Androgen Receptor Splice Variants Research Articles

The association between androgen receptor splice variant 7 status and prognosis of metastatic castration-resistant prostate cancer: A systematic review and meta-analysis.

Recent studies have found that metastatic castrated-resistant prostate cancer (mCRPC) with positive androgen receptor splice variant 7 (AR-V7) may have poor prognosis during endocrine or chemotherapy treatment, but the specific mechanism was still unclear. We had finished literature search in March 2019 from PubMed, Web of Science database, and Embase. The final results were presented in this research. The pooled results showed that AR-V7 status predicted pooled PSA-PFS (HR=4.31, 95% CI: 2.57-7.24, p<.001), rPFS (HR=2.39, 95% CI: 1.28-4.48, p=.006) and OS (HR=4.27, 95% CI: 3.22-5.66, p<.001) in mCRPC patients after endocrine or chemotherapy treatment. Subgroup analysis of different treatments revealed that mCRPC patients treated with chemotherapy had significant association between positive AR-V7 and OS (HR=2.82, 95% CI: 1.72-4.62, p<.001), and also during endocrine therapy (HR=4.78, 95% CI: 3.33-6.86, p<.001). Our study demonstrated that AR-V7-positive mCRPC patients may have worse prognosis. AR-V7 may be an independent prognostic factor for endocrine therapy or chemotherapy in patients with mCRPC.

PIM-1 Is Overexpressed at a High Frequency in Circulating Tumor Cells from Metastatic Castration-Resistant Prostate Cancer Patients.

PIM-1 is an oncogene involved in cell cycle progression, cell growth, cell survival and therapy resistance, activated in many types of cancer, and is now considered as a very promising target for cancer therapy. We report for the first time that PIM-1 is overexpressed in circulating tumor cells (CTCs) from metastatic castration-resistant prostate cancer patients (mCRPC). We first developed and validated a highly sensitive RT-qPCR assay for quantification of PIM-1 transcripts. We further applied this assay to study PIM-1 expression in EpCAM(+) CTC fraction isolated from 64 peripheral blood samples of 50 mCRPC patients. CTC enumeration in all samples was performed using the FDA-cleared CellSearch® system. PIM-1 overexpression was detected in 24/64 (37.5%) cases, while in 20/24 (83.3%) cases that were positive for PIM-1 expression, at least one CTC/7.5 mL PB was detected in the CellSearch®. Our data indicate that PIM-1 overexpression is observed at high frequency in CTCs from mCRPC patients and this finding, in combination with androgen receptor splice variant 7 (AR-V7) expression in CTCs, suggest its potential role as a very promising target for cancer therapy. We strongly believe that PIM-1 overexpression in EpCAM(+) CTC fraction merits to be further evaluated and validated as a non-invasive circulating tumor biomarker in a large and well-defined patient cohort with mCRPC.

SUN-LB136 A Comparison of Androgen Receptor Splice Variant, AR-V7, and Glucocorticoid Receptor Activity in Prostate Cancer

