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SignificanceOlaparib, a PARP inhibitor, is effective against various cancers, including prostate cancer. However, resistance to olaparib poses a significant challenge. This study uncovers that mitochondrial alterations and PINK1 gene overexpression contribute to this resistance in prostate cancer cells. Enhanced mitochondrial functionality and increased PINK1 expression in olaparib-resistant cells underscore the importance of targeting mitochondrial dynamics and PINK1 to develop more effective treatments for overcoming olaparib resistance in prostate cancer.

Olaparib is a pioneering PARP inhibitor (PARPi) approved for treating castration-resistant prostate cancer (CRPC) tumors harboring DNA repair defects, but clinical resistance has been documented. To study acquired resistance, we developed Olaparib-resistant (OlapR) cell lines through chronic Olaparib treatment of LNCaP and C4-2B cell lines. Here, we found that IGFBP3 is highly expressed in acquired (OlapR) and intrinsic (Rv1) models of Olaparib resistance. We show that IGFBP3 expression promotes Olaparib resistance by enhancing DNA repair capacity through activation of EGFR and DNA-PKcs. IGFBP3 depletion enhances efficacy of Olaparib by promoting DNA damage accumulation and subsequently, cell death in resistant models. Mechanistically, we show that silencing IGFBP3 or EGFR expression reduces cell viability and resensitizes OlapR cells to Olaparib treatment. Inhibition of EGFR by Gefitinib suppressed growth of OlapR cells and improved Olaparib sensitivity, thereby phenocopying IGFBP3 inhibition. Collectively, our results highlight IGFBP3 and EGFR as critical mediators of Olaparib resistance.

Current common treatments for castration-resistant prostate cancer (CRPC) typically belong to one of three major categories: next-generation anti-androgen therapies (NGAT) including enzalutamide, abiraterone acetate, apalutamide, and darolutamide; taxane therapy represented by docetaxel; and PARP inhibitors (PARPi) like olaparib. Although these treatments have shown efficacy and have improved outcomes for many patients, some do not survive due to the emergence of therapeutic resistance. The clinical landscape is further complicated by limited knowledge about how the sequence of treatments impacts the development of therapeutic cross-resistance in CRPC. We have developed multiple CRPC models of acquired therapeutic resistance cell sublines from C4-2B cells. These include C4-2B MDVR, C4-2B AbiR, C4-2B ApaR, C4-2B DaroR, TaxR, and 2B-olapR, which are resistant to enzalutamide, abiraterone, apalutamide, darolutamide, docetaxel, and olaparib, respectively. These models are instrumental for analyzing gene expression and assessing responses to various treatments. Our findings reveal distinct cross-resistance characteristics among NGAT-resistant cell sublines. Specifically, resistance to enzalutamide induces resistance to abiraterone and vice versa, while maintaining sensitivity to taxanes and olaparib. Conversely, cells with acquired resistance to docetaxel exhibit cross-resistance to both cabazitaxel and olaparib but retain sensitivity to NGATs like enzalutamide and abiraterone. OlapR cells, significantly resistant to olaparib compared to parental cells, are still responsive to NGATs and docetaxel. Moreover, OlapR models display cross-resistance to other clinically relevant PARP inhibitors, including rucaparib, niraparib, and talazoparib. RNA-sequencing analyses have revealed a complex network of altered gene expressions that influence signaling pathways, energy metabolism, and apoptotic signaling, pivotal to cancer's evolution and progression. The data indicate that resistance mechanisms are distinct among different drug classes. Notably, NGAT-resistant sublines exhibited a significant downregulation of androgen-regulated genes, contrasting to the stable expression noted in olaparib and docetaxel-resistant sublines. These results may have clinical implications by showing that treatments of one class can be sequenced with those from another class, but caution should be taken when sequencing drugs of the same class.

