Aberrant Androgen Receptor Research Articles

Altered mRNA expression due to insertion or substitution of thymine at position +3 of two splice-donor sites in the androgen receptor gene.

We have discovered two types of 5' intronic gene mutation that impair androgen receptor (AR) mRNA expression severely, and cause complete androgen insensitivity. Labium majus skin fibroblasts (LMSF) hemizygous for each mutation had negligible specific androgen binding, and did not react to an antibody against an N-terminal peptide of the AR. Both mutations were detected by direct sequencing of exons PCR-amplified with flanking primers. One mutation is an adenine to thymine transversion at position +3 of the intron 6 splice-donor site. Using LMSF mRNA, RT-PCR of a portion of the AR androgen-binding domain yielded a small amount of a 302-bp mutant fragment instead of a 433-bp wild-type product. Sequencing established that exon 5 was followed, out of frame, by exon 7: exon 6 was skipped. The other mutation is a thymine insertion at the +3 position of the intron 1 donor-splice site. RT-PCR and sequencing revealed a small amount of normal-size mRNA with normal exon 1-exon 2 splicing. Quantitative RT-PCR on mutant LMSF showed AR mRNA levels were well below 10% of normal; hence, most of the aberrant AR mRNA resulting from each mutation is probably unstable. The misbehavior caused by these two mutations indicates that in the AR the splice-donor site +3 adenine is critical; indeed, 57% of eukaryotic introns have adenine in the +3 position, while only 2% have thymine.

Plasma androgen receptor and serum chromogranin A in advanced prostate cancer

Recently, mixed forms between adenocarcinoma and neuroendocrine prostate cancer (NEPC) have emerged that are characterized by persistent androgen receptor (AR)-signalling and elevated chromogranin A (CgA) levels. The main aim of this study was to analyze castration-resistant prostate cancer (CRPC) patients treated with abiraterone or enzalutamide, assessing progression-free/overall survival (PFS/OS) in association with circulating AR and CgA. AR aberrations were analyzed by droplet digital PCR in pre-treatment plasma samples collected from two biomarker protocols [197 patients from a retrospective study (REC 2192/2013) and 59 from a prospective trial (REC 6798/2015)]. We subdivided patients into three groups according to CgA by receiver-operating characteristic (ROC) curves. In the primary cohort, plasma AR gain and mutations (p.L702H/p.T878A) were detected in 78 (39.6%) and 16 (8.1%) patients, respectively. We observed a significantly worse PFS/OS in patients with higher-CgA than in patients with normal-CgA, especially those with no AR-aberrations. Multivariable analysis showed AR gain, higher-CgA and LDH levels as independent predictors of PFS [hazard ratio (HR) = 2.16, 95% confidence interval (95% CI) 1.50–3.12, p < 0.0001, HR = 1.73, 95% CI 1.06–2.84, p = 0.026, and HR = 2.13, 95% CI 1.45–3.13, p = 0.0001, respectively) and OS (HR = 1.72, 95% CI 1.15–2.57, p = 0.008, HR = 3.63, 95% CI 2.13–6.20, p < 0.0001, and HR = 2.31, 95% CI 1.54–3.48, p < 0.0001, respectively). These data were confirmed in the secondary cohort. Pre-treatment CgA detection could be useful to identify these mixed tumors and would seem to have a prognostic role, especially in AR-normal patients. This association needs further evaluation in larger prospective cohorts.

Targeting Semaphorin 3C in Prostate Cancer With Small Molecules.

Despite the amenability of early-stage prostate cancer to surgery and radiation therapy, locally advanced and metastatic prostate cancer is clinically problematic. Chemical castration is often used as a first-line therapy for advanced disease, but progression to the castration-resistant prostate cancer phase occurs with dependable frequency, largely through mutations to the androgen receptor (AR), aberrant AR signaling, and AR-independent mechanisms, among other causes. Semaphorin 3C (SEMA3C) is a secreted signaling protein that is essential for cardiac and neuronal development and has been shown to be regulated by the AR, to drive epithelial-to-mesenchymal transition and stem features in prostate cells, to activate receptor tyrosine kinases, and to promote cancer progression. Given that SEMA3C is linked to several key aspects of prostate cancer progression, we set out to explore SEMA3C inhibition by small molecules as a prospective cancer therapy. A homology-based SEMA3C protein structure was created, and its interaction with the neuropilin (NRP)-1 receptor was modeled to guide the development of the corresponding disrupting compounds. Experimental screening of 146 in silico‒identified molecules from the National Cancer Institute library led to the discovery of four promising candidates that effectively bind to SEMA3C, inhibit its association with NRP1, and attenuate prostate cancer growth. These findings provide proof of concept for the feasibility of inhibiting SEMA3C with small molecules as a therapeutic approach for prostate cancer.

