Androgen-responsive Prostate Cancer Cell Research Articles

Abstract B044: Exosomal cancer-type SLCO1B3 and ABCC3 as potential biomarkers of castration resistant prostate cancer

Abstract Extracellular vesicles (EVs) provide a minimally invasive source of tumor-specific markers for liquid biopsy that can be utilized for cancer detection, diagnosis, or monitoring progression. Our laboratory has previously demonstrated the endogenous expression of all transporter systems in the common prostate cancer cell lines (LNCaP, 22Rv1, PC3, DU-145) using a medium throughput TaqMan-based qRT-PCR assay. We identified the two most differentially expressed transporter genes in castration-resistant prostate cancer cells (CRPC) as the cancer-type solute carrier organic anion transporter family 1B3 (ct-SLCO1B3) and the ATP Binding Cassette Subfamily Member C (ABBC3). Both transporters have been investigated as EV-derived tumor markers; however, their detection in prostate cancer-derived EVs remains to be determined. This study aims to investigate whether ct-SLCO1B31B3 and ABCC3 mRNA exists in prostate cancer-derived EVs and can be further detected using plasma specimens. We isolated EVs from prostate cancer (LNCaP, 22Rv1, PC3, DU-145) cell line supernatant and extracted exosomal RNA for qPCR analyses. Both ct-SLCO1B3 and ABBC3 transcripts were only detected in CRPC-derived EV cells vs androgen-responsive prostate cancer cells. To explore the possibility that both transcripts in CRPC-derived EVs can be detected in plasma, we isolated plasma EVs derived from human prostate cancer xenograft mice, and then performed digital droplet PCR analysis. We demonstrated that ct-SLCO1B3 and ABBC3 are detectable in the DU-145 xenograft mouse model, both in tissues and in plasma-derived EVs. Our results provide evidence that ct-SLCO1B3 and ABCC3 exist in EVs and can be detected (at least) in mouse plasma, potentially supporting their roles as novel, plasma-based biomarkers for monitoring prostate cancer progression. Citation Format: Erica L. Beatson, Kristi Y. Lee, Martina A. Knechel, Elijah R. Sommer, Roger Depaz, Emily N. Risdon, Andres F. Leon, Jonathan D. Strope, Douglas K. Price, Cindy H. Chau, William D. Figg. Exosomal cancer-type SLCO1B3 and ABCC3 as potential biomarkers of castration resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B044.

Clinical and molecular significance of the RNA m6A methyltransferase complex in prostate cancer.

N6-methyladenosine (m6A) is the most abundant internal mRNA modification and is dynamically regulated through distinct protein complexes that methylate, demethylate, and/or interpret the m6A modification. These proteins, and the m6A modification, are involved in the regulation of gene expression, RNA stability, splicing and translation. Given its role in these crucial processes, m6A has been implicated in many diseases, including in cancer development and progression. Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in men and recent studies support a role for m6A in PCa. Despite this, the literature currently lacks an integrated analysis of the expression of key components of the m6A RNA methyltransferase complex, both in PCa patients and in well-established cell line models. For this reason, this study used immunohistochemistry and functional studies to investigate the mechanistic and clinical significance of the METTL3, METTL14, WTAP and CBLL1 components of the m6A methyltransferase complex in PCa specimens and cell lines. Expression of METTL3 and CBLL1, but not METTL14 and WTAP, was associated with poorer PCa patient outcomes. Expression of METTL3, METTL14, WTAP and CBLL1 was higher in PCa cells compared with non-malignant prostate cells, with the highest expression seen in castrate-sensitive, androgen-responsive PCa cells. Moreover, in PCa cell lines, expression of METTL3 and WTAP was found to be androgen-regulated. To investigate the mechanistic role(s) of the m6A methyltransferase complex in PCa cells, short hairpin RNA (shRNA)-mediated knockdown coupled with next generation sequencing was used to determine the transcriptome-wide roles of METTL3, the catalytic subunit of the m6A methyltransferase complex. Functional depletion of METTL3 resulted in upregulation of the androgen receptor (AR), together with 134 AR-regulated genes. METTL3 knockdown also resulted in altered splicing, and enrichment of cell cycle, DNA repair and metabolic pathways. Collectively, this study identified the functional and clinical significance of four essential m6A complex components in PCa patient specimens and cell lines for the first time. Further studies are now warranted to determine the potential therapeutic relevance of METTL3 inhibitors in development to treat leukaemia to benefit patients with PCa.