The next-generation antiandrogen drugs such as enzalutamide and abiraterone extend survival times and improve quality of life in patients with advanced prostate cancer. However, resistance to both drugs occurs frequently through mechanisms that are incompletely understood. Wnt signaling, particularly through Wnt5a, plays vital roles in promoting prostate cancer progression and induction of resistance to enzalutamide and abiraterone. Development of novel strategies targeting Wnt5a to overcome resistance is an urgent need. In this study, we demonstrated that Wnt5a/FZD2-mediated noncanonical Wnt pathway is overexpressed in enzalutamide-resistant prostate cancer. In patient databases, both the levels of Wnt5a and FZD2 expression are upregulated upon the development of enzalutamide resistance and correlate with higher Gleason score, biochemical recurrence, and metastatic status, and with shortened disease-free survival duration. Blocking Wnt5a/FZD2 signal transduction not only diminished the activation of noncanonical Wnt signaling pathway, but also suppressed the constitutively activated androgen receptor (AR) and AR variants. Furthermore, we developed a novel bioengineered BERA-Wnt5a siRNA construct and demonstrated that inhibition of Wnt5a expression by the BERA-Wnt5a siRNA significantly suppressed tumor growth and enhanced enzalutamide treatment in vivo. These results indicate that Wnt5a/FZD2 signal pathway plays a critical role in promoting enzalutamide resistance, and targeting this pathway by BERA-Wnt5a siRNA can be developed as a potential therapy to treat advanced prostate cancer.

PARP inhibition represents the dawn of precision medicine for treating prostate cancer. Despite this advance, questions remain regarding the use of PARP inhibitors (PARPi) for the treatment of this disease, including (i) how specifically do PARPi-sensitive tumor cells respond to treatment, and (ii) how does PARPi resistance develop? To address these questions, we characterized response to olaparib in sensitive LNCaP and C4-2B cells and developed two olaparib-resistant derivative cell line models from each, termed LN-OlapR and 2B-OlapR, respectively. OlapR cells possess distinct morphology from parental cells and display robust resistance to olaparib and other clinically relevant PARPis, including rucaparib, niraparib, and talazoparib. In LNCaP and C4-2B cells, we found that olaparib induces massive DNA damage, leading to activation of the G2-M checkpoint, activation of p53, and cell-cycle arrest. Furthermore, our data suggest that G2-M checkpoint activation leads to both cell death and senescence associated with p21 activity. In contrast, both LN-OlapR and 2B-OlapR cells do not arrest at G2-M and display a markedly blunted response to olaparib treatment. Interestingly, both OlapR cell lines harbor increased DNA damage relative to parental cells, suggesting that OlapR cells accumulate and manage persistent DNA damage during acquisition of resistance, likely through augmenting DNA repair capacity. Further impairing DNA repair through CDK1 inhibition enhances DNA damage, induces cell death, and sensitizes OlapR cells to olaparib treatment. Our data together further our understanding of PARPi treatment and provide a cellular platform system for the study of response and resistance to PARP inhibition.

Resistance to androgen receptor (AR) targeted therapies remains as the main reason for most prostate cancer related deaths. Lineage plasticity resulting in altered, treatment insensitive prostate tumor cell phenotypes such neuroendocrine differentiated prostate cancer is a common manifestation within resistant tumors upon AR-targeted therapies. The mechanisms responsible for lineage plasticity in prostate cancer remain incompletely understood. Here we demonstrate that the enzalutamide resistant MDVR cell line possesses lineage plastic characteristics associated with overexpression of the Wnt transporter Wntless (WLS). Furthermore, we present evidence that overexpression of WLS is common in varying cell line models of lineage plastic prostate cancer, is higher in neuroendocrine patient samples, and positively correlates with the neuroendocrine marker SYP in clinical data. Targeting WLS in lineage plastic cellular models reduces viability and represses lineage plasticity associated gene expression. Our study provides insight into the importance of WLS to the development of lethal resistant prostate cancer and provides a potential target for the treatment of advanced disease.

BackgroundTreatment-emergent neuroendocrine prostate cancer (NEPC) after androgen receptor (AR) targeted therapies is an aggressive variant of prostate cancer with an unfavorable prognosis. The underlying mechanisms for early neuroendocrine differentiation are poorly defined and diagnostic and prognostic biomarkers are needed.MethodsWe performed transcriptomic analysis on the enzalutamide-resistant prostate cancer cell line C4-2B MDVR and NEPC patient databases to identify neural lineage signature (NLS) genes. Correlation of NLS genes with clinicopathologic features was determined. Cell viability was determined in C4-2B MDVR and H660 cells after knocking down ARHGEF2 using siRNA. Organoid viability of patient-derived xenografts was measured after knocking down ARHGEF2.ResultsWe identify a 95-gene NLS representing the molecular landscape of neural precursor cell proliferation, embryonic stem cell pluripotency, and neural stem cell differentiation, which may indicate an early or intermediate stage of neuroendocrine differentiation. These NLS genes positively correlate with conventional neuroendocrine markers such as chromogranin and synaptophysin, and negatively correlate with AR and AR target genes in advanced prostate cancer. Differentially expressed NLS genes stratify small-cell NEPC from prostate adenocarcinoma, which are closely associated with clinicopathologic features such as Gleason Score and metastasis status. Higher ARGHEF2, LHX2, and EPHB2 levels among the 95 NLS genes correlate with a shortened survival time in NEPC patients. Furthermore, downregulation of ARHGEF2 gene expression suppresses cell viability and markers of neuroendocrine differentiation in enzalutamide-resistant and neuroendocrine cells.ConclusionsThe 95 neural lineage gene signatures capture an early molecular shift toward neuroendocrine differentiation, which could stratify advanced prostate cancer patients to optimize clinical treatment and serve as a source of potential therapeutic targets in advanced prostate cancer.