Down-Regulation of the Androgen Receptor by G-Quadruplex Ligands Sensitizes Castration-Resistant Prostate Cancer Cells to Enzalutamide.

Stabilization of the G-quadruplexes (G4s) within the androgen receptor (AR) gene promoter to block transcription may represent an innovative approach to interfere with aberrant AR signaling in castration resistant prostate cancer (CRPC). A library of differently functionalized naphthalene diimides (NDIs) was screened for their ability to stabilize AR G4s: the core-extended NDI (7) stood out as the most promising ligand. AR-positive cells were remarkably sensitive to 7 in comparison to AR-negative CRCP or normal prostate epithelial cells; 7 induced remarkable impairment of AR mRNA and protein amounts and significant perturbations in the expression levels of KLK3 and of genes involved in the activation of AR program via feedback mechanisms. Moreover, 7 synergistically interacted with Enzalutamide, an inhibitor of AR signaling used in second-line therapies. Overall, our data show that stabilization of AR G4s may represent an alternative treatment options for CRPC and other malignancies relying on aberrant androgen signaling.

Collateral resistance to taxanes in enzalutamide-resistant prostate cancer through aberrant androgen receptor and its variants.

Currently, the optimal sequential use of androgen receptor (AR) axis‐targeted agents and taxane chemotherapies remains undetermined. We aimed to elucidate the resistance status between taxanes and enzalutamide, and the functional role of the AR axis. Enzalutamide‐resistant 22Rv1 cells showed collateral resistance to taxanes, including docetaxel and cabazitaxel. However, taxane‐resistant cells showed no collateral resistance to enzalutamide; taxane‐resistant cells expressed comparable protein levels of full‐length AR and AR variants. Knockdown of both full‐length AR and AR variants rendered cells sensitive to taxanes, whereas knockdown of AR variants sensitized cells to enzalutamide, but not to taxanes. In contrast, overexpression of full‐length AR rendered cells resistant to taxanes. Consistently, the prostate‐specific antigen response and progression‐free survival in docetaxel chemotherapy were worse in cases with prior use of ARAT agents compared with cases without. Collateral resistance to taxanes was evident after obtaining enzalutamide resistance, and aberrant AR signaling might be involved in taxane resistance.

Abstract A067: Targeting the bromodomain and extra-terminal (BET) family proteins and beyond in metastatic castration-resistant prostate cancer (mCRPC): Overcoming aberrant androgen receptor (AR) signaling

