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Targeting androgen signaling with the second-generation anti-androgen drugs, such as enzalutamide (Enza), abiraterone (Abi), apalutamide (Apal), and darolutamide (Daro), is the mainstay for the treatment of castration-resistant prostate cancer (CRPC). While these treatments are effective initially, resistance occurs frequently. Continued expression of androgen receptor (AR) and its variants such as AR-V7 despite AR-targeted therapy contributes to treatment resistance and cancer progression in advanced CRPC patients. This highlights the need for new strategies blocking continued AR signaling. Here, we identify a novel AR/AR-V7 degrader (ARVib) and found that ARVib effectively degrades AR/AR-V7 protein and attenuates AR/AR-V7 downstream target gene expression in prostate cancer cells. Mechanistically, ARVib degrades AR/AR-V7 protein through the ubiquitin-proteasome pathway mediated by HSP70/STUB1 machinery modulation. ARVib suppresses HSP70 expression and promotes STUB1 nuclear translocation, where STUB1 binds to AR/AR-V7 and promotes its ubiquitination and degradation. ARVib significantly inhibits resistant prostate tumor growth and improves enzalutamide treatment in vitro and in vivo. These data suggest that ARVib has potential for development as an AR/AR-V7 degrader to treat resistant CRPC.

Targeting androgen signaling with the second-generation anti-androgen drugs, such as enzalutamide (Enza), abiraterone (Abi), apalutamide (Apal), and darolutamide (Daro), is the mainstay for the treatment of castration-resistant prostate cancer (CRPC). While these treatments are effective initially, resistance occurs frequently. Continued expression of androgen receptor (AR) and its variants such as AR-V7 despite AR-targeted therapy contributes to treatment resistance and cancer progression in advanced CRPC patients. This highlights the need for new strategies blocking continued AR signaling. Here, we identify a novel AR/AR-V7 degrader (ARVib) and found that ARVib effectively degrades AR/AR-V7 protein and attenuates AR/AR-V7 downstream target gene expression in prostate cancer cells. Mechanistically, ARVib degrades AR/AR-V7 protein through the ubiquitin-proteasome pathway mediated by HSP70/STUB1 machinery modulation. ARVib suppresses HSP70 expression and promotes STUB1 nuclear translocation, where STUB1 binds to AR/AR-V7 and promotes its ubiquitination and degradation. ARVib significantly inhibits resistant prostate tumor growth and improves enzalutamide treatment in vitro and in vivo. These data suggest that ARVib has potential for development as an AR/AR-V7 degrader to treat resistant CRPC.

The mechanisms resulting in resistance to next-generation antiandrogens in castration-resistant prostate cancer are incompletely understood. Numerous studies have determined that constitutively active androgen receptor (AR) signaling or full-length AR bypass mechanisms may contribute to the resistance. Previous studies established that AKR1C3 and AR-V7 play important roles in enzalutamide and abiraterone resistance. In the present study, we found that AKR1C3 increases AR-V7 expression in resistant prostate cancer cells through enhancing protein stability via activation of the ubiquitin-mediated proteasome pathway. AKR1C3 reprograms AR signaling in enzalutamide-resistant prostate cancer cells. In addition, bioinformatical analysis of indomethacin-treated resistant cells revealed that indomethacin significantly activates the unfolded protein response, p53, and apoptosis pathways, and suppresses cell-cycle, Myc, and AR/ARV7 pathways. Targeting AKR1C3 with indomethacin significantly decreases AR/AR-V7 protein expression in vitro and in vivo through activation of the ubiquitin-mediated proteasome pathway. Our results suggest that the AKR1C3/AR-V7 complex collaboratively confers resistance to AR-targeted therapies in advanced prostate cancer.

Protein homeostasis (proteostasis) is a potential mechanism that contributes to cancer cell survival and drug resistance. Constitutively active androgen receptor (AR) variants confer anti-androgen resistance in advanced prostate cancer. However, the role of proteostasis involved in next generation anti-androgen resistance and the mechanisms of AR variant regulation are poorly defined. Here we show that the ubiquitin-proteasome-system (UPS) is suppressed in enzalutamide/abiraterone resistant prostate cancer. AR/AR-V7 proteostasis requires the interaction of E3 ubiquitin ligase STUB1 and HSP70 complex. STUB1 disassociates AR/AR-V7 from HSP70, leading to AR/AR-V7 ubiquitination and degradation. Inhibition of HSP70 significantly inhibits prostate tumor growth and improves enzalutamide/abiraterone treatments through AR/AR-V7 suppression. Clinically, HSP70 expression is upregulated and correlated with AR/AR-V7 levels in high Gleason score prostate tumors. Our results reveal a novel mechanism of anti-androgen resistance via UPS alteration which could be targeted through inhibition of HSP70 to reduce AR-V7 expression and overcome resistance to AR-targeted therapies.