Increased cytokine gene expression and cognition risk associated with androgen deprivation therapy.

BackgroundAndrogen deprivation therapy (ADT) is a standard treatment modality for locally advanced, high‐risk, and metastatic hormone‐sensitive prostate cancer. Long‐term ADT treatment likely develops side‐effects that include changes in cognition or onset of dementia. However, the molecular understanding of this effect remains elusive. We attempt to establish a link between ADT and changes in cognitive function using patient databases and bioinformatics analyses.MethodsGene expression profiling was performed using RNA sequencing data from Alzheimer patient cohort and compared with the data from advanced‐stage prostate cancer patients receiving neoadjuvant antiandrogen therapy. Differentially expressed genes (DEGs) were analyzed using the Ingenuity knowledge database.ResultsA total of 1952 DEGs in the Alzheimer patient cohort and 101 DEGs were identified in ADT treated prostate cancer patients. Comparing both data sets provided a subset of 33 commonly expressed genes involving cytokine‐cytokine signaling with an over representation of cytokine‐cytokine receptor interaction, inflammatory cytokines, signaling by interleukins together with alterations in the circulating lymphocyte repertoire, adaptive immune responses, regulation of cytokine production, and changes in T‐cell subsets. Additionally, lipopolysaccharide, tumor necrosis factor, and toll‐like receptors were identified as upstream transcriptional regulators of these pathways. The most commonly expressed genes viz. IL‐17A, CCL2, IL‐10, IL‐6, IL‐1RN, LIF/LIFR were further validated by quantitative RT‐PCR exhibited higher expression in antiandrogen treated neuronal, glial, and androgen‐responsive prostate cancer cells, compared to no‐androgen antagonist treatment.ConclusionsOur findings suggest that changes in cytokine signaling under the influence of ADT in prostate cancer patients may be linked with cognitive impairment presenting new avenues for diagnostic and therapeutic development in combating brain deficits.

Effects of MTX-23, a Novel PROTAC of Androgen Receptor Splice Variant-7 and Androgen Receptor, on CRPC Resistant to Second-Line Antiandrogen Therapy

Although second-line antiandrogen therapy (SAT) is the standard of care in men with castration-resistant prostate cancer (CRPC), resistance inevitably occurs. One major proposed mechanism of resistance to SAT involves the emergence of androgen receptor (AR) splice variant-7, AR-V7. Recently, we developed MTX-23 using the principle of proteolysis targeting chimera (PROTAC) to target both AR-V7 and AR-full length (AR-FL). MTX-23 has been designed to simultaneously bind AR's DNA binding domain (DBD) and the Von Hippel-Lindau (VHL) E3 ubiquitin ligase. Immunoblots demonstrated that MTX-23's degradation concentration 50% (DC50) for AR-V7 and AR-FL was 0.37 and 2 μmol/L, respectively. Further studies revealed that MTX-23 inhibited prostate cancer cellular proliferation and increased apoptosis only in androgen-responsive prostate cancer cells. The antiproliferative effect of MTX-23 was partially reversed when either AR-V7 or AR-FL was overexpressed and was completely abrogated when both were overexpressed. To assess the potential therapeutic value of MTX-23, we next generated 12 human prostate cancer cell lines that are resistant to the four FDA-approved SAT agents-abiraterone, enzalutamide, apalutamide, and darolutamide. When resistant cells were treated with MTX-23, decreased cellular proliferation and reduced tumor growth were observed both in vitro and in mice. These results collectively suggest that MTX-23 is a novel PROTAC small molecule that may be effective against SAT-resistant CRPC by degrading both AR-V7 and AR-FL.