Prostate Cancer (PCa) is an androgen dependent disease and patients with metastatic PCa are treated with androgen deprivation therapy (ADT). Although most tumors respond initially, tumors become resistant and are termed castration resistant prostate cancer (CRPC). There is compelling evidence that most of these tumors retain androgen receptor (AR) dependence and some data to suggest that, in some cases, the glucocorticoid receptor (GR) substitutes for AR. AR, itself, is re-activated through a variety of mechanisms including the expression of constitutively active AR splice variants that lack the ligand binding domain (LBD) of AR. Expression of one variant, AR-V7, which contains the amino-terminal domain and DNA binding domain of AR and 16 unique amino-acids, has been correlated with resistance to second line ADT. Although there has been some debate regarding the role of AR-V7, whether it is only a partial substitute for AR or has unique activities, our studies of engineered cell lines treated to express levels of AR-V7 equivalent to AR, clearly show that while the AR isoforms have common targets, they each also have unique targets. Consistent with this, the cistromes of the two show many unique sites as well as common sites. AR-V7 binding is enriched near the transcription start site (TSS) and we have identified a novel de novo binding motif. These findings suggest the possibility of developing a gene signature unique to AR-V7.Because GR activity in PCa has also been suggested as an escape mechanism in response to ADT, and GR binds to the same consensus response elements, we sought to identify a GR signature in PCa, to compare it with the AR and AR-V7 signatures, and to ask whether the AR-V7 and/or GR signatures are enriched in CRPC. Because much of the gene signatures are cell line dependent, we sought to compare GR and AR-V7 action in cells that express both. LN-95 cells express both AR-V7 and GR. MDA-PCa-2b cells, a cell line derived from an African American patient, expresses GR, but not AR-V7. The parental line was infected with a lentivirus that expresses AR-V7 in response to doxycycline. Transcriptomes were determined for AR, GR, and AR-V7 in these lines using RNA-Seq. Overall the magnitude of regulation of gene expression was generally lower than in the LNCaP AR-V7 and VCaP-AR-V7 lines, but there was good overlap of the MDA-PCa-2b AR-V7 regulated with the LNCaP AR-V7 regulated genes. Genes induced by GR overlapped with AR and isoform common genes, but did not overlap with AR-V7 specific genes. A comparison of AR-V7 specific genes common to the LNCaP and VCaP models as well as to publicly available data sets for LN-95 and 22RV1 AR-V7 signatures, show a strong correlation with CRPC compared to primary tumors when analyzed in the Grasso data set.

IKBKE activity enhances AR levels in advanced prostate cancer via modulation of the Hippo pathway

Resistance to androgen receptor (AR) targeting therapeutics in prostate cancer (PC) is a significant clinical problem. Mechanisms by which this is accomplished include AR amplification and expression of AR splice variants, demonstrating that AR remains a key therapeutic target in advanced disease. For the first time we show that IKBKE drives AR signalling in advanced PC. Significant inhibition of AR regulated gene expression was observed upon siRNA-mediated IKBKE depletion or pharmacological inhibition due to inhibited AR gene expression in multiple cell line models including a LNCaP derivative cell line resistant to the anti-androgen, enzalutamide (LNCaP-EnzR). Phenotypically, this resulted in significant inhibition of proliferation, migration and colony forming ability suggesting that targeting IKBKE could circumvent resistance to AR targeting therapies. Indeed, pharmacological inhibition in the CWR22Rv1 xenograft mouse model reduced tumour size and enhanced survival. Critically, this was validated in patient-derived explants where enzymatic inactivation of IKBKE reduced cell proliferation and AR expression. Mechanistically, we provide evidence that IKBKE regulates AR levels via Hippo pathway inhibition to reduce c-MYC levels at cis-regulatory elements within the AR gene. Thus, IKBKE is a therapeutic target in advanced PC suggesting repurposing of clinically tested IKBKE inhibitors could be beneficial to castrate resistant PC patients.

Recent Advances in Androgen-Receptor Splicing Variants Related to the Mechanism and Treatment of Castration-Resistant Prostate Cancer

Recent Advances in Androgen-Receptor Splicing Variants Related to the Mechanism and Treatment of Castration-Resistant Prostate Cancer

Preclinical Study Using ABT263 to Increase Enzalutamide Sensitivity to Suppress Prostate Cancer Progression Via Targeting BCL2/ROS/USP26 Axis Through Altering ARv7 Protein Degradation.

Background: The recently developed antiandrogen, Enzalutamide (Enz), has reformed the standard of care for castration resistant prostate cancer (CRPC) patients. However, Enz-resistance inevitably emerges despite success of Enz in prolonging CRPC patients’ survival. Here we found that Enz-resistant prostate cancer (PCa) cells had higher BCL2 expression. We aimed to test whether targeting BCL2 would influence Enz sensitivity of prostate cancer (PCa) and identify the potential mechanism. Methods: The study was designed to target Enz-induced BCL2 with inhibitor ABT263 and test Enz sensitivity in Enz-resistant PCa cells by MTT assay. Cellular reactive oxygen species (ROS) levels were detected with dihydroethidium staining, and in vitro deubiquitinating enzyme activity assay was used to evaluate ubiquitin specific protease 26 (USP26) activity. Results: ABT263 could increase Enz sensitivity in both Enz-sensitive and Enz-resistant PCa cells via inducing ROS generation. Elevated cellular ROS levels might then inhibit USP26 activity to increase the ubiquitination of androgen receptor (AR) and AR splice variant 7 (ARv7) and their ubiquitin/proteasome-dependent degradation, which contributed to the increase of Enz sensitivity. In vivo mouse model also demonstrates that ABT263 will suppress the PCa progression. Conclusion: This study demonstrated that targeting Enz-induced BCL2 with inhibitor ABT263 could increase Enz sensitivity in both Enz-sensitive and Enz-resistant PCa cells through induction of cellular ROS levels and suppression of USP26 activity with a consequent increase of ubiquitin/proteasome-dependent degradation of AR and ARv7 protein expression.