Docetaxel and cabazitaxel-based taxane chemotherapy are critical components in the management of advanced prostate cancer. However, their efficacy is hindered due to de novo presentation with or the development of resistance. Characterizing models of taxane-resistant prostate cancer will lead to creation of strategies to overcome insensitivity. We have previously characterized docetaxel-resistant C4-2B and DU145 cell line derivatives, TaxR and DU145-DTXR, respectively. In the present study, we characterize cabazitaxel-resistant derivative cell lines created from chronic cabazitaxel exposure of TaxR and DU145-DTXR cells, CabR and CTXR, respectively. We show that CabR and CTXR cells are robustly resistant to both taxanes but retain sensitivity to antiandrogens. Both CabR and CTXR cells possess increased expression of ABCB1, which is shown to mediate resistance to treatment. Interestingly, we also present evidence for coordinated overexpression of additional genes present within the 7q21.12 gene locus where ABCB1 resides. This locus, known as the ABCB1 amplicon, has been demonstrated to be amplified in multidrug-resistant tumor cells, but little is known regarding its role in prostate cancer. We show that two ABCB1-amplicon genes other than ABCB1, RUNDC3B and DBF4, promote cellular viability and treatment resistance in taxane-resistant prostate cancer models. We present evidence that coordinated amplification of ABCB1-amplicon genes is common in a subset of prostate cancer patients. These data together suggest that ABCB1-amplicon activation plays a critical role in taxane resistance.

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.

PurposeMost patients with prostate cancer receiving enzalutamide or abiraterone develop resistance. Clinical evidence indicates that serum levels of dehydroepiandrosterone sulfate (DHEAS) and biologically active DHEA remain in the high range despite antiandrogen treatment. The conversion of DHEAS into DHEA by steroid sulfatase (STS) may contribute to sustained intracrine androgen synthesis. Here, we determine the contribution of STS to treatment resistance and explore the potential of targeting STS to overcome resistance in prostate cancer.Experimental designSTS expression was examined in patients and cell lines. In vitro, STS activity and expression were modulated using STS-specific siRNA or novel STS inhibitors (STSi). Cell growth, colony formation, androgen production, and gene expression were examined. RNA-sequencing analysis was conducted on VCaP cells treated with STSi. Mice were treated with STSis with or without enzalutamide to determine their effects in vivo.ResultsSTS is overexpressed in patients with castration-resistant prostate cancer (CRPC) and resistant cells. STS overexpression increases intracrine androgen synthesis, cell proliferation, and confers resistance to enzalutamide and abiraterone. Inhibition of STS using siRNA suppresses prostate cancer cell growth. Targeting STS activity using STSi inhibits STS activity, suppresses androgen receptor transcriptional activity, and reduces the growth of resistant C4-2B and VCaP prostate cancer cells. STSis significantly suppress resistant VCaP tumor growth, decrease serum PSA levels, and enhance enzalutamide treatment in vitro and in vivo.ConclusionsThese studies suggest that STS drives intracrine androgen synthesis and prostate cancer proliferation. Targeting STS represents a therapeutic strategy to treat CRPC and improve second-generation antiandrogen therapy.

The next-generation antiandrogen drugs, XTANDI (enzalutamide), ZYTIGA (abiraterone acetate), ERLEADA (apalutamide) and NUBEQA (darolutamide) extend survival times and improve quality of life in patients with advanced prostate cancer. Despite these advances, resistance occurs frequently and there is currently no definitive cure for castration-resistant prostate cancer. Our previous studies identified that similar mechanisms of resistance to enzalutamide or abiraterone occur following treatment and cross-resistance exists between these therapies in advanced prostate cancer. Here, we show that enzalutamide- and abiraterone-resistant prostate cancer cells are further cross-resistant to apalutamide and darolutamide. Mechanistically, we have determined that the AKR1C3/AR-V7 axis confers this cross-resistance. Knockdown of AR-V7 in enzalutamide-resistant cells resensitize cells to apalutamide and darolutamide treatment. Furthermore, targeting AKR1C3 resensitizes resistant cells to apalutamide and darolutamide treatment through AR-V7 inhibition. Chronic apalutamide treatment in C4-2B cells activates the steroid hormone biosynthesis pathway and increases AKR1C3 expression, which confers resistance to enzalutamide, abiraterone, and darolutamide. In conclusion, our results suggest that apalutamide and darolutamide share similar resistant mechanisms with enzalutamide and abiraterone. The AKR1C3/AR-V7 complex confers cross-resistance to second-generation androgen receptor-targeted therapies in advanced prostate cancer.