A phase I, dose-expansion cohort study on the safety of a cannabidiol for biochemical recurrence in prostate cancer patients.

TPS263 Background: Cannabinoids, widely used in pain control, as an appetite stimulant, and anti-emetic in cancer patients, may play role as an anti-carcinogenic agent. A preclinical study demonstrated that cannabinoid receptors (CB1 and CB2) were highly expressed in human androgen-responsive prostate cancer (PCa) cells (LNCaP) as compared to normal prostate epithelial cells (PrEC). When these two cell lines were treated with a potent cannabinoid receptor agonist, PCa cells showed a decrease in prostate-specific antigen (PSA) protein expression and increased apoptosis in a dose and time-dependent manner with no effect seen in PrEC cells. An FDA-approved cannabidiol (CBD) agent, is derived from Cannabis sativa L. plants. Extracts from these plants are processed to yield pure CBD and contain less than 0.5% THC. We will expand these preclinical studies with a phase I trial. Methods: This trial is an open-label, single-center, phase I dose escalation study followed by dose expansion to evaluate acute toxicity, long-term safety and tolerability, and preliminary antitumor activity of cannabidiols in patients with biochemically recurrent (BCR) PC after primary definitive local therapy and prostate-specific antigen doubling time ≤ 12 months. The dose escalation will be determined by a Bayesian Optimal Interval design and the target dose-limiting toxicity rate is set at 30%. An additional 6-9 patients will be enrolled as an expansion cohort once the maximum tolerated dose is determined. Patients will be treated for a total 90 days with a 10 day taper. The primary objective is to evaluate the acute toxicity and long-term safety and tolerability of cannabidiols in patients with BCR. The secondary objective includes measuring changes in serial PSA levels, PSA velocity, and testosterone levels and to assess health-related quality of life (EORTC QLQ-C30 and QLQ-PR 25) from baseline throughout the treatment period. CB receptor 1 and 2 expression levels by immunohistochemistry staining will be evaluated from archival prostatectomy specimens if available as an exploratory objective. This trial opened on 7.28.2020 and has started enrollment. Clinical trial information: NCT04428203.

Generation of Phenothiazine with Potent Anti-TLK1 Activity for Prostate Cancer Therapy.

SummaryThrough in vitro kinase assays and docking studies, we report the synthesis and biological evaluation of a phenothiazine analog J54 with potent TLK1 inhibitory activity for prostate cancer (PCa) therapy. Most PCa deaths result from progressive failure in standard androgen deprivation therapy (ADT), leading to metastatic castration-resistant PCa. Treatments that can suppress the conversion to mCRPC have high potential to be rapidly implemented in the clinics. ADT results in increased expression of TLK1B, a key kinase upstream of NEK1 and ATR and mediating the DNA damage response that typically results in temporary cell-cycle arrest of androgen-responsive PCa cells, whereas its abrogation leads to apoptosis. We studied J54 as a potent inhibitor of this axis and as a mediator of apoptosis in vitro and in LNCaP xenografts, which has potential for clinical investigation in combination with ADT. J54 has low affinity for the dopamine receptor in modeling and competition studies and weak detrimental behavioral effects in mice and C. elegans.

Abstract 1467: Metabolic reprogramming fuels prostate cancer cells towards enzalutamide resistance