Abstract Despite robust responses to androgen deprivation therapy and AR targeting therapies (including abiraterone and enzalutamide), nearly all cases of advanced prostate cancer progress to lethal mCRPC and develop therapeutic resistance. This progression is associated with persistent AR signaling, in part due to expression of constitutively active AR splice variants that include AR variant 7 (AR-V7). We show that AR-V7 expression in patient biopsies (protein) and circulating tumor cells (RNA) associates with poor outcome from mCRPC. Therapies that regulate AR-V7 and induce robust anticancer responses are required to confirm the clinical importance of AR-V7 in mCRPC. One such promising approach, currently in clinical trials, is inhibitors of BET family proteins, which include BRD2, BRD3, and BRD4. Preclinical studies have shown that BRD4 binds to AR at the androgen response element and facilitates the recruitment of the transcriptional machinery. We show that BRD4 protein expression increases as patients develop mCRPC and at diagnosis associates with patient outcome and more advanced disease. In addition, through RNAseq analysis we show that expression levels of BRD2, 3, and 4 in mCRPC associated with degree of AR activity. We, and others, have shown that the use of BET inhibitors (BETi) in vitro on AR/AR-V7 expressing cell lines not only decreased AR activity but also preferentially decreased the production on AR-V7 mRNA. In light of BETi having efficacy against BRD2, 3, and 4, and all isoforms being expressed in mCRPC, we explored the effect of genetic knockdown of each isoform. We show that BETi treatment is sufficient to decrease AR-V7 mRNA and protein in CRPC cell lines. Moreover, we demonstrate that BRD4 knockdown, and to a greater extent, the combination of BRD2, 3, and 4 knockdown blocked AR and AR-V7 signaling. Furthermore, C-MYC knockdown did not recapitulate the effect of BETi and led to an increase in AR signaling. Consistent with these findings, BETi and the combination of BRD2, 3, and 4 knockdown reduced the growth of CRPC cell lines. To further investigate whether BETi is sufficient to inhibit AR-V7 production in patients with mCRPC, we treated patient-derived organoids (PDOs) and a mouse xenograft (PDX) grown from patient metastatic biopsies who had progressed on enzalutamide and/or abiraterone. In this study 4 out of 9 PDOs were sensitive to BETi. Consistent with cell culture experiments, BETi treatment of PDO and PDX led to downregulation of both AR-V7 mRNA and protein expression, and growth inhibition. In light of the pleotropic effects of BETi on cancer cell biology and potential for treatment-related toxicities, we explored whether we could identify critical factors for BETi mediated AR-V7 regulation in CRPC. The ability of BETi to regulate AR-V7 may suggest an effect of BETi on pre-mRNA splicing of AR resulting in the observed decrease of AR-V7 expression. RNAseq analysis of the AR-V7 expressing CRPC cell line LNCaP95 treated with BETi demonstrated an increase in total splicing. Despite this, focused analysis of splicing factors and spliceosome components identified a subset of eight splicing factors being downregulated by BETi treatment, including one yet-uncharacterized factor (splicing factor B; Sf-B) that is crucial for AR-V7 expression and LNCaP95 cell growth. In addition, mCRPC patients who express high levels of Sf-B had a significantly poorer outcome and the protein structure of Sf-B is druggable using the drug discovery knowledgebase canSAR. Based on our results, we propose that inhibition of Sf-B may lead to decreased splicing and expression of AR-V7; providing a novel approach to target AR-V7 in mCRPC. Citation Format: Adam Sharp, Jon Welti, Wei Yuan, Ines Figueiredo, Veronica Gil, Daniel Nava Rodrigues, Maryou Lambros, Eleanor Knight, Jian Ning, Jeff Francis, David Dolling, Lorna Pope, Antje Neeb, Gunther Boysen, Yezi Zhu, Mateus Crespo, Alec Paschalis, Jun Luo, Stephen Plymate, Bissan Al-Lazikani, Amanda Swain, Johann de Bono. Targeting the bromodomain and extra-terminal (BET) family proteins and beyond in metastatic castration-resistant prostate cancer (mCRPC): Overcoming aberrant androgen receptor (AR) signaling [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A067.

Abstract IA18: Sensitivity and resistance biomarkers to PARP inhibitors in Mcrpc

A better understanding of the molecular underpinnings of prostate cancer renders the opportunity for more precise patient care by individualizing therapeutic strategies according to predictive biomarkers of benefit. Until now, clinical management of patients with prostate cancer, either localized or metastatic, has not yet incorporated precision medicine strategies. To pursue this, a new classification of the disease needs to be based on molecular features that are clinically relevant. Several studies have now confirmed genomic defects in the DNA damage response pathways (DDR), and primarily in the homologous recombination mediated repair pathway (HR), as a cornerstone of prostate cancer biology. This is particularly relevant in patients with metastatic castration-resistant prostate cancer (mCRPC). Among this population with lethal disease, around 20-25% of cases present genomic loss (by pathogenic mutations or by homozygous deletion) of at least one gene involved in HR, and in half of this cases the mutation is inherited, which makes these findings also relevant for patients’ relatives, considering how some of this events are linked to increased risk of prostate and others cancers. The HR gene most commonly lost in advanced prostate cancer is BRCA2 (8-12% in different series), followed by ATM (4-6%), CDK12 (3-5%), BRCA1, and PALB2 ( PARP-1 and 2 are key proteins in DNA single-strand break repair and a therapeutic target using a family of drugs (PARP inhibitors) already approved for the treatment of ovarian cancer associated to HR defects, primarily BRCA2 mutations. We completed a first stage of a phase II adaptive clinical trial evaluating the antitumor activity of the PARP inhibitor olaparib in mCRPC (TOPARP), as well as investigating predictive biomarkers associated with response to PARP inhibition. In a cohort of 49 evaluable patients who previously to docetaxel (49/49) and abiraterone and/or enzalutamide (48/49), 16 (32%) achieved a response to olaparib, using the trial preplanned definition of response (radiologic response by RECIST and/or 50% PSA fall and/or CTC conversion from >5 to On one hand, it is possible that different genes have a different weight in determining HR proficiency, and hence, synthetic lethality with PARP inhibition. It is possible that loss of some genes may be partially or totally compensated for by other proteins, whereas loss of some other genes may be more catastrophic for the tumor cell. On the other hand, it is also feasible that the presence of secondary biologic events in prostate cancer, such as AR aberrations, TP53/Rb1 loss, or others, may modulate the impact of HR defects. Analytical validation and clinical qualification of functional assays as predictive biomarkers of PARP inhibitor sensitivity would help towards integrating complex genomic, transcriptome, and epigenetics data with regards to defining the optimal predictive biomarkers for treatment stratification. The complex interaction between androgen receptor (AR) signaling and DDR may be relevant for PARP inhibition stratification. Preclinical studies suggest that blocking AR may render the cell sensitive to PARP inhibition; however, the impact of these findings on therapeutic strategies in the clinic is still not clear and further studies in localized and advanced disease are needed. Previously, a synthetic lethal interaction for PARP inhibition with PTEN loss or with the presence of ERG rearrangements in prostate cancer has been postulated from preclinical studies; to date, these data have not translated into clinical data supporting that these are optimal biomarkers to stratify patients for PARP inhibitors. It is possible these data are, however, very relevant to designing rational combination trials. Lastly, HR gene mutations are also relevant towards secondary resistance mechanisms to PARP inhibitors, which further supports their relevance in determining sensitivity. In a follow-up study we identified how in patients with germline or somatic BRCA2 and PALB2 mutations responding to olaparib, secondary genomic events (basically small fragment deletions) emerged in the region of the original mutation, reverting the gene reading frame to normal. These events occur in a polyclonal manner and may be restoring HR function. These have now been confirmed by other studies in prostate, breast, and ovarian cancer. Citation Format: Joaquin Mateo. Sensitivity and resistance biomarkers to PARP inhibitors in Mcrpc [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr IA18.