Protein homeostasis (proteostasis) is a potential mechanism that contributes to cancer cell survival and drug resistance. Constitutively active androgen receptor (AR) variants confer anti-androgen resistance in advanced prostate cancer. However, the role of proteostasis involved in next generation anti-androgen resistance and the mechanisms of AR variant regulation are poorly defined. Here we show that the ubiquitin-proteasome-system (UPS) is suppressed in enzalutamide/abiraterone resistant prostate cancer. AR/AR-V7 proteostasis requires the interaction of E3 ubiquitin ligase STUB1 and HSP70 complex. STUB1 disassociates AR/AR-V7 from HSP70, leading to AR/AR-V7 ubiquitination and degradation. Inhibition of HSP70 significantly inhibits prostate tumor growth and improves enzalutamide/abiraterone treatments through AR/AR-V7 suppression. Clinically, HSP70 expression is upregulated and correlated with AR/AR-V7 levels in high Gleason score prostate tumors. Our results reveal a novel mechanism of anti-androgen resistance via UPS alteration which could be targeted through inhibition of HSP70 to reduce AR-V7 expression and overcome resistance to AR-targeted therapies.

Activation of the androgen receptor (AR) and its splice variants is linked to advanced prostate cancer and drives resistance to antiandrogens. The roles of AR and AR variants in the development of resistance to androgen deprivation therapy (ADT) and bicalutamide treatment, however, are still incompletely understood. To determine whether AR variants play a role in bicalutamide resistance, we developed bicalutamide-resistant LNCaP cells (LNCaP-BicR) and found that these resistant cells express significantly increased levels of AR variants, particularly AR-V7, both at the mRNA and protein levels. Exogenous expression of AR-V7 in bicalutamide-sensitive LNCaP cells confers resistance to bicalutamide treatment. Knockdown of AR-V7 in bicalutamide- and enzalutamide-resistant CWR22Rv1, enzalutamide-resistant C4-2B (C4-2B MDVR), and LNCaP-BicR cells reversed bicalutamide resistance. Niclosamide, a potent inhibitor of AR variants, significantly enhanced bicalutamide treatment. Niclosamide and bicalutamide combination treatment not only suppressed AR and AR variants expression and inhibited their recruitment to the PSA promoter, but also significantly induced apoptosis in bicalutamide- and enzalutamide-resistant CWR22Rv1 and C4-2B MDVR cells. In addition, combination of niclosamide with bicalutamide inhibited the growth of enzalutamide-resistant tumors. In summary, our results demonstrate that AR variants, particularly AR-V7, drive bicalutamide resistance and that targeting AR-V7 with niclosamide can resensitize bicalutamide-resistant cells to bicalutamide treatment. Furthermore, combination of niclosamide with bicalutamide inhibits enzalutamide resistant tumor growth, suggesting that the combination of niclosamide and bicalutamide could be a potential cost-effective strategy to treat advanced prostate cancer in patients, including those who fail to respond to enzalutamide therapy. Mol Cancer Ther; 16(8); 1521-30. ©2017 AACR.

Abiraterone suppresses intracrine androgen synthesis via inhibition of CYP17A1. However, clinical evidence suggests that androgen synthesis is not fully inhibited by abiraterone and the sustained androgen production may lead to disease relapse. In the present study, we identified AKR1C3, an important enzyme in the steroidogenesis pathway, as a critical mechanism driving resistance to abiraterone through increasing intracrine androgen synthesis and enhancing androgen signaling. We found that overexpression of AKR1C3 confers resistance to abiraterone while downregulation of AKR1C3 resensitizes resistant cells to abiraterone treatment. In abiraterone-resistant prostate cancer cells, AKR1C3 is overexpressed and the levels of intracrine androgens are elevated. In addition, AKR1C3 activation increases intracrine androgen synthesis and enhances androgen receptor (AR) signaling via activating AR transcriptional activity. Treatment of abiraterone-resistant cells with indomethacin, an AKR1C3 inhibitor, overcomes resistance and enhances abiraterone therapy both in vitro and in vivo by reducing the levels of intracrine androgens and diminishing AR transcriptional activity. These results demonstrate that AKR1C3 activation is a critical mechanism of resistance to abiraterone through increasing intracrine androgen synthesis and enhancing androgen signaling. Furthermore, this study provides a preclinical proof-of-principle for clinical trials investigating the combination of targeting AKR1C3 using indomethacin with abiraterone for advanced prostate cancer. Mol Cancer Ther; 16(1); 35-44. ©2016 AACR.