The value of AR-V7 in predicting therapeutic efficiency of abiraterone for CRPC patients

Objective Determine the relationship between AR-V7 expression and treatment efficiency of abiraterone. Methods A prospective cohort study was conducted to detect CTCs AR-V7 in newly diagnosed CRPC patients, aged ≥18 years who were admitted to the urologic department of Peking University cancer hospital from January 2016 to January 2019, with one or more systemic metastases. All the patients intended to be treated with abiraterone. According to the AR-V7 status, patients were divided into 2 groups (AR-V7 positive and negative). PSA decline time, PSA PFS, clinical status PFS, imaging PFS and CSS are analyzed and compared by t-test and Chi-square between 2 groups. Result 49 patients were AR-V7 negative and 28 were AR-V7 positive. Compared with AR-V7 positive patients, PSA decline time (72.04±66.92 vs. 190.11±102.44, P=0.000), PSA non-response rate (6.12% vs. 21.4%, P=0.040) are significantly lower in AR-V7 negative patients. PSA PFS(489.17±269.39 vs. 130.56±120, P=0.010), Clinical PFS (551.91±322.05 vs. 261.44±200.85, P=0.018), and Imaging PFS (523.7±223.28 vs. 247.56±202.80, P=0.003) are significantly longer in AR-V7 negative patients. Conclusion Expression status of AR-V7 is related to the response of abiraterone treatment and the prognosis of the patients. Key words: Prostatic neoplasms; Castration resistant prostate cancer; Androgen receptor splice variant 7(AR-V7); Abiraterone

Glycogen-rich Clear Cell Carcinoma of the Breast: A Comprehensive Review.

Glycogen-rich clear cell carcinoma (GRCC) is a very rare form of primary breast cancer (<0.1% of all breast cancers). It is characterized by the presence of neoplastic cells with a glycogen-abundant clear cytoplasm (the Periodic Acid Schiff-positive, diastase-sensitive). The expression of steroid receptors (estrogen and progesterone receptors) has been variably reported (35% to 100% of the cases), whereas most studies reported low human epidermal growth factor receptor 2 positivity in GRCC. High androgen receptor positivity without androgen receptor splice variant-7 was reported in one recent study. Although sparse, the preliminary theranostic data on GRCC indicate the potential of targeted treatments in selected cases (antiandrogen, PIK3CA, and immune checkpoint inhibitors). Because of its rarity, the prognosis for GRCC patients remains controversial. Herein, we comprehensively appraise the epidemiological, morphologic, molecular, and clinical characteristics of this rare mammary malignancy.

Androgen receptor variant-driven prostate cancer II: advances in clinical investigation.