Constitutively active androgen receptor splice variants AR-V3, AR-V7 and AR-V9 are co-expressed in castration-resistant prostate cancer metastases

BackgroundA significant subset of prostate cancer (PC) patients with a castration-resistant form of the disease (CRPC) show primary resistance to androgen receptor (AR)-targeting drugs developed against CRPC. As one explanation could be the expression of constitutively active androgen receptor splice variants (AR-Vs), our current objectives were to study AR-Vs and other AR aberrations to better understand the emergence of CRPC.MethodsWe analysed specimens from different stages of prostate cancer by next-generation sequencing and immunohistochemistry.ResultsAR mutations and copy number variations were detected only in CRPC specimens. Genomic structural rearrangements of AR were observed in 5/30 metastatic CRPC patients, but they were not associated with expression of previously known AR-Vs. The predominant AR-Vs detected were AR-V3, AR-V7 and AR-V9, with the expression levels being significantly higher in CRPC cases compared to prostatectomy samples. Out of 25 CRPC metastases that expressed any AR variant, 17 cases harboured expression of all three of these AR-Vs. AR-V7 protein expression was highly heterogeneous and higher in CRPC compared to hormone-naïve tumours.ConclusionsAR-V3, AR-V7 and AR-V9 are co-expressed in CRPC metastases highlighting the fact that inhibiting AR function via regions common to all AR-Vs is likely to provide additional benefit to patients with CRPC.

Abstract 1796: Repurposing CRISPR to study aberrant AR signaling in models of castration-resistant prostate cancer

Abstract Prostate cancer (PC) is currently the most commonly diagnosed noncutaneous cancer affecting men worldwide. Despite the initial response to androgen ablation, patients relapse with tumors becoming refractory to clinically approved anti-androgens, resulting invariably in castration-resistant PC (CRPC). In CRPC, androgen receptor (AR) signaling is inappropriately restored. Resistance to available hormone therapies in PC has been linked to aberrant splicing of nascent AR transcripts and subsequent expression of constitutively active AR splice variants. Although we have information on the expression of receptor variants in advanced disease, the mechanisms that regulate these aberrantly functioning proteins are markedly understudied. Therefore, a better understanding of how splice variants are generated and function in this disease setting is critical for the development of more refined targeted therapies. By modeling clinically relevant AR splice variants, we will identify and exploit mechanisms of receptor regulation for advanced prostate cancer therapy. The CRISPR-Cas9 technology has been exploited to generate a CWR22Rv1 derivative cell line that lacks AR-FL and hence only expresses AR-Vs. This has allowed us to study the function of AR-Vs in an AR-FL-free background and therefore attribute the observed outcomes exclusively to AR splice variants. In addition, a novel CRISPR-based approach, currently developed in our lab, allows selective targeting of a catalytically inactive form of Cas9 (dead Cas9, dCas9) fused to a FLAG tag, to native chromatin regions and subsequent isolation of protein/RNA complexes associated with these sites. We have taken advantage of spatial mRNA splicing and transcription coupling in order to isolate splicing factors responsible for the generation of the AR variant 7 (AR-V7) in the CWR22Rv1 cell line and its AR-FL CRISPR knockout derivative. Three gRNAs were designed against the AR cryptic exon 3 genomic region. Targeting of FLAG-tagged dCas9 to that site was exploited for in situ capture and isolation of endogenous splicing factors specific to AR-V7, upon rapid FLAG immunoprecipitation and characterization by mass spectrometry. Identification of the AR-V7 spliceosome will fill a critical knowledge gap in our understanding of how AR-Vs are generated and provide key targets for future therapeutic intervention in advanced PC patients. Citation Format: Evangelia E. Kounatidou, Craig Robson, Luke Gaughan. Repurposing CRISPR to study aberrant AR signaling in models of castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1796.