Considerable evidence from both clinical and experimental studies suggests that androgen receptor variants, particularly androgen receptor variant 7 (AR-V7), are critical in the induction of resistance to enzalutamide and abiraterone. In this study, we investigated the role of AR-V7 in the cross-resistance of enzalutamide and abiraterone and examined if inhibition of AR-V7 can improve abiraterone treatment response. We found that enzalutamide-resistant cells are cross-resistant to abiraterone, and that AR-V7 confers resistance to abiraterone. Knock down of AR-V7 by siRNA in abiraterone resistant CWR22Rv1 and C4-2B MDVR cells restored their sensitivity to abiraterone, indicating that AR-V7 is involved in abiraterone resistance. Abiraterone resistant prostate cancer cells generated by chronic treatment with abiraterone showed enhanced AR-V7 protein expression. Niclosamide, an FDA-approved antihelminthic drug that has been previously identified as a potent inhibitor of AR-V7, re-sensitizes resistant cells to abiraterone treatment in vitro and in vivo. In summary, this preclinical study suggests that overexpression of AR-V7 contributes to resistance to abiraterone, and supports the development of combination of abiraterone with niclosamide as a potential treatment for advanced castration resistant prostate cancer.

Considerable evidence from both clinical and experimental studies suggests that androgen receptor variants, particularly androgen receptor variant 7 (AR-V7), are critical in the induction of resistance to enzalutamide and abiraterone. In this study, we investigated the role of AR-V7 in the cross-resistance of enzalutamide and abiraterone and examined if inhibition of AR-V7 can improve abiraterone treatment response. We found that enzalutamide-resistant cells are cross-resistant to abiraterone, and that AR-V7 confers resistance to abiraterone. Knock down of AR-V7 by siRNA in abiraterone resistant CWR22Rv1 and C4-2B MDVR cells restored their sensitivity to abiraterone, indicating that AR-V7 is involved in abiraterone resistance. Abiraterone resistant prostate cancer cells generated by chronic treatment with abiraterone showed enhanced AR-V7 protein expression. Niclosamide, an FDA-approved antihelminthic drug that has been previously identified as a potent inhibitor of AR-V7, re-sensitizes resistant cells to abiraterone treatment in vitro and in vivo. In summary, this preclinical study suggests that overexpression of AR-V7 contributes to resistance to abiraterone, and supports the development of combination of abiraterone with niclosamide as a potential treatment for advanced castration resistant prostate cancer.

The introduction of enzalutamide and abiraterone has led to improvement in the treatment of metastatic castration-resistant prostate cancer. However, acquired resistance to enzalutamide and abiraterone therapies frequently develops within a short period in many patients. In the present study, we developed enzalutamide-resistant prostate cancer cells in an effort to understand the mechanisms of resistance. Global gene-expression analysis showed that the steroid biosynthesis pathway is activated in enzalutamide-resistant prostate cancer cells. One of the crucial steroidogenic enzymes, AKR1C3, was significantly elevated in enzalutamide-resistant cells. In addition, AKR1C3 is highly expressed in metastatic and recurrent prostate cancer and in enzalutamide-resistant prostate xenograft tumors. LC/MS analysis of the steroid metabolites revealed that androgen precursors such as cholesterol, DHEA and progesterone, as well as androgens are highly upregulated in enzalutamide-resistant prostate cancer cells compared to the parental cells. Knockdown of AKR1C3 expression by shRNA or inhibition of AKR1C3 enzymatic activity by indomethacin resensitized enzalutamide-resistant prostate cancer cells to enzalutamide treatment both in vitro and in vivo. In contrast, overexpression of AKR1C3 confers resistance to enzalutamide. Furthermore, the combination of indomethacin and enzalutamide resulted in significant inhibition of enzalutamide-resistant tumor growth. These results suggest that AKR1C3 activation is a critical resistance mechanism associated with enzalutamide resistance; targeting intracrine androgens and AKR1C3 will overcome enzalutamide resistance and improve survival of advanced prostate cancer patients.

