expression of AR-responsive genes, PSA and NKX3.1. CONCLUSIONS: These findings demonstrate that overexpression of NF-κB2/p52 induces castration-resistant growth and aberrant activation of the AR in LNCaP cells. NF-κB2/p52 may promote castration resistant progression by activating the AR under conditions of androgen deprivation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1709.