Abstract Androgen deprivation therapy is standard care for advanced-stage prostate cancer. Enzalutamide (Xtandi®), a second-generation antiandrogen is the most clinically prescribed drug that has prolonged survival in men. However, this response is temporary and results in the rapid emergence of resistance. The underlying mechanisms of enzalutamide resistance remain unclear. Emerging evidence suggests that metabolic reprogramming mediates resistance to standard-of-care therapies. To identify the molecular underpinnings of enzalutamide resistance, we performed Next-Gen Sequencing (NGS), analyzing global metabolic changes in enzalutamide-resistant clones utilizing two androgen-responsive human prostate cancer cells and comparing them with parental counterparts. Enzalutamide-resistant LNCaP and C4-2B cells were generated from 20µM enzalutamide exposed for 6 months and maintained in media containing 5µM drug, referred to as LNCaP/C42B ENZU cells. Gene set enrichment analysis of differentially expressed genes (DEGs) demonstrated an overlap between genes of metabolic signaling pathways together with fatty acid signaling, fatty acid β oxidation, drug resistance, bile acid biosynthesis, lipid metabolism, and peroxisome signaling. Interestingly, NGS data of LNCaP-ENZU cells showed a higher number of DEGs genes associated with metabolic reprogramming, compared to C42B-ENZU cells. Analysis of LNCaP-ENZU cells exhibited more than 10 fold higher (log2 fold) expression of XIST, AKT3, ZNF655, IRS4, HOXB3, FBN2, FHL1, GSTP1, VCAN, KIAA0408 genes; whereas ZNF544, KLK2, CSMD1, ZG16B, SPDEF, AR, C1R1, FOLH1, HISTIHIB, and TNPR222 were downregulated (-10 to -12 log2 fold), compared to parental counterpart. The majority of these genes belong to the metabolic regulatory network. Furthermore, genes viz. AKT3, HOXB3, and KIA0048 RALYL and KLK2 exhibited interaction with androgen receptor (AR), and may influence the ligand-binding domain of AR resulting in stimulation of AR/AR targeted genes. Q-PCR data were in agreement with NGS data where the signature genes associated with the metabolic synthesis of fatty acid, lipid and glucose were overexpressed in LNCaP-ENZU cells. In particular, fatty acid synthesis genes viz. ACADL, ACSL4, FHL2, and SLC27A6 were high up to 14.7, 15.8, 6.02 and 17.8 log2 fold value. Expression of solute carrier SLC12A5, SLC13A4, SLC24A4, SLC30A3, SLC35D1, and SLC47A1 genes showed 1195, 5, 20.01, 4.6, 9.5, and 4.3 log2 fold higher expression, which function in increasing the nucleotide-sugar transmembrane transporter activity. Expression of lipid-associated genes viz. ABCB4, CYP2D6, CYP4X1, CYP39A1, and SLC25A17 exhibited 30, 25.6, 25.5, 8.4, and 17.5 log2 fold higher expression. These gene expression correlated with higher-end products of TCA cycle, glucose metabolism, citric acid and lactate production in LNCaP ENZU-resistant cells. Taken together, our findings demonstrate molecular alterations after enzalutamide exposure may provide fuel to the cells through metabolic reprogramming. Clinically, targeting the aforementioned genes associated with metabolic reprogramming may lead to control of the enzalutamide-mediated recurrence of prostate cancer. Citation Format: Shiv S. Verma, Eswar Shankar, Rajnee Kanwal, Ricky Chan, Sanjay Gupta. Metabolic reprogramming fuels prostate cancer cells towards enzalutamide resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1467.

‘MUSCADINE GRAPE SKIN EXTRACT INHIBITS ANDROGEN‐INDEPENDENT PROSTATE CANCER CELL GROWTH, INDUCING CELL‐CYCLE ARREST, AND DECREASING MIGRATION BY TARGETING HEAT SHOCK PROTEIN 40