The prognostic significance of androgen receptor and β-catenin immunohistochemical expression in urothelial carcinoma with and without detrusor muscle invasion from an Egyptian institution.

Androgen receptor (AR) activation plays an important role in the promotion and progression of urothelial tumorigenesis. Also, dysregulation of the Wnt/β-catenin signaling pathway has also been linked to bladder cancer growth. However, cross talk between the two pathways in remains unclear in bladder cancer. This study investigated the prognostic significance of AR and β-catenin expression and their relationship to different clinic-pathological parameters, recurrence free and progression free survival. 106 urothelial carcinoma cases were used to study the immunohistochemical expression of AR and β-catenin. Log-Rank test to compare survival between groups. Androgen receptor positivity was in 37 (34.9%) cases. Both aberrant β-catenin and AR positivity were associated with higher tumor grade (p = 0.033 and p = 0.037 respectively) and muscle invasion (p = 0.007 and p = 0.039 respectively). Aberrant β-catenin only showed statistically significant association with tumor diameter (p = 0.036), tumor stage (p = 0.038), LN metastasis (p = 0.001), tumor recurrence (p = 0.026) and tumor progression (p = 0.01). Cases with aberrant β-catenin showed higher AR positivity (p = 0.018). Our study highlighted important information about the link between Wnt/β-catenin and AR pathway during the progression of transitional urothelial carcinoma with aberrant β-catenin could be used as a prognostic marker.

Quercetin Targets hnRNPA1 to Overcome Enzalutamide Resistance in Prostate Cancer Cells.

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.

Inhibitors of the androgen receptor N‑terminal domain : Therapies targeting the Achilles' heel of various androgen receptor molecules in advanced prostate cancer

Although prostate cancer responds well to primary endocrine therapies, tumor progression with castration resistant tumor cells almost invariably occurs within afew years. Unfortunately, some CRPC patients do not respond to second-line therapies with abiraterone or enzalutamide. Moreover, patients who initially responded well to second-line hormone therapy develop resistance to abiraterone and/or enzalutamide within ashort period of time. Besides an increase of intracellular androgen receptor (AR) levels, the predominant resistance mechanisms include AR aberrations (point mutations, AR splice variants) occurring predominantly at the androgen or ligand binding domain of the AR. The following review delineates recent progress in the development of AR inhibitors that do not depend on androgen binding and represent aputative third generation of AR inhibitors.

Abstract 2036: In vitro and in vivo effect of carmustine and selenite combination on EGFR signaling in androgen-independent prostate cancer