Approximately 10-30% of men with mCRPC will test positive for AR-V7 using one of two analytically and clinically validated circulating tumor cell (CTC)-based assays. These men have poor outcomes with approved AR-targeting therapies but may retain sensitivity to chemotherapy. Here, we discuss the clinical implications of testing and strategies that may benefit AR splice variant (AR-V)-positive men and discuss whether such variants are passengers or drivers of aggressive clinical behavior. We conducted a systemic review of the literature, covering updates since our 2016 review on androgen receptor variants in mCRPC, outcomes, and existing and novel approaches to therapy. We provide an expert opinion about management strategies for AR-V7-positive men and key unanswered research questions. AR-V7-positive men, defined by Epic nuclear protein detection or the modified AdnaTest mRNA detection in CTCs, identify a subset of men with mCRPC that have a low probability of response to AR-targeting therapy with short progression-free and overall survival in multivariable analyses. AR-variants do not exist in isolation, but rather in the context of a complex, heterogeneous, and evolving mCRPC genome and phenotype as well as patient-specific clinical heterogeneity, and multiple mechanisms of resistance likely exist in patients regardless of AR-V7 detection. Efforts to develop broader resistance assays are needed, and effective treatment strategies beyond taxanes are needed to address the causal driver role of AR-variants and to benefit patients with AR-V-expressing prostate cancer. CTC AR-V7 detection using the AdnaTest mRNA or Epic nuclear protein assays represents the first analytically and prospective clinically validated liquid biopsy assays that may inform treatment decisions in men with mCRPC, particularly after failure of first-line AR-therapy. The importance of AR-variants is likely to increase with the earlier use of AR-targeting strategies in other settings, and novel interventions for these men are needed.

Preliminary results of a prospective assessment of androgen receptor splice variants in mCRPC patients undergoing androgen receptor targeted agents.

246 Background: Different treatment options for metastatic castrate resistant prostate cancer (mCRPC) are currently available, including Androgen Receptor Targeted Agents (ARTA) and Taxane based chemotherapy. However, selection criteria aiming to drive the therapeutic choice in this setting are lacking, and no standardized therapeutic sequence is validated. In our institution, a prospective study testing predictive value of circulating tumor cells (CTC) in patients (pts) receiving a first line ARTA for mCRPC is currently ongoing (PRIMERA). Here, we present preliminary results from the first enrolled cohort. Methods: Pts affected by mCRPC eligible for Androgen Deprivation Therapy (ADT) plus ARTA were prospectively enrolled and underwent baseline determination of androgen-receptor splice variant 7 (AR-V7) and androgen-receptor-full length (ARFL) variants on CTC at baseline (ARTA start). Fisher Exact Test was conducted to explore the correlation between baseline features and CTC detection rate. Results: Overall, 26 pts were enrolled and baseline results are available for 13 pts. CTC were found in 5 (38.5%) pts. No significant difference was reported in terms of CTC detection probability for pts with high vs low burden of disease according to CHARTEED definition (p=0.42), neither for pts with PSA at CRPC diagnosis &lt; or &gt; 10 ng/ml, (p=0.2). However, a PSA &gt; 10 ng/ml at ADT start in metastatic hormone sensitive setting (mHSPC) was significantly related to CTC detection probability (p=0.01). AR-V7 variants were found in 1 out 5 (20%) CTC-positive pts. Conclusions: PSA at ADT start may significantly be related to CTC detection rate. CTC detection could be particularly useful in pts with a high burden of disease at the beginning of systemic therapy. Metastatic disease could remain dormant until development of CRPC status, with consequent release of increased amount of CTC, more likely to be detected in this setting. AR-V7 expression was found in 20% of CTC positive pts, in line with literature data.[Table: see text]

Single-arm, phase II study to evaluate the safety and efficacy of sacituzumab govitecan in patients with metastatic castration-resistant prostate cancer who have progressed on second generation AR-directed therapy.