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.

Castration-resistant prostate cancer remains as an incurable disease. Exploiting DNA damage repair defects via inhibition of poly (ADP-ribose) polymerase (PARP) is becoming an attractive therapeutic option. The TOPARP-A clinical trial demonstrated that the PARP inhibitor olaparib may be an effective strategy for treating prostate cancer. However, several unanswered questions regarding the use of olaparib remain: 1) How do we best stratify patients for olaparib treatment? 2) Where do we place olaparib in the treatment sequence paradigm? 3) Is there cross-resistance between olaparib and currently used therapies? Here, we tested putative cross-resistance between current therapies and olaparib in treatment-resistant castration-resistant prostate cancer models. Docetaxel-resistant cells exhibited robust resistance to olaparib which could be attributed to blunted PARP trapping in response to olaparib treatment. Upregulated ABCB1 mediates cross-resistance between taxanes and olaparib, which can be overcome through decreasing ABCB1 expression or inhibiting ABCB1 using elacridar or enzalutamide. We also show that combining olaparib with enzalutamide is more effective in olaparib-sensitive cells than either single agent. Our results demonstrate that cross-resistance between olaparib and other therapies could blunt response to treatment and highlight the need to develop strategies to maximize olaparib efficacy.

BackgroundDe-regulation of Wnt signaling pathways has been shown to be associated with progression of castration-resistant prostate cancer and more recently, studies indicate that both canonical and non-canonical Wnt pathways may mediate resistance to anti-androgen therapies such as enzalutamide. However, the mechanisms by which Wnt signaling is altered in prostate cancer remain poorly understood. Wnt pathway function begins with Wnt biogenesis and secretion from Wnt signal sending cells. While previous studies have investigated downstream mechanisms of Wnt pathway alterations in prostate cancer, little is known on the role of Wnt secretion mediating proteins. Wntless (WLS) is thought to be essential for the secretion of all Wnts. In this study, we sought to understand the role of WLS in prostate cancer.MethodsRNA-seq and gene set enrichment analysis were used to understand expression profile changes in enzalutamide-resistant C4-2B-MDVR (MDVR) cells versus parental C4-2B cells. Quantitative-PCR and western blot were used to confirm RNA-seq data and to assess expression changes of gene targets of interest. Rv1 cells were used as a separate model of enzalutamide-resistant prostate cancer. RNAi was used to inhibit WLS expression. Cell viability, colony formation, and PSA ELISA assays were used to assess cell growth and survival.ResultsTranscriptomic profiling revealed enriched Wnt pathway signatures in MDVR versus parental C4-2B cells. We further show that MDVR cells upregulate Wnt signaling and overexpress WLS. Inhibition of WLS decreases Wnt signaling, markedly attenuates prostate cancer cell viability, induces apoptosis, and re-sensitizes enzalutamide-resistant cells to enzalutamide treatment. Lastly, we show that inhibition of WLS reduces AR and AR-variants expression and downstream signaling.ConclusionsOur findings support a role for WLS in the progression of prostate cancer to a treatment-resistant state. Further efforts to understand Wnt signaling pathway alterations in this disease may lead to the development of novel treatments.