Abstract Introduction: Despite the use of androgen deprivation therapy, the majority of prostate cancer patients will progress to castration resistant disease within 2-3 years, driven by aberrant androgen receptor activation. We have previously shown that combination of carmustine and selenite effectively induces apoptosis by completely reducing AR and AR-variants in AR-dependent castration resistant prostate cancer cells both in vitro and in vivo. Although studies have demonstrated that AR signaling is a central mechanism of castration resistant prostate cancer progression, the cell growth mediated by AR-independent signaling is also operative. EGFR signaling has been implicated in the survival, invasion, and metastasis of prostate cancer cells in an AR-independent manner. Therefore, in this study, we tested whether the combination of carmustine and selenite could inhibit EGFR signaling, induce apoptosis, and inhibit growth of AR-independent prostate cancer cells in an in vitro cell culture and in vivo xenograft models. Methods: AR negative PC-3 prostate cancer cell line was used to study the effect of carmustine and selenite alone or in combination on EGFR signaling. Cell viability, proliferation, and apoptosis in the presence or absence of EGF (50ng/ml) were determined. Dose and time dependent effect of EGF on the activation of EGFR was studied. The carmustine and/or selenite effect on EGF stimulated phospho-EGFR, and its downstream signaling pathways, such as AKT and MAPK, were studied in comparison with inhibitors of EGFR (AG1478) or PI3 kinase (LY294002) or MAPK (PD98059). The in vivo effect of carmustine and selenite was examined on PC-3 tumor growth in athymic nude mice. Results: Combination of carmustine and selenite treatment in the presence of EGF markedly suppressed cell viability and proliferation of PC-3 cells. EGF exposure increased phospho-EGFR (Tyr845, Tyr992, Tyr1068, and Tyr1045), pAkt (Ser473), and pERK1/2 (Thr204/Tyr202) protein expression levels in PC-3 cells in a dose and time dependent manner. The combination treatment exhibited a strong inhibitory effect on EGF stimulated EGFR, Akt, and ERK1/2 phosphorylation. Combination treatment was able to induce apoptosis even in the presence of EGF. Consistent with in vitro results, combination treatment for 3 weeks reduced PC-3 xenograft tumors in nude mice by 88% without any toxicity. Whereas individual agent treatment showed only partial effect both in vitro and in vivo. Conclusions: Our pre-clinical data demonstrate that the combination treatment of carmustine and selenite substantially inhibits EGFR signaling, induces apoptosis, and reduces tumor growth of AR-independent prostate cancer cells in vitro and in vivo. Our novel findings suggest that the combination of carmustine and selenite is an effective therapeutic agent for successful treatment, survival, and improved quality of life for patients with prostate cancer. Citation Format: Vijayalakshmi Thamilselvan, Mani Menon, Sivagnanam Thamilselvan. In vitro and in vivo effect of carmustine and selenite combination on EGFR signaling in androgen-independent prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2036. doi:10.1158/1538-7445.AM2017-2036

Combination of Carmustine and Selenite Inhibits EGFR Mediated Growth Signaling in Androgen-Independent Prostate Cancer Cells.

Although aberrant androgen receptor (AR) signaling is a central mechanism for castration resistant prostate cancer (CRPC) progression, AR-independent growth signaling is also present in CRPC. The current therapeutic options for patients with CRPC are limited and new drugs are desperately needed to eliminate these crucial growth signaling pathways. We have previously shown that combination of carmustine and selenite effectively induces apoptosis and growth inhibition by targeting AR and AR-variants in CRPC cells. High levels of EGFR expression present in the CRPC cells mediates the cell proliferation via AR-independent growth signaling mechanisms. Therefore, in this study, we investigated whether the combination of carmustine and selenite could inhibit EGFR mediated growth signaling and induce apoptosis in androgen independent-AR negative prostate cancer cells. EGF exposure dose and time dependently increased phospho-EGFR (Tyr845, Tyr1068, and Tyr1045), pAkt (Ser473), and pERK1/2 (Thr204/Tyr202) protein expression levels in AIPC cells. Combination of carmustine and selenite treatment markedly suppressed EGF-stimulated proliferation and survival of AIPC cells and effectively induced apoptosis. The ROS generated by the combination of carmustine and selenite exhibited a strong inhibition on EGF stimulated EGFR and its downstream signaling molecules such as Akt, NF-kB, ERK1/2, and Cyclin D1. Individual agent treatment showed only partial effect. Overall, our findings demonstrated that the combination of carmustine and selenite treatment dramatically inhibits EGFR signaling, proliferation, and induces apoptosis in AIPC cells, suggesting a potential candidate for the treatment of CRPC. The results of the study further suggest that the combination of carmustine and selenite treatment can overcome EGFR mediated AR-independent growth response in CRPC during anti-androgen therapy. J. Cell. Biochem. 118: 4331-4340, 2017. © 2017 Wiley Periodicals, Inc.

Polyphenic trait promotes liver cancer in a model of epigenetic instability in mice.