TPS251 Background: In patients with mCRPC, androgen receptor (AR) pathway inhibitors such as enzalutamide or abiraterone acetate are often used as first-line systemic therapy. After progression on either of these inhibitors, the likelihood of response to the other is significantly lower than in the first-line setting. Multiple resistance mechanisms have been identified, including the emergence of AR splice variants. Trop2 is a cell-surface antigen that is expressed in &gt; 70% of prostate cancers and is enriched in mCRPC expressing AR splice variants. Sacituzumab govitecan (SG; IMMU-132) is a novel antibody drug conjugate composed of humanized anti-Trop2 antibody conjugated to SN-38 payload by hydrolyzable linker. SG has potential to be an effective therapy after progression on initial AR pathway inhibitors to meet this significant unmet clinical need. Methods: This is a single-arm, open-label, multicenter phase 2 trial (NCT03725761) to determine the efficacy and safety of SG. The primary endpoint is PSA response rate (≥50% PSA decline at or within 9 weeks of starting treatment); secondary endpoints include rPFS and overall survival. 55 subjects with mCRPC who have progressed on second-generation AR-directed therapy in either the hormone sensitive or CRPC setting will be included. Fresh or archived metastatic site biopsy is required. Subjects will be treated with SG IV at 10 mg/kg on Day 1 and Day 8 of 21-day cycles until disease progression. Subjects who receive ≥1 SG dose and have ≥1 PSA post-dose assessment will be included in final efficacy analysis. Circulating tumor cell biomarker studies will be performed throughout treatment and at progression to investigate potential mechanisms of response/resistance to SG. Tumor biopsies at baseline and after 3 therapy cycles will evaluate genomic alterations that predict treatment response. Subjects who remain progression free will undergo repeated evaluations of extent of disease every 9 weeks. The trial is actively accruing. Managed by: Prostate Cancer Clinical Trials Consortium; Funding: Immunomedics; Support: Prostate Cancer Foundation. Clinical trial information: NCT03725761.

IND candidate EPI-7386 as an N-terminal domain androgen receptor inhibitor in development for the treatment of prostate cancer.

142 Background: The androgen receptor (AR) pathway drives most metastatic castration-resistant prostate cancers (mCRPC) even in late stages of the disease. Anti-androgen resistance mechanisms include AR gene amplification, C-terminal ligand-binding domain (LBD) mutations and expression of constitutively active AR splice variants lacking the LBD (e.g. AR-V7). Selective inhibition of the N-terminal domain (NTD) of the AR can inhibit its’ transcriptional activity even in the presence of LBD-driven resistance. In a phase I trial, the first-generation AR NTD inhibitor (aniten) EPI-002, demonstrated minor PSA declines in mCRPC patients. The efficacy and safety profile of second generation aniten IND-candidate EPI-7386 will be presented. Methods: Novel aniten chemical structures were developed to increase molecule potency using a wide variety of CRPC models. Similarly, the stability and selectivity of the molecule were characterized with screening and functional assays. IND-enabling studies further support the compounds’ safety profile. Results: EPI-7386 demonstrated a 20-fold improvement in AR-driven cellular potency compared to EPI-002, while being highly stable in human hepatocytes and across animal species. In vitro proliferation assays demonstrated on-target activity across a panel of prostate cancer cell lines, with activity in AR-V7-driven cellular models. EPI-7386 was able to induce tumor regression in CRPC xenografts and show superiority to enzalutamide (ENZ) in ENZ resistant models. In addition, the combination of ENZ with EPI-7386 demonstrated a more robust antitumor response. IND-enabling studies demonstrate a safe and well-tolerated profile supporting an IND filing in 1Q 2020. Pharmacodynamic markers specific to anitens will be presented, in addition to early clinical development plans. Conclusions: The next generation aniten compound EPI-7386 is more active and metabolically stable than EPI-002. It demonstrated potential as a single agent in overcoming anti-androgen clinical resistance as well as in combination therapy in earlier stages of the disease. The clinical strategy supporting the development of this new generation of aniten will be discussed.

Exosomal TUBB3 mRNA expression level to predict prognosis for abiraterone treatment in metastatic prostate patients.