Current treatments for castration resistant prostate cancer (CRPC) largely fall into two classes: androgen receptor (AR)-targeted therapies such as the next-generation antiandrogen therapies (NGAT), enzalutamide and abiraterone, and taxanes such as docetaxel and cabazitaxel. Despite improvements in outcomes, patients still succumb to the disease due to the development of resistance. Further complicating the situation is lack of a well-defined treatment sequence and potential for cross-resistance between therapies. We have developed several models representing CRPC with acquired therapeutic resistance. Here, we utilized these models to assess putative cross-resistance between treatments. We find that resistance to enzalutamide induces resistance to abiraterone and vice versa, but resistance to neither alters sensitivity to taxanes. Acquired resistance to docetaxel induces cross-resistance to cabazitaxel but not to enzalutamide or abiraterone. Correlating responses with known mechanisms of resistance indicates that AR variants are associated with resistance to NGATs, whereas the membrane efflux protein ABCB1 is associated with taxane resistance. Mechanistic studies show that AR variant-7 (AR-v7) is involved in NGAT resistance but not resistance to taxanes. Our findings suggest the existence of intra cross-resistance within a drug class (i.e., within NGATs or within taxanes), whereas inter cross-resistance between drug classes does not develop. Furthermore, our data suggest that resistance mechanisms differ between drug classes. These results may have clinical implications by showing that treatments of one class can be sequenced with those of another, but caution should be taken when sequencing similar classed drugs. In addition, the development and use of biomarkers indicating resistance will improve patient stratification for treatment. Mol Cancer Ther; 17(10); 2197-205. ©2018 AACR.

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.

Prostate cancer remains dependent on androgen receptor signaling even after castration. Aberrant androgen receptor signaling in castration-resistant prostate cancer is mediated by mechanisms such as alterations in the androgen receptor and activation of interacting signaling pathways. Clinical evidence confirms that resistance to the next-generation antiandrogen, enzalutamide, may be mediated to a large extent by alternative splicing of the androgen receptor to generate constitutively active splice variants such as AR-V7. The splice variants AR-V7 and ARv567es have been implicated in the resistance to not only enzalutamide, but also to abiraterone and other conventional therapeutics such as taxanes. Numerous studies, including ours, suggest that splicing factors such as hnRNPA1 promote the generation of AR-V7, thus contributing to enzalutamide resistance in prostate cancer cells. In the present study, we discovered that quercetin, a naturally occurring polyphenolic compound, reduces the expression of hnRNPA1, and consequently, that of AR-V7. The suppression of AR-V7 by quercetin resensitizes enzalutamide-resistant prostate cancer cells to treatment with enzalutamide. Our results indicate that quercetin downregulates hnRNPA1 expression, downregulates the expression of AR-V7, antagonizes androgen receptor signaling, and resensitizes enzalutamide-resistant prostate cancer cells to enzalutamide treatment in vivo in mouse xenografts. These findings demonstrate that suppressing the alternative splicing of the androgen receptor may have important implications in overcoming the resistance to next-generation antiandrogen therapy. Mol Cancer Ther; 16(12); 2770-9. ©2017 AACR.

Advancements in research have added several new therapies for castration-resistant prostate cancer (CRPC), greatly augmenting our ability to treat patients. However, CRPC remains an incurable disease due to the development of therapeutic resistance and the existence of cross-resistance between available therapies. Understanding the interplay between different treatments will lead to improved sequencing and the creation of combinations that overcome resistance and prolong survival. Whether there exists cross-resistance between docetaxel and the next-generation taxane cabazitaxel is poorly understood. In this study, we use C4-2B and DU145 derived docetaxel-resistant cell lines to test response to cabazitaxel. Our results demonstrate that docetaxel resistance confers cross-resistance to cabazitaxel. We show that increased ABCB1 expression is responsible for cross-resistance to cabazitaxel and that inhibition of ABCB1 function through the small-molecule inhibitor elacridar resensitizes taxane-resistant cells to treatment. In addition, the antiandrogens bicalutamide and enzalutamide, previously demonstrated to be able to resensitize taxane-resistant cells to docetaxel through inhibition of ABCB1 ATPase activity, are also able to resensitize resistant cells to cabazitaxel treatment. Finally, we show that resensitization using an antiandrogen is far more effective in combination with cabazitaxel than docetaxel. Collectively, these results address key concerns in the field, including that of cross-resistance between taxanes and highlighting a mechanism of cabazitaxel resistance involving ABCB1. Furthermore, these preclinical studies suggest the potential in using combinations of antiandrogens with cabazitaxel for increased effect in treating advanced CRPC. Mol Cancer Ther; 16(10); 2257-66. ©2017 AACR.

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.