Previously we demonstrated that muscadine grape skin extract (MSKE), a natural product, significantly inhibited androgen‐responsive prostate cancer cell growth by inducing apoptosis through the targeting of survival pathways. However, the therapeutic effect of MSKE on more aggressive androgen‐independent prostate cancer remains unknown. This study examined the effects of MSKE treatment in metastatic prostate cancer using complementary PC‐3 cells and xenograft model. MSKE significantly inhibited PC‐3 human prostate cancer cell tumor growth in vitro and in vivo. The growth‐inhibitory effect of MSKE appeared to be through the induction of cell‐cycle arrest. This induction was accompanied by a reduction in the protein expression of Hsp40 and cell‐cycle regulation proteins, cyclin D1 and NF‐kBp65. In addition, MSKE induced p21 expression independent of wild‐type p53 induced protein expression. Moreover, we demonstrate that MSKE significantly inhibited cell migration in PC‐3 prostate cancer cells. Overall, these results demonstrate that MSKE inhibits prostate tumor growth and migration, and induces cell‐cycle arrest by targeting Hsp40 and proteins involved in cell‐cycle regulation and proliferation. This suggests that MSKE may also be explored either as a neo‐adjuvant or therapeutic for castration resistant prostate cancer.

Abstract 1264: Design, synthesis and biological evaluation of new phenothiazine derivatives as potential Tousled-like kinase 1 inhibitors in prostate cancer treatment

Abstract Introduction: The standard therapy for advanced Prostate Cancer (PCa) consists of anti-androgens which provide respite from the disease progression, yet ultimately fail and result in the incurable phase of the disease: mCRPC. Targeting PCa cells before their progression to mCRPC would significantly improve the outcome. Untoward toxicity limits the combination therapies targeting the DNA Damage Response (DDR), and hence the goal of clinical trials is to target the DDR more specifically. Androgen deprivation therapy (ADT) in LNCaP cells results in the increased expression of TLK1B, a critical kinase upstream of NEK1 and ATR, thereby mediating a DDR that typically causes a temporary cell cycle arrest of androgen-responsive PCa cells. Following the DNA damage, the addition of a TLK1 specific inhibitor, thioridazine (THD), impairs ATR and Chk1 activation, establishing the existence of an ADT>TLK1>NEK1>ATR>Chk1 DDR pathway, while its abrogation, leads to apoptosis. However, THD is a known anti-psychotic and has undesirable side-effects. Hence, there is a compelling need to design and develop next-generation TLK1 inhibitors to circumvent the adverse effects and advance them in the clinic. Methods: We performed immunoblotting of the tumour tissue phosphoproteins (pATR, pChk1 and pNEK1) and immunohistochemistry analysis of the tissue sections from the LNCaP xenograft models. To identify and develop new potent inhibitors against TLK1, we employed an in-silico homology modelling and molecular docking approach. Based on the protein-ligand binding interactions and the docking score, a handful of compounds were shortlisted, synthesised and screened for the TLK1 inhibition potential in-vitro and using cell-based assays. Results: Our experimental data revealed that the pATR, pChk1, pNEK1, Ki-67 and PCNA were remarkably inhibited when treated with THD in combination with an anti-androgen drug, Bicalutamide (BIC). Moreover, it also induced apoptosis and increased DNA damage as demonstrated by the cleaved PARP, Caspase 3 and γH2AX levels respectively. The new inhibitor screening assay showed J54 compound to be most potent and inhibitory with a logIC50 of 1.1µM. J54 binds to the protein’s allosteric site noncompetitively with ATP and interacts with His504 and Gly630 with a corresponding docking score of -6.736. J54 is found to be non-toxic to normal cells and also suppresses the growth of androgen-dependent colonies of LNCaP cells cultured with BIC. Conclusion: Our preliminary work suggests that targeting the TLK1/NEK1 axis with specific TLK1 inhibitors might be an effective therapy for PCa in combination with standard care, ADT. Citation Format: Siddhant Bhoir, Javeena Hussain, Vibha Singh, Rupesh Chikhale, Richard Bryce, Sivapriya Kirubakaran, Arrigo DeBenedetti. Design, synthesis and biological evaluation of new phenothiazine derivatives as potential Tousled-like kinase 1 inhibitors in prostate cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1264.

Targeting the TLK1/NEK1 DDR axis with Thioridazine suppresses outgrowth of androgen independent prostate tumors.