244 Background: TUBB3 (Class III β-tubulin), a dynamic component of microtubules, is considered a prognostic biomarker of tumor aggressiveness and poor survival. Previous studies have researched into its indication of resistance to chemotherapeutic drugs. However, its association with abiraterone, a next-generation androgen-axis targeting drug in prostate cancer has not been studied yet. In this study, we aimed to find out if exosomal TUBB3 expression could predict the efficacy of abiraterone in metastatic prostate cancer patients. Methods: Blood samples of metastatic prostate cancer patients during different disease stages were collected. We isolated the exosomes and extracted the RNA for analysis of TUBB3 by ddPCR. Absolute target mRNA concentration was measured by ddPCR as copies per milliliter (copies/20ul). Clinical data were collected for all patients. Primary study endpoint was progression-free survival (PFS). Statistical analyses were performed with SPSS 25.0. Results: A total of 54 metastatic prostate cancer patients who have received abiraterone treatment were enrolled. Baseline characteristics are comparable between high (≥10 copies/20ul) and low (&lt;10 copies/20ul) TUBB3 expression groups. Univariate analyses revealed that baseline hemoglobin≤120g/L, baseline sodium concentration &gt;140 mmol/L, baseline potassium concentration &gt;4.39 mmol/L and high exosomal TUBB3 expression were associated with shorter PFS, while only baseline sodium concentration and exosomal TUBB3 expression were independent prognosticators in multivariate analyses. In subsequently subgroup analyses, high exosomal TUBB3 expression demonstrates particularly high hazard ratio in multiple subgroups (patients younger than 70 years old, without neuroendocrine differentiation and immunohistochemical androgen-receptor splice variant 7 expression, with baseline PSA &gt;100 ng/mL, and patients receiving abiraterone as first-line therapy). Conclusions: Exosomal TUBB3 expression has potential in making treatment plan and predict prognosis in metastatic prostate cancer patients. High exosomal TUBB3 expression indicates poor response to abiraterone, especially as first line therapy.

Novel approach to therapeutic targeting of castration-resistant prostate cancer

Development of resistance to anti-androgen therapy limits the usefulness of second-generation androgen receptor (AR) antagonists including enzalutamide and abiraterone in castration resistant prostate cancer (CRPC) patients. Recent genomic studies reveal that AR-regulated genes contribute to CRPC emergence. Several reasons for the development of resistance towards anti-androgens have been hypothesized, including intracellular testosterone production, androgen overexpression, somatic mutations of AR resulting in a gain of function, constitutive activation of AR splice variants, imbalance in AR regulators, and bypass of AR in CRPC progression. Recent findings suggest that epigenetic alterations are involved in the deregulation of AR signaling. Overexpression of enhancer of zeste homolog 2 (EZH2), the enzymatic member of the polycomb repressor complex PRC2, has emerged as a key activator of AR in CRPC. Studies indicate that overabundance of EZH2 in localized prostate tumors increases the risk of biochemical recurrence after surgery, as it activates AR by enhancing methylation, resulting in the suppression of tumor suppressor genes and activation of oncogenes. This apparent association between EZH2 and AR in activating target genes by cooperative recruitment might play a critical role in the emergence of CRPC. Our hypothesis is that combination treatment targeting EZH2 and AR may be a novel efficacious therapeutic regime for the treatment of castrate resistant prostate cancer, and we propose to investigate this possibility.

Transcriptionally Active Androgen Receptor Splice Variants Promote Hepatocellular Carcinoma Progression

Owing to the marked sexual dimorphism of hepatocellular carcinoma (HCC), sex hormone receptor signaling has been implicated in numerous aspects of liver cancer pathogenesis. We sought to reconcile the clear contribution of androgen receptor (AR) activity that has been established in preclinical models of HCC with the clinical failure of AR antagonists in patients with advanced HCC by evaluating potential resistance mechanisms to AR-targeted therapy. The AR locus was interrogated for resistance-causing genomic modifications using publicly available primary HCC datasets (1,019 samples). Analysis of HCC tumor and cell line RNA-seq data revealed enriched expression of constitutively active, treatment-refractory AR splice variants (AR-SV). HCC cell lines expressed C-terminal-truncated AR-SV; 28 primary HCC samples abundantly expressed AR-SV. Low molecular weight AR species were nuclear localized and constitutively active. Furthermore, AR/AR-SV signaling promoted AR-mediated HCC cell progression and conferred resistance to AR antagonists. Ligand-dependent and -independent AR signaling mediated HCC epithelial-to-mesenchymal transition by regulating the transcription factor SLUG. These data suggest that AR-SV expression in HCC drives HCC progression and resistance to traditional AR antagonists. Novel therapeutic approaches that successfully target AR-SVs may be therapeutically beneficial for HCC. SIGNIFICANCE: Treatment-refractory, constitutively active androgen receptor splice variants promote hepatocellular carcinoma progression by regulating the epithelial-to-mesenchymal transition pathway.