BackgroundDocetaxel is one of the primary drugs used for treating castration resistant prostate cancer (CRPC). Unfortunately, over time patients invariably develop resistance to docetaxel therapy and their disease will continue to progress. The mechanisms by which resistance develops are still incompletely understood. This study seeks to determine the involvement of miRNAs, specifically miR-181a, in docetaxel resistance in CRPC.MethodsReal-time PCR was used to measure miR-181a expression in parental and docetaxel resistant C4-2B and DU145 cells (TaxR and DU145-DTXR). miR-181a expression was modulated in parental or docetaxel resistant cells by transfecting them with miR-181a mimics or antisense, respectively. Following transfection, cell number was determined after 48 h with or without docetaxel. Cross resistance to cabazitaxel induced by miR-181a was also determined. Western blots were used to determine ABCB1 protein expression and rhodamine assays used to assess activity. Phospho-p53 expression was assessed by Western blot and apoptosis was measured by ELISA in C4-2B TaxR and PC3 cells with inhibited or overexpressed miR-181a expression with or without docetaxel.ResultsmiR-181a is significantly overexpressed in TaxR and DU145-DTXR cells compared to parental cells. Overexpression of miR-181a in parental cells confers docetaxel and cabazitaxel resistance and knockdown of miR-181a in TaxR cells re-sensitizes them to treatment with both docetaxel and cabazitaxel. miR-181a was not observed to impact ABCB1 expression or activity, a protein which was previously demonstrated to be highly involved in docetaxel resistance. Knockdown of miR-181a in TaxR cells induced phospho-p53 expression. Furthermore, miR-181a knockdown alone induced apoptosis in TaxR cells which could be further enhanced by the addition of DTX.ConclusionsOverexpression of mir-181a in prostate cancer cells contributes to their resistance to docetaxel and cabazitaxel and inhibition of mir-181a expression can restore treatment response. This is due, in part, to modulation of p53 phosphorylation and apoptosis.

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.

BackgroundProstate cancer (PCa) is androgen-dependent initially and progresses to a castration-resistant state after androgen deprivation therapy. Treatment options for castration-resistant PCa include the potent second-generation anti-androgen enzalutamide or CYP17A1 inhibitor abiraterone. Recent clinical observations point to the development of resistance to these therapies which may be mediated by constitutively active alternative splice variants of the androgen receptor (AR).MethodsSensitivity of LNCaP cells overexpressing Lin28 (LN-Lin28) to enzalutamide, abiraterone, or bicalutamide was compared to that of control LN-neo cells using cell growth assays, proliferation assays using MTT, anchorage-dependent clonogenic ability assays and soft agar assays. Ability of LN-Lin28 cells to maintain AR activation after treatment with enzalutamide, abiraterone, or bicalutamide was tested using immunofluorescence, Western blotting, ChIP assays, and qRT-PCR. Importance of Lin28 in enzalutamide resistance was assessed by the downregulation of Lin28 expression in C4-2B and 22Rv1 cells chronically treated with enzalutamide. Requirement for sustained AR signaling in LN-Lin28 cells was examined by the downregulation of either full length AR or AR-V7 using siRNA.ResultsWe show that Lin28 promotes the development of resistance to currently used targeted therapeutics by enhancing the expression of AR splice variants such as AR-V7. PCa cells overexpressing Lin28 exhibit resistance to treatment with enzalutamide, abiraterone, or bicalutamide. Downregulation of Lin28 resensitizes enzalutamide-resistant PCa cells to enzalutamide treatment. We also show that the upregulation of splicing factors such as hnRNPA1 by Lin28 may mediate the enhanced generation of AR splice variants in Lin28-expressing cells.ConclusionsOur findings suggest that Lin28 plays a key role in the acquisition of resistance to AR-targeted therapies by PCa cells and establish the importance of Lin28 in PCa progression.

PurposePrevious studies show that inhibition of ABCB1 expression overcomes acquired docetaxel resistance in C4-2B-TaxR cells. In this study, we examined whether antiandrogens, such as bicalutamide and enzalutamide, could inhibit ABCB1 activity and overcome resistance to docetaxel.Experimental designABCB1 efflux activity was determined using a rhodamine efflux assay. ABCB1 ATPase activity was determined by Pgp-Glo assay systems. The effects of the antiandrogens bicalutamide and enzalutamide on docetaxel sensitivity were determined by cell growth assays and tumor growth in vivo.ResultsWe found that bicalutamide and enzalutamide inhibit ABCB1 ATP-binding cassette transporter activity through blocking ABCB1 efflux activity. Bicalutamide inhibited ABCB1 efflux activity by 40%, whereas enzalutamide inhibited ABCB1 efflux activity by approximately 60%. Both bicalutamide and enzalutamide inhibit ABCB1 ATPase activity. In addition, bicalutamide and enzalutamide inhibit ABCB1 efflux activity and desensitize docetaxel-resistant and androgen receptor (AR)-negative DU145 cells. Combination of bicalutamide with docetaxel had a significant antitumor effect in both AR-positive and AR-negative docetaxel-resistant xenograft models, suggesting that bicalutamide desensitizes docetaxel-resistant cells to docetaxel treatment independent of AR status.ConclusionsWe identified a novel mechanism of action for antiandrogens such as bicalutamide and enzalutamide as inhibitors of ABCB1 efflux and ATPase activity. Bicalutamide and enzalutamide desensitize docetaxel-resistant prostate cancer cells to docetaxel treatment independent of AR status. These studies may lead to the development of combinational therapies with bicalutamide/enzalutamide and docetaxel as effective regimens to treat advanced prostate cancer independent of AR status, and possibly other types of cancer.