Standard therapy for advanced Prostate Cancer (PCa) consists of antiandrogens, which provide respite from disease progression, but ultimately fail resulting in the incurable phase of the disease: mCRPC. Targeting PCa cells before their progression to mCRPC would greatly improve the outcome. Combination therapy targeting the DNA Damage Response (DDR) has been limited by general toxicity, and a goal of clinical trials is how to target the DDR more specifically. We now show that androgen deprivation therapy (ADT) of LNCaP cells results in increased expression of TLK1B, a key kinase upstream of NEK1 and ATR and mediating the DDR that typically results in a temporary cell cycle arrest of androgen responsive PCa cells. Following DNA damage, addition of the TLK specific inhibitor, thioridazine (THD), impairs ATR and Chk1 activation, establishing the existence of a ADT > TLK1 > NEK1 > ATR > Chk1, DDR pathway, while its abrogation leads to apoptosis. Treatment with THD suppressed the outgrowth of androgen‐independent (AI) colonies of LNCaP and TRAMP‐C2 cells cultured with bicalutamide. Moreover, THD significantly inhibited the growth of several PCa cells in vitro (including AI lines). Administration of THD or bicalutamide was not effective at inhibiting long‐term tumor growth of LNCaP xenografts. In contrast, combination therapy remarkably inhibited tumor growth via bypass of the DDR. Moreover, xenografts of LNCaP cells overexpressing a NEK1‐T141A mutant were durably suppressed with bicalutamide. Collectively, these results suggest that targeting the TLK1/NEK1 axis might be a novel therapy for PCa in combination with standard of care (ADT).

Muscadine grape skin extract inhibits prostate cancer cells by inducing cell-cycle arrest, and decreasing migration through heat shock protein 40

Previously we demonstrated that muscadine grape skin extract (MSKE), a natural product, significantly inhibited androgen-responsive prostate cancer cell growth by inducing apoptosis through the targeting of survival pathways. However, the therapeutic effect of MSKE on more aggressive androgen-independent prostate cancer remains unknown. This study examined the effects of MSKE treatment in metastatic prostate cancer using complementary PC-3 cells and xenograft model. MSKE significantly inhibited PC-3 human prostate cancer cell tumor growth in vitro and in vivo. The growth-inhibitory effect of MSKE appeared to be through the induction of cell-cycle arrest. This induction was accompanied by a reduction in the protein expression of Hsp40 and cell-cycle regulation proteins, cyclin D1 and NF-kBp65. In addition, MSKE induced p21 expression independent of wild-type p53 induced protein expression. Moreover, we demonstrate that MSKE significantly inhibited cell migration in PC-3 prostate cancer cells. Overall, these results demonstrate that MSKE inhibits prostate tumor growth and migration, and induces cell-cycle arrest by targeting Hsp40 and proteins involved in cell-cycle regulation and proliferation. This suggests that MSKE may also be explored either as a neo-adjuvant or therapeutic for castration resistant prostate cancer.

Cordyceps sinensis Promotes the Growth of Prostate Cancer Cells

Background: This study aims to test whether Cordyceps sinensis (CS), the most expensive Asian nutrient supplement might stimulate growth of prostate cancer cells. Methods: Impact of CS on growth of prostate cancer was determined in vivo and in vitro.Results: Firstly, the serum testosterone level was significantly elevated in mice fed CS. Prostate glands were significantly enlarged (weight index 0.53 ± 0.04 mg/g vs. 0.31 ± 0.04 mg/g, P = 0.006). Furthermore, cell viability was increased twofold in the androgen-responsive prostate cancer cell line (VCaP) after CS treatment. This promoting effect disappeared after bicalutamide was added. In addition, serum prostate-specific antigen (PSA) in mice bearing VCaP xenografts was significantly elevated (0.66 ± 0.04 ng/ml vs. 0.26 ± 0.06 ng/ml, P < 0.001) after treatment with CS. Finally, VCaP tumors in mice treated with CS grew much faster (479.2 ± 78.74 mm3 vs. 283 ± 58.97 mm3, P = 0.074). However, the above promoting effects of CS were not observed in parallel studies using the PC-3 cell line which lacks AR expression.Conclusions: These results suggest that CS promotes growth of prostate cancer cells by increasing production of testosterone and stimulating the AR-dependent pathway. Additional studies are required to see whether CS is safely consumed by patients with prostate cancer.