Hepatocellular carcinoma (HCC) represents the fifth‐most common form of cancer worldwide and carries a high mortality rate attributed to lack of effective treatment. Males are 8 times more likely to develop HCC than females, an effect largely driven by sex hormones, albeit through still poorly understood mechanisms. We previously identified TRIM28 (tripartite protein 28), a scaffold protein capable of recruiting a number of chromatin modifiers, as a crucial mediator of sexual dimorphism in the liver. Trim28hep–/– mice display sex‐specific transcriptional deregulation of a wide range of bile and steroid metabolism genes and development of liver adenomas in males. We now demonstrate that obesity and aging precipitate alterations of TRIM28‐dependent transcriptional dynamics, leading to a metabolic infection state responsible for highly penetrant male‐restricted hepatic carcinogenesis. Molecular analyses implicate aberrant androgen receptor stimulation, biliary acid disturbances, and altered responses to gut microbiota in the pathogenesis of Trim28hep–/–‐associated HCC. Correspondingly, androgen deprivation markedly attenuates the frequency and severity of tumors, and raising animals under axenic conditions completely abrogates their abnormal phenotype, even upon high‐fat diet challenge. Conclusion: This work underpins how discrete polyphenic traits in epigenetically metastable conditions can contribute to a cancer‐prone state and more broadly provides new evidence linking hormonal imbalances, metabolic disturbances, gut microbiota, and cancer. (Hepatology 2017;66:235–251).

Sigma1 Targeting to Suppress Aberrant Androgen Receptor Signaling in Prostate Cancer.

Suppression of androgen receptor (AR) activity in prostate cancer by androgen depletion or direct AR antagonist treatment, although initially effective, leads to incurable castration-resistant prostate cancer (CRPC) via compensatory mechanisms including resurgence of AR and AR splice variant (ARV) signaling. Emerging evidence suggests that Sigma1 (also known as sigma-1 receptor) is a unique chaperone or scaffolding protein that contributes to cellular protein homeostasis. We reported previously that some Sigma1-selective small molecules can be used to pharmacologically modulate protein homeostasis pathways. We hypothesized that these Sigma1-mediated responses could be exploited to suppress AR protein levels and activity. Here we demonstrate that treatment with a small-molecule Sigma1 inhibitor prevented 5α- dihydrotestosterone-mediated nuclear translocation of AR and induced proteasomal degradation of AR and ARV, suppressing the transcriptional activity and protein levels of both full-length and splice-variant AR. Consistent with these data, RNAi knockdown of Sigma1 resulted in decreased AR levels and transcriptional activity. Furthermore, Sigma1 physically associated with ARV7 and ARv567es as well as full-length AR. Treatment of mice xenografted with ARV-driven CRPC tumors with a drug-like small-molecule Sigma1 inhibitor significantly inhibited tumor growth associated with elimination of AR and ARV7 in responsive tumors. Together, our data show that Sigma1 modulators can be used to suppress AR/ARV-driven prostate cancer cells via regulation of pharmacologically responsive Sigma1-AR/ARV interactions, both in vitro and in vivoCancer Res; 77(9); 2439-52. ©2017 AACR.

Circulating androgen receptor and serum chromogranin A in castration-resistant prostate cancers (CRPC) patients treated with abiraterone and enzalutamide.

160 Background: Circulating aberrations (copy number/point mutation] of androgen receptor ( AR) as well as serum chromogranin A (CgA) have been associated to treatment response and clinical outcome in CRPC patients (pts) treated with abiraterone (abi)/enzalutamide(enza). Methods: We determined AR aberrations by digital PCR, Taqman and targeted-next generation sequencing on pre-treatment plasma from 169 CRPC pts treated with abi 1g/od (n = 96) and prednisone 5mg/bid or enza 160mg/od (n = 73) (Romanel, 2015; Salvi, 2015; Salvi, 2016). All pts were subdivided into 3 groups using receiver-operating characteristic (ROC) curves (Burgio, 2014; Conteduca, 2014) to evaluate properly the impact of CgA levels: serum CgA level was normal when &lt; 120 ng/ml (group A,n = 76); within 3 times the upper normal value(UNV) when between 120 and 360(group B,n = 57), more than 3 times UNV when &gt; 360(group C,n = 36). The primary endpoint was the correlation between AR aberrations and CgA level. The secondary endpoints were progression-free/overall survival (PFS/OS) and PSA response rate (RR) ( &gt; 50% PSA decline after 12 weeks of therapy) stratified by AR status and CgA level. Results: We observed AR gain in 57(33.7%) of 169 pts, 29(30.2%) of 96 pts treated with abi and 28(38.3%) of 73 treated with enza. Circulating AR status was independent from baseline CgA levels in all pts (p = 0.36). In only AR-normal pts, we showed a significant survival difference among 3 CgA groups (P &lt; 0.0001), particularly Group C vs. A+B [HR (hazard ratio) 5.1, 95%CI (confidence interval) 2.37-10.96 and HR 3.7 95%CI 1.79-7.69, respectively for PFS/OS]. We reported no difference of PFS/OS in AR-gained pts (P = 0.6 and P = 0.1, respectively with HR~0.6 in all CgA groups). In addition, group C vs. A+B had a significant PSA RR [P = 0.03 odds ratio (OR) 2.7 95%CI 1.02-7.30] in AR-normal pts. No PSA RR difference was observed in AR-gained pts (P = 1.0 OR 0.97 95%CI 0.18-5.26). Conclusions: In CRPC pts treated with abi/enza, baseline CgA levels may be an additional predictive/prognostic factor, especially in a particular set of CRPC pts with normal plasma AR status and worse treatment outcome. This association needs a prospective larger study.