Regression of castration-resistant prostate cancer by a novel compound QW07 targeting androgen receptor N-terminal domain.

Androgen deprivation therapy (ADT) via surgical or chemical castration frequently fails to halt lethal castration-resistant prostate cancer (CRPC), which is induced by multiple mechanisms involving constitutive androgen receptor (AR) splice variants, AR mutation, and/or de novo androgen synthesis. The AR N-terminal domain (NTD) possesses most transcriptional activity and is proposed as a potential target for CRPC drug development. We constructed a screening system targeting AR-NTD transcription activity to screening a compound library and identified a novel small molecule compound named QW07. The function evaluation and mechanism investigation of QW07 were carried out in vitro and in vivo. QW07 bound to AR-NTD directly, blocked the transactivation of AR-NTD, blocked interactions between co-regulatory proteins and androgen response elements (AREs), inhibited the expression of genes downstream of AR, and inhibited prostate cancer growth in vitro and in vivo. QW07 was demonstrated as an AR-NTD-specific antagonist with the potential to inhibit both canonical and variant-mediated AR signaling to regress the CRPC xenografts and is proposed as a lead compound for a specific antagonist targeting AR-NTD.

A candidate androgen signalling signature predictive of response to abiraterone acetate in men with metastatic castration-resistant prostate cancer

BackgroundThe unmet need for predictive biomarkers emerged from the unpredictable pattern of response to androgen signalling inhibition in metastatic castration-resistant prostate cancer (mCRPC). Here, we report on the testing of a previously identified candidate androgen signalling signature associated with response to androgen signalling inhibition. Patients and methodsWe report on the outcome of the first module of a phase II trial on abiraterone acetate (AA) followed by combination with dasatinib or sunitinib. Bone marrow biopsies (BMBs) with matched bone marrow aspirate and blood samples were collected at baseline and upon progression. End-points included assessment of a prespecified molecular signature consisting of nuclear androgen receptor (AR) overexpression, cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17) expression, and AR-C-/N terminal expression ratio of ≥0.8 by immunohistochemistry (IHC) in patients with benefit versus primary resistance to AA (i.e. progression within 4 months). Tumour markers also included v-ets avian erythroblastosis virus E26 oncogene homologue (ERG), androgen receptor splice variant (ARV7) by IHC and steroids by liquid chromatography-tandem mass spectrometry. ResultsOf 170 patients accrued from 03/2011 to 02/2015, 44 (26%) were primary resistant to AA. Forty-eight patients had tumour infiltrated BMB at baseline. Pretreatment androgen signalling signature was linked to benefit from AA (p < 0.001). Presence of ERG was associated with benefit (p = 0.05), whereas nuclear ARV7 presence and 20 or more bone lesions at baseline with primary resistance (p = 0.04 and p = 0.0006, respectively). ConclusionTesting of a prespecified androgen signalling signature was highly supportive of its predictive value in maximal androgen deprivation strategies in mCRPC. Further validation is under way. Trial registrationClinicalTrials.gov NCT01254864.

Bardoxolone-Methyl (CDDO-Me) Suppresses Androgen Receptor and Its Splice-Variant AR-V7 and Enhances Efficacy of Enzalutamide in Prostate Cancer Cells.

Androgen receptor (AR) signaling is fundamental to prostate cancer (PC) progression, and hence, androgen deprivation therapy (ADT) remains a mainstay of treatment. However, augmented AR signaling via both full length AR (AR-FL) and constitutively active AR splice variants, especially AR-V7, is associated with the recurrence of castration resistant prostate cancer (CRPC). Oxidative stress also plays a crucial role in anti-androgen resistance and CRPC outgrowth. We examined whether a triterpenoid antioxidant drug, Bardoxolone-methyl, known as CDDO-Me or RTA 402, can decrease AR-FL and AR-V7 expression in PC cells. Nanomolar (nM) concentrations of CDDO-Me rapidly downregulated AR-FL in LNCaP and C4-2B cells, and both AR-FL and AR-V7 in CWR22Rv1 (22Rv1) cells. The AR-suppressive effect of CDDO-Me was evident at both the mRNA and protein levels. Mechanistically, acute exposure (2 h) to CDDO-Me increased and long-term exposure (24 h) decreased reactive oxygen species (ROS) levels in cells. This was concomitant with an increase in the anti-oxidant transcription factor, Nrf2. The anti-oxidant N-acetyl cysteine (NAC) could overcome this AR-suppressive effect of CDDO-Me. Co-exposure of PC cells to CDDO-Me enhanced the efficacy of a clinically approved anti-androgen, enzalutamide (ENZ), as evident by decreased cell-viability along with migration and colony forming ability of PC cells. Thus, CDDO-Me which is in several late-stage clinical trials, may be used as an adjunct to ADT in PC patients.