Abstract B008: PMEPA1 isoform specific regulation of androgen and TGF beta signaling in prostate cancer

Abstract Introduction and Objectives: The dysfunctions of androgen receptor (AR) and TGF-β signaling contribute to prostate tumorigenesis and cancer progression. Our laboratory has identified PMEPA1 as an androgen responsive gene with prostate abundance, as well as PMEPA1/NEDD4 and AR negative feedback loop in the regulation of AR levels in prostate cancer cells. In addition, it has also been established by other groups that PMEPA1 inhibits TGF-β signaling via a similar negative feedback loop as a TGF-β responsive gene. Five isoforms are transcribed from distinct promoters within the PMEPA1 locus. PMEPA1 isoforms were shown to have variations at the N-terminus of the protein. This study focuses on understanding of the expression and biologic functions of PMEPA1 isoforms in CaP. Methods: The PMEPA1 isoforms were evaluated for expressions in multiple CaP cell lines, trensfectant derivatives, and prostate tumors. LNCaP cells were treated with R1881 (0, 0.1, 1.0 nM) and DU-145 and PC-3 cells were treated with TGF-β (0, 5 and 25 ng/ml) for 24 hours. PMEPA1 isoform specific plasmids and siRNAs were transfected into LNCaP, DU-145, and PC-3 cells individually. Cell proliferation was analyzed by cell counting, cell plating efficiency assay, and soft agar assay. The protein levels of PMEPA1 isoforms, AR, PSA, TGF-β receptor I, Smad2 were detected by immunoblotting, and the transcript levels of KLK3 (PSA), TGF-β responsive genes including THBS1, NEDD9 and COL1A1 were evaluated by QRT-PCR. The TGF-β signaling activity was measured by SMAD reported dual-luciferase assay. The transcript levels of PMEPA1-252, PMEPA3-287-STAG1, AR, PSA in 120 matched human benign and malignant frozen tissue were evaluated with Q-PCR. Results: The expressions of PMEPA1-252 and PMEPA2-344 isoforms were restricted to androgen-responsive prostate cancer cells in comparison to broader expression pattern of other isoforms (PMEPA3-287/STAG1, PMEPA4-259 and PMEPA5-237). The expression of PMEPA1-2 was androgen regulated, whereas expression of PMEPA 3-5 was regulated by TGF-β. Only PMEPA1-252 inhibited cell growth of LNCaP, DU-145 and PC-3 cells. In contrast, PMEPA2-4 promoted cell growth of DU-145 and PC-3 cells. Only PMEPA1-252 mediated AR protein degradation and inhibited AR signaling. In contrast, PMEPA3-287-STAG1 and PMEPA4-259 inhibited TGF-β signaling luciferase activity and transcript levels of THBS1, NEDD9 and COL1A1. All PMEPA1 isoforms were found to have no effects on protein level of TGF-β receptor I. Moreover, the deletion mutants of N-terminus and transmembrane domains interrupted the isoform specific inhibitory effects on AR/TGF-β signaling. The transcript level of PMEPA1-252 was higher than PMEPA3-287-STAG1 in benign prostate tissue. Different from PMEPA1-252, the expression of PMEPA3-287-STAG1 was not found to decrease in prostate tumor tissue. There was no significant correlation between the expression of PMEPA1-252 and PMEPA3-287-STAG1 in prostate tissue. The transcript of PMEPA1-252 was associated with PSA, but the expression of PMEPA3-287-STAG1 was found not to be associated with PSA or AR in CaP cells. Conclusions: The PMEPA isoforms appear to underscore distinct biologic functions in the context of androgen and TGF-β signaling. Intensively studied PMEPA1-252 was specific for AR degradation in prostate cancer cells and was consistent with previous observations of association of AR upregulation with loss of PMEPA1 in prostate cancer. The roles of PMEPA1 isoforms need to be better defined in prostate cancer and other cancers. Funding: This study was supported by CPDR, USUHS, HU0001-10-2-0002 to I.L.R. Citation Format: Hua Li, Shashwat Sharad, Lakshmi Ravindranath, Gyorgy Petrovics, Yongmei Chen, Alagarsamy Srinivasan, Inger Rosner, Albert Dobi, Shiv Srivastava. PMEPA1 isoform specific regulation of androgen and TGF beta signaling in prostate cancer [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 B008.