The impact of TAS3681, a new type of androgen receptor antagonist, on aberrant AR signaling that drives tumor resistance to AR-targeted therapies by down-regulating full length and splice variant AR.

197 Background: The treatment of castration-resistant prostate cancer (CRPC) has changed dramatically with the use of two new therapies, enzalutamide and abiraterone, directed at the androgen receptor (AR) signaling axis. However, eventually almost all patients relapse after these agents because of acquired resistance by a variety of mechanisms such as AR overexpression, the occurrence of AR mutations, or the induction of AR splice variants that confer ligand independent AR transactivation. TAS3681 is an oral pure AR antagonist with AR down-regulating activity. In this study, we investigated the effect of TAS3681 on several issues related to resistance to current AR-targeted therapy. Methods: VCaP cells transfected with wild-type AR-expressing vector, which overexpress AR, were treated with TAS3681 in the presence of androgen. Cells were harvested and luciferase activity was determined. For the analysis of F876L mutant AR, LNCaP cells stably expressing F876L AR protein were incubated with TAS3681 in the presence of androgen. The WST-8 assay was performed to measure cell viability. Prostate cancer cells expressing AR-v7 were treated with TAS3681, and full length (FL)-AR and AR-v7 protein levels were determined by Western blot. TAS3681 was orally dosed to confirm AR down-regulation in tumor and evaluate antitumor efficacy in AR-v7 positive CRPC tumor xenograft mouse model. Results: In prostate cancer (PCa) cells which overexpress AR, in contrast to enzalutamide, TAS3681 effectively suppressed AR transactivation and cell proliferation via AR down-regulation. TAS3681 effectively antagonized F876L mutant AR which confers resistance to cell growth inhibition by enzalutamide. Moreover, TAS3681 reduced both FL-AR and AR-v7 protein level in PCa cells in vitro and in vivo, and showed strong antitumor efficacy in AR-v7 positive xenografts. Conclusions: TAS3681 disrupts the aberrant AR signaling via a novel mechanism of action: pure AR antagonism plus down-regulation of full-length and splice variant AR. These findings suggest that TAS3681 has the potential to overcome resistance to current AR pathway target drugs.

Identification of miR-30b-3p and miR-30d-5p as direct regulators of androgen receptor signaling in prostate cancer by complementary functional microRNA library screening.

The Androgen Receptor (AR) plays a key role in prostate biology and in the progression of prostate cancer (PCa) to castration resistance. The role of microRNAs (miRNAs) in aberrant AR signaling have not been fully characterized. Here we screened a library of 810 miRNA mimics to identify miRNAs that alter AR activity in complementary functional assays including protein lysate microarray (LMA) quantification of AR and PSA protein levels, AR transcriptional reporter activity, and AR-positive PCa cell viability. Candidate AR-regulating miRNAs were verified through AR transcriptional reporter and cell viability assays. MiRNA binding sites were found within the AR 3′-untranslated region (UTR) and within the AR and AR-V7 coding regions. MiRNA activity was characterized by western blotting, 3′-UTR reporter assay, and AR-GFP and AR-V7-GFP reporter assays. Results uncovered miR-30 family members as direct AR inhibitors. Inhibition of endogenous miR-30b-3p and miR-30d-5p enhanced AR expression and androgen-independent cell growth. Droplet digital RT-PCR quantification of miR-30c-5p and miR-30d-5p revealed significantly reduced levels in metastatic castration resistant PCa (CRPC), when compared to healthy prostate tissues. MiR-30d-5p levels were inversely correlated with AR activity, as measured by PSA mRNA, in metastatic CRPC. Collectively, these studies provide a comprehensive evaluation of AR-regulating miRNAs in PCa.

Characterising mechanisms of aberrant androgen receptor signalling in advanced prostate cancer

Characterising mechanisms of aberrant androgen receptor signalling in advanced prostate cancer