Targeting Androgen Receptor Activation Function-1 with EPI to Overcome Resistance Mechanisms in Castration-Resistant Prostate Cancer.

Persistent androgen receptor (AR) transcriptional activity is clinically evident in castration-resistant prostate cancer (CRPC). Therefore, AR remains as a viable therapeutic target for CRPC. All current hormonal therapies target the C-terminus ligand-binding domain (LBD) of AR. By using EPI to target AR activation function-1 (AF-1), in the N-terminal domain that is essential for AR transactivation, we evaluate the ability of EPI to overcome several clinically relevant AR-related mechanisms of resistance. To study the effect of EPI on AR transcriptional activity against overexpressed coactivators, such as SRC1-3 and p300, luciferase reporter assays were performed using LNCaP cells. AR-negative COS-1 cells were employed for reporter assays to examine whether the length of polyglutamine tract affects inhibition by EPI. The effect of EPI on constitutively active AR splice variants was studied in LNCaP95 cells, which express AR-V7 variant. To evaluate the effect of EPI on the proliferation of LNCaP95 cells, we performed in vitro BrdUrd incorporation assay and in vivo studies using xenografts in mice. EPI effectively overcame several molecular alterations underlying aberrant AR activity, including overexpressed coactivators, AR gain-of-function mutations, and constitutively active AR-V7. EPI inhibited AR transcriptional activity regardless of the length of polyglutamine tract. Importantly, EPI significantly inhibited the in vitro and in vivo proliferation of LNCaP95 prostate cancer cells, which are androgen independent and enzalutamide resistant. These findings support EPI as a promising therapeutic agent to treat CRPC, particularly against tumors driven by constitutively active AR splice variants that are resistant to LBD-targeting drugs. Clin Cancer Res; 22(17); 4466-77. ©2016 AACRSee related commentary by Sharp et al., p. 4280.

Yang YC ，Banuelos CA ，Mawji NR ，Wang J ，Kato M ，Haile S ，McEwan IJ ，Plymate S ，Sadar MD ... -  《-》

Combined N-terminal androgen receptor and autophagy inhibition increases the antitumor effect in enzalutamide sensitive and enzalutamide resistant prostate cancer cells.

Multiple androgen receptor (AR)-dependent and -independent resistance mechanisms limit the efficacy of current castration-resistant prostate cancer (CRPC) treatment. Novel N-terminal domain (NTD) binding AR-targeting compounds, including EPI-001 (EPI), have the promising ability to block constitutively active splice variants, which represent a major resistance mechanism in CRPC. Autophagy is a conserved lysosomal degradation pathway that acts as survival mechanism in cells exposed to anticancer treatments. We hypothesized, that promising NTD-AR treatment may upregulate autophagy and that a combination of NTD-AR and autophagy inhibition might therefore increase antitumor effects. AR-expressing prostate cancer cell lines (LNCaP, LNCaP-EnzR) were treated with different concentrations of EPI (10, 25, 50 μM) and in combination with the autophagy inhibitors chloroquine (CHQ, 20 μM) or 3-methyladenine (3-MA, 5 mM). Cell proliferation was assessed by WST-1-assays after 1 and 7 days. Ethidium bromide and Annexin V were used to measure viability and apoptosis on day 7 after treatment. Autophagosome formation was detected by AUTOdot staining. In addition, autophagic activity was monitored by immunocytochemistry and Western blot (WES) for the expression of ATG5, Beclin1, LC3-I/II and p62. Treatment with EPI resulted in a dose-dependent reduction of cell growth and increased apoptosis in both cancer cell lines on day 7. In addition, EPI treatment demonstrated an upregulated autophagosome formation in LNCaP and LNCaP-EnzR cells. Assessment of autophagic activity by immunocytochemistry and WES revealed an increase of ATG5 and LC3-II expression and a decreased p62 expression in all EPI-treated cells. A combined treatment of EPI with autophagy inhibitors led to a further significant reduction of cell viability in both cell lines. Our results demonstrate that NTD targeting AR inhibition using EPI leads to an increased autophagic activity in LNCaP and LNCaP-EnzR prostate cancer cells. A combination of NTD AR blockage with simultaneous autophagy inhibition increases the antitumor effect of EPI in prostate cancer cells. Double treatment may offer a promising strategy to overcome resistance mechanisms in advanced prostate cancer.

Kranzbühler B ，Salemi S ，Mortezavi A ，Sulser T ，Eberli D ... -  《-》

Cotargeting Androgen Receptor Splice Variants and mTOR Signaling Pathway for the Treatment of Castration-Resistant Prostate Cancer.