Androgen receptor degraders overcome common resistance mechanisms developed during prostate cancer treatment

Androgen receptor (AR) antagonists, such as enzalutamide, have had a major impact on the treatment of metastatic castration-resistant prostate cancer (CRPC). However, even with the advent of AR antagonist therapies, patients continue to develop resistance, and new strategies to combat continued AR signalling are needed. Here, we develop AR degraders using PROteolysis TArgeting Chimeric (PROTAC) technology in order to determine whether depletion of AR protein can overcome mechanisms of resistance commonly associated with current AR-targeting therapies. ARD-61 is the most potent of the AR degraders and effectively induces on-target AR degradation with a mechanism consistent with the PROTAC design. Compared to clinically-approved AR antagonists, administration of ARD-61 in vitro and in vivo results in more potent anti-proliferative, pro-apoptotic effects and attenuation of downstream AR target gene expression in prostate cancer cells. Importantly, we demonstrate that ARD-61 functions in enzalutamide-resistant model systems, characterized by diverse proposed mechanisms of resistance that include AR amplification/overexpression, AR mutation, and expression of AR splice variants, such as AR-V7. While AR degraders are unable to bind and degrade AR-V7, they continue to inhibit tumor cell growth in models overexpressing AR-V7. To further explore this, we developed several isogenic prostate cell line models in which AR-V7 is highly expressed, which also failed to influence the cell inhibitory effects of AR degraders, suggesting that AR-V7 is not a functional resistance mechanism for AR antagonism. These data provide compelling evidence that full-length AR remains a prominent oncogenic driver of prostate cancers which have developed resistance to AR antagonists and highlight the clinical potential of AR degraders for treatment of CRPC.

Targeting GRP78-dependent AR-V7 protein degradation overcomes castration-resistance in prostate cancer therapy.

Rationale: Androgen receptor splice variant 7 (AR-V7) is a leading cause of the development of castration-resistant prostate cancer (CRPC). However, the regulation and function of AR-V7 at levels of post-translational modifications in prostate cancer therapy remain poorly understood. Here, we conducted a library screen of natural products to identify potential small molecules responsible for AR-V7 protein degradation in human prostate cancer cell lines.Methods: A natural product library was used to screen the inhibitor of AR-V7. Co-IP and biomass spectrum assays were used to identify the AR-V7-interacting proteins, whereas western blot, confocal microscopy, RNA interfering, and gene transfection were used to validate these interactions. Cell viability, EDU staining, and colony formation assays were employed to detect cell growth and proliferation. Flowcytometry assays were used to detect the distribution of cell cycle. Mouse xenograft models were used to study the anti-CRPC effects in vivo.Results: This screen identified rutaecarpine, one of the major components of the Chinese medicine Evodia rutaecarpa, as a novel chemical that selectively induces AR-V7 protein degradation via K48-linked ubiquitination. Mechanically, this effect relies on rutaecarpine inducing the formation of a GRP78-AR-V7 protein complex, which further recruits the E3 ligase SIAH2 to directly promote the ubiquitination of AR-V7. Consequently, the genetic and pharmacological activation of the GRP78-dependent AR-V7 protein degradation restores the sensitivity of castration-resistant prostate cancer to anti-androgen therapy in cell culture and animal models.Conclusions: These findings not only provide a new approach for overcoming castration-resistance in prostate cancer therapy, but also increase our understanding about the interplay between molecular chaperones and ubiquitin ligase in shaping protein stability.