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.

Castration-resistant prostate cancer (CRPC) remains dependent on androgen receptor (AR) signaling. Alternative splicing of the AR to generate constitutively active, ligand-independent variants is one of the principal mechanisms that promote the development of resistance to next-generation antiandrogens such as enzalutamide. Here, we demonstrate that the splicing factor heterogeneous nuclear RNA-binding protein A1 (hnRNPA1) plays a pivotal role in the generation of AR splice variants such as AR-V7. hnRNPA1 is overexpressed in prostate tumors compared with benign prostates, and its expression is regulated by NF-κB2/p52 and c-Myc. CRPC cells resistant to enzalutamide exhibit higher levels of NF-κB2/p52, c-Myc, hnRNPA1, and AR-V7. Levels of hnRNPA1 and AR-V7 are positively correlated with each other in prostate cancer. The regulatory circuit involving NF-κB2/p52, c-Myc, and hnRNPA1 plays a central role in the generation of AR splice variants. Downregulation of hnRNPA1 and consequently of AR-V7 resensitizes enzalutamide-resistant cells to enzalutamide, indicating that enhanced expression of hnRNPA1 may confer resistance to AR-targeted therapies by promoting the generation of splice variants. These findings may provide a rationale for cotargeting these pathways to achieve better efficacy through AR blockade.

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.

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 enzalutamide.

Cancer cells reprogram their metabolic pathways to facilitate fast proliferation. Previous studies have shown that overexpression of NF-κB2/p52 (p52) in prostate cancer cells promotes cell growth and leads to castration resistance through aberrant activation of androgen receptor (AR). In addition, these cells become resistant to enzalutamide. In this study, we investigated the effects of p52 activation on glucose metabolism and on response to enzalutamide therapy. Data analysis of gene expression arrays showed that genes including GLUT1 (SLC2A1), PKM2, G6PD, and ME1 involved in the regulation of glucose metabolism were altered in LNCaP cells overexpressing p52 compared with the parental LNCaP cells. We demonstrated an increased amount of glucose flux in the glycolysis pathway, as well as the pentose phosphate pathway (PPP) upon p52 activation. The p52-overexpressing cells increase glucose uptake and are capable of higher ATP and lactate production compared with the parental LNCaP cells. The growth of p52-overexpressing cells depends on glucose in the culture media and is sensitive to glucose deprivation compared with the parental LNCaP cells. Targeting glucose metabolism by the glucose analog 2-deoxy-d-glucose synergistically inhibits cell growth when combined with enzalutamide, and resensitizes p52-overexpressing cells to enzalutamide treatment. These results suggest that p52 modulates glucose metabolism, enhances glucose flux to glycolysis and PPPs, thus facilitating fast proliferation of the cells. Co-targeting glucose metabolism together with AR axis synergistically inhibits cell growth and restores enzalutamide-resistant cells to enzalutamide treatment.

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.

IntroductionTreatment of primary prostate cancer (CaP) is the withdrawal of androgens. However, CaP eventually progresses to grow in a castration-resistant state due to aberrant activation of androgen receptor (AR). Understanding the mechanisms leading to the aberrant activation of AR is critical to develop effective therapy. We have previously identified Rho GDP Dissociation Inhibitor alpha (GDIα) as a novel suppressor in prostate cancer. In this study, we examine the effect of GDIα on AR signaling in prostate cancer cells.MethodsGDIα was transiently or stably transfected into several prostate cancer cell lines including LNCaP, C4-2, CWR22Rv1, and DU145. The regulation of AR expression by GDIα was analyzed by qRT-PCR and Western blot. AR activity was measured by luciferase reporter assays and electrophoretic mobility shift analysis (EMSA). Immunofluorescence assay was performed to study AR nuclear translocation. The interaction between GDIα and AR was examined by co-immunoprecipitation assays.ResultsIn this study, we have identified GDIα as a negative regulator of AR signaling pathway. Overexpression of GDIα downregulates AR expression at both mRNA and protein levels. Overexpression of GDIα is able to prevent AR nuclear translocation and inhibit transactivation of AR target genes. Co-immunoprecipitation assays showed that GDIα physically interacts with the N-terminal domain of AR.ConclusionsGDIα suppresses AR signaling through inhibition of AR expression, nuclear translocation, and recruitment to androgen-responsive genes. GDIα regulatory pathway may play a critical role in regulating AR signaling and prostate cancer growth and progression.