PurposeIt is known that over expression of IL6 in prostate cancer cells confer enzalutamide resistance and that this may occur through constitutive Stat3 activation. Additionally, recent pre-clinical studies suggested enzalutamide might have the potential adverse effect of inducing metastasis of prostate cancer cells via Stat3 activation. This study is aimed to target Stat3 activation and improve enzalutamide therapy.Experimental designSensitivity of prostate cancer cells to enzalutamide was tested using cell growth assays and clonogenic assays. Wound healing and invasion assays were performed to determine cell migration and invasion in vitro. Quantitative reverse transcription-PCR, ELISA and Western blotting were performed to detect expression levels of PSA, c-Myc, survivin, Stat3, and AR. ChIP assay was performed to examine recruitment of AR to the PSA promoter.ResultsIn the present study, we found niclosamide, a previously identified novel inhibitor of androgen receptor variant (AR-V7), inhibited Stat3 phosphorylation, and expression of downstream target genes. Niclosamide synergistically reversed enzalutamide resistance in prostate cancer cells and combination treatment of niclosamide with enzalutamide significantly induced cell apoptosis and inhibited cell growth, colony formation, cell migration and invasion. Knock down of Stat3 abrogated enzalutamide resistance resulting in reduced recruitment of AR to the PSA promoter in prostate cancer cells expressing IL6. Moreover, niclosamide reversed enzalutamide resistance by down-regulating Stat3 target gene expression Stat3and abrogating recruitment of AR to PSA promoter resulting in PSA inhibition.ConclusionsThis study demonstrated the IL6-Stat3-AR axis in prostate cancer is one of the crucial mechanisms of enzalutamide resistance. Niclosamide has the potential to target the IL6-Stat3-AR pathway to overcome enzalutamide resistance and inhibit migration and invasion in advanced prostate cancer.

PurposeEnzalutamide, a second-generation antiandrogen, was recently approved for the treatment of castration-resistant prostate cancer (CRPC) in patients who no longer respond to docetaxel. Despite these advances that provide temporary respite, resistance to enzalutamide occurs frequently. Androgen receptor (AR) splice variants such as AR-V7 have recently been shown to drive castration-resistant growth and resistance to enzalutamide. This study was designed to identify inhibitors of AR variants and test its ability to overcome resistance to enzalutamide.Experimental designThe drug screening was conducted using luciferase activity assay to determine the activity of AR-V7 after treatment with the compounds in the Prestwick Chemical Library, which contains about 1,120 FDA-approved drugs. The effects of the identified inhibitors on AR-V7 activity and enzalutamide sensitivity were characterized in CRPC and enzalutamide-resistant prostate cancer cells in vitro and in vivo.ResultsNiclosamide, an FDA-approved antihelminthic drug, was identified as a potent AR-V7 inhibitor in prostate cancer cells. Niclosamide significantly downregulated AR-V7 protein expression by protein degradation through a proteasome-dependent pathway. Niclosamide also inhibited AR-V7 transcription activity and reduced the recruitment of AR-V7 to the PSA promoter. Niclosamide inhibited prostate cancer cell growth in vitro and tumor growth in vivo. Furthermore, the combination of niclosamide and enzalutamide resulted in significant inhibition of enzalutamide-resistant tumor growth, suggesting that niclosamide enhances enzalutamide therapy and overcomes enzalutamide resistance in CRPC cells.ConclusionsNiclosamide was identified as a novel inhibitor of AR variants. Our findings offer preclinical validation of niclosamide as a promising inhibitor of AR variants to treat, either alone or in combination with current antiandrogen therapies, patients with advanced prostate cancer, especially those resistant to

PurposeUse of enzalutamide has improved the treatment of advanced prostate cancer. However, resistance to enzalutamide can develop frequently in initial responders. This study aimed to test whether overexpression of IL-6 and constitutive activation of Stat3 in prostate cancer cells increase resistance to enzalutamide.Experimental designSensitivity of prostate cancer cells to enzalutamide was tested using cell growth assays and clonogenic assays. Quantitative reverse transcription-PCR, ELISA, and Western blotting were performed to detect expression levels of IL-6, c-Myc, survivin, and AR. Expression of Stat3 was downregulated using siRNA specific to Stat3. ChIP assay was performed to examine recruitment of AR to the PSA promoter.ResultsProstate cancer cells expressing autocrine IL-6 are resistant to enzalutamide and autocrine IL-6 leads to constitutive activation of Stat3 and its target genes. Down regulation of Stat3 led to an increase in sensitivity of prostate cancer cells to enzalutamide. Overexpression of constitutively active Stat3 in prostate cancer cells induced resistance to enzalutamide treatment. Constitutively active Stat3 also enhanced the recruitment of AR to PSA promoter which could not be disrupted by enzalutamide. The Stat3 inhibitor AG490 reversed enzalutamide resistance in prostate cancer cells, while combination treatment with enzalutamide and AG490 significantly inhibited cell growth and induced cell apoptosis.ConclusionsThis study demonstrates that the autocrine IL-6 pathway induces enzalutamide resistance in prostate cancer cells via the constitutive activation of Stat3. Co-targeting IL6-Stat3 pathway with enzalutamide may be utilized for treatment of advanced prostate cancer.