The PI3K/Akt/mTOR pathway is activated in most castration-resistant prostate cancers (CRPC). Transcriptionally active androgen receptor (AR) plays a role in the majority of CRPCs. Therefore, cotargeting full-length (FL) AR and PI3K/Akt/mTOR signaling has been proposed as a possible, more effective therapeutic approach for CRPC. However, truncated AR-splice variants (AR-V) that are constitutively active and dominant over FL-AR are associated with tumor progression and resistance mechanisms in CRPC. It is currently unknown how blocking the PI3K/Akt/mTOR pathway impacts prostate cancer driven by AR-Vs. Here, we evaluated the efficacy and mechanism of combination therapy to block mTOR activity together with EPI-002, an AR N-terminal domain (NTD) antagonist that blocks the transcriptional activities of FL-AR and AR-Vs in models of CRPC. To determine the functional roles of FL-AR, AR-Vs, and PI3K/Akt/mTOR pathways, we employed EPI-002 or enzalutamide and BEZ235 (low dose) or everolimus in human prostate cancer cells that express FL-AR or FL-AR and AR-Vs (LNCaP95). Gene expression and efficacy were examined in vitro and in vivo EPI-002 had antitumor activity in enzalutamide-resistant LNCaP95 cells that was associated with decreased expression of AR-V target genes (e.g., UBE2C). Inhibition of mTOR provided additional blockade of UBE2C expression. A combination of EPI-002 and BEZ235 decreased the growth of LNCaP95 cells in vitro and in vivo Cotargeting mTOR and AR-NTD to block transcriptional activities of FL-AR and AR-Vs provided maximum antitumor efficacy in PTEN-null, enzalutamide-resistant CRPC. Clin Cancer Res; 22(11); 2744-54. ©2015 AACR.

Kato M ，Banuelos CA ，Imamura Y ，Leung JK ，Caley DP ，Wang J ，Mawji NR ，Sadar MD ... -  《-》

An androgen receptor N-terminal domain antagonist for treating prostate cancer.

Myung JK ，Banuelos CA ，Fernandez JG ，Mawji NR ，Wang J ，Tien AH ，Yang YC ，Tavakoli I ，Haile S ，Watt K ，McEwan IJ ，Plymate S ，Andersen RJ ，Sadar MD ... -  《-》

Bruceantin targets HSP90 to overcome resistance to hormone therapy in castration-resistant prostate cancer.

Rationale: Aberrant androgen receptor (AR) signaling via full-length AR (AR-FL) and constitutively active AR variant 7 (AR-V7) plays a key role in the development of castration-resistant prostate cancer (CRPC) and resistance to hormone therapies. Simultaneous targeting of AR-FL and AR-V7 may be a promising strategy to overcome resistance to hormone therapy. This study aimed to identify novel drug candidates co-targeting AR-FL and AR-V7 activities and elucidate their molecular mechanism of anti-CRPC activities. Methods: Using a CRPC cell-based reporter assay system, we screened a small library of antimalarial agents to explore the possibility of repositioning them for CRPC treatment and identified bruceantin (BCT) as a potent anti-CRPC drug candidate. A series of cell-based, molecular, biochemical, and in vivo approaches were performed to evaluate the therapeutic potential and molecular mechanism of BCT in CRPC. These approaches include reporter gene assays, cell proliferation, RNA-seq, qRT-PCR, mouse xenografts, co-immunoprecipitation, GST pull-down, immobilized BCT pull-down, molecular modeling, and bioinformatic analyses. Results: We identified BCT as a highly potent inhibitor co-targeting AR-FL and AR-V7 activity. BCT inhibits the transcriptional activity of AR-FL/AR-V7 and downregulates their target genes in CRPC cells. In addition, BCT efficiently suppresses tumor growth and metastasis of CRPC cells. Mechanistically, BCT disrupts the interaction of HSP90 with AR-FL/AR-V7 by directly binding to HSP90 and inhibits HSP90 chaperone function, leading to degradation of AR-FL/AR-V7 through the ubiquitin-proteasome system. Clinically, HSP90 expression is upregulated and correlated with AR/AR-V7 levels in CRPC. Conclusion: Our findings suggest that BCT could serve as a promising therapeutic candidate against CRPC and highlight the potential benefit of targeting AR-FL/AR-V7-HSP90 axis to overcome resistance caused by aberrant AR-FL/AR-V7 signaling.

Moon SJ ，Jeong BC ，Kim HJ ，Lim JE ，Kim HJ ，Kwon GY ，Jackman JA ，Kim JH ... -  《Theranostics》