Docetaxel is the first-line standard treatment for castration-resistant prostate cancer. However, relapse eventually occurs due to the development of resistance to docetaxel. To unravel the mechanism of acquired docetaxel resistance, we established docetaxel-resistant prostate cancer cells, TaxR, from castration-resistant C4-2B prostate cancer cells. The IC50 for docetaxel in TaxR cells was about 70-fold higher than parental C4-2B cells. Global gene expression analysis revealed alteration of expression of a total of 1,604 genes, with 52% being upregulated and 48% downregulated. ABCB1, which belongs to the ATP-binding cassette (ABC) transporter family, was identified among the top upregulated genes in TaxR cells. The role of ABCB1 in the development of docetaxel resistance was examined. Knockdown of ABCB1 expression by its specific shRNA or inhibitor resensitized docetaxel-resistant TaxR cells to docetaxel treatment by enhancing apoptotic cell death. Furthermore, we identified that apigenin, a natural product of the flavone family, inhibits ABCB1 expression and resensitizes docetaxel-resistant prostate cancer cells to docetaxel treatment. Collectively, these results suggest that overexpression of ABCB1 mediates acquired docetaxel resistance and targeting ABCB1 expression could be a potential approach to resensitize docetaxel-resistant prostate cancer cells to docetaxel treatment.

Resistance of prostate cancer cells to the next-generation antiandrogen enzalutamide may be mediated by a multitude of survival signaling pathways. In this study, we tested whether increased expression of NF-κB2/p52 induces prostate cancer cell resistance to enzalutamide and whether this response is mediated by aberrant androgen receptor (AR) activation and AR splice variant production. LNCaP cells stably expressing NF-κB2/p52 exhibited higher survival rates than controls when treated with enzalutamide. C4-2B and CWR22Rv1 cells chronically treated with enzalutamide were found to express higher levels of NF-κB2/p52. Downregulation of NF-κB2/p52 in CWR22Rv1 cells chronically treated with enzalutamide rendered them more sensitive to cell growth inhibition by enzalutamide. Analysis of the expression levels of AR splice variants by quantitative reverse transcription PCR and Western blotting revealed that LNCaP cells expressing p52 exhibit higher expression of AR splice variants. Downregulation of expression of NF-κB2/p52 in VCaP and CWR22Rv1 cells by short hairpin RNA abolished expression of splice variants. Downregulation of expression of either full-length AR or the splice variant AR-V7 led to an increase in sensitivity of prostate cancer cells to enzalutamide. These results collectively demonstrate that resistance to enzalutamide may be mediated by NF-κB2/p52 via activation of AR and its splice variants.

PurposeProstate cancer (PCa) is characterized by deregulated expression of several tumor suppressor or oncogenic miRNAs. The objective of this study was the identification and characterization of miR-let-7c as a potential tumor suppressor in PCa.Experimental designLevels of expression of miR-let-7c were examined in human PCa cell lines and tissues using qRT-PCR and in situ hybridization. Let-7c was overexpressed or suppressed to assess the effects on the growth of human PCa cell lines. Lentiviral-mediated re-expression of let-7c was utilized to assess the effects on human PCa xenografts.ResultsWe identified miR-let-7c as a potential tumor suppressor in PCa. Expression of let-7c is downregulated in castration-resistant prostate cancer (CRPC) cells. Overexpression of let-7c decreased while downregulation of let-7c increased cell proliferation, clonogenicity and anchorage-independent growth of PCa cells in vitro. Suppression of let-7c expression enhanced the ability of androgen-sensitive PCa cells to grow in androgen-deprived conditions in vitro. Reconstitution of Let-7c by lentiviral-mediated intratumoral delivery significantly reduced tumor burden in xenografts of human PCa cells. Furthermore, let-7c expression is downregulated in clinical PCa specimens compared to their matched benign tissues, while the expression of Lin28, a master regulator of let-7 miRNA processing, is upregulated in clinical PCa specimens.ConclusionsThese results demonstrate that microRNA let-7c is downregulated in PCa and functions as a tumor suppressor, and is a potential therapeutic target for PCa.