Abstract B48: Effect of concomitant CYP3A5-modulating drugs on androgen-receptor signaling in African American men undergoing androgen deprivation therapy

Abstract Introduction: In a recent study we have established that CYP3A5 augments nuclear translocation of androgen receptor (AR) and promotes prostate cancer (PC) growth. CYP3A isoforms are responsible for metabolizing almost 50% of the commonly prescribed drugs, many of which are CYP3A5 inhibitors and inducers. PC patients are elderly with comorbidities and are often prescribed concomitant medications while undergoing androgen-deprivation therapy (ADT), the most preferred treatment for PC. CYP3A5 is polymorphic, and African Americans (AA, 73%) express higher levels of CYP3A5 due to presence of wild-type CYP3A5 (*1/*1) compared to Caucasians (95%) expressing the mutant (*3/*3) producing only 13% of the active full-length CYP3A5 protein. Due to the expression of wild-type CYP3A5 in AA, concomitant medications modulating CYP3A5 activity can drastically alter AR activity and ADT efficacy in these patients. Our current work is focused on deciphering the mechanism of how commonly used CYP3A5-modulating medications can alter AR signaling in AA carrying the wild-type CYP3A5. Method: We genotyped the androgen-responsive prostate cancer cell lines to determine their CYP3A5 status using TaqMan® Drug Metabolism assay. We used cDNA from cells treated with either nontarget or CYP3A5 siRNA in RT2 profiler PCR arrays to identify set of genes in AR signaling pathway that are affected by CYP3A5 inhibition. Confocal microscopy was used to study AR nuclear localization after exposure to commonly used CYP3A5 inducer/inhibitor drugs. Cell fractionation experiments were used to further confirm the effect of these drugs on AR signaling. Luciferase-based reporter assay was used to test effect of CYP3A5-modulating drugs on AR activity. Results: Our genotyping assay revealed that MDA PCa 2b (African American) carries *1/*3 CYP3A5 gene compared to LNCaP (Caucasian) carrying *3/*3 genotype. To compare the differences due to CYP3A5 genotype on AR signaling, we selected MDAPCa2b and LNCaP cells for further studies. Consistent with earlier observations with LNCaP, MDA PCa 2b cell lines also showed decreased nuclear translocation of AR with CYP3A5 siRNA in both confocal and cell fractionation experiments. We also observed blocking of AR nuclear localization with azamulin, a CYP3A-specific inhibitor in both cell lines. The q-PCR-based profiler assay revealed a set of nine AR-regulated genes (SCL45A3, FKBP5, NCAPD3, MYC, MME, ELL2, PIK3R3, HPRT1, and SPDEF) downregulated with CYP3A5 siRNA treatment with a p value of ≤0.005 in MDA PCa 2b cells. MYC is known to be involved in cell cycle progression and apoptosis whereas PIK3R3 codes for the regulatory subunit of PI3 kinase regulating cell growth. SPDEF codes for a transcription factor promoting PSA known to aid in the cell growth. Downregulation of these genes with CYP3A5 siRNA treatment indicates that CYP3A5 modifier medications can alter AR downstream signaling regulating growth. We tested some commonly prescribed drugs that are either CYP inhibitors (amiodarone, ritonavir) or inducers (phenytoin, rifampicin) and evaluated their ability to alter AR signaling. The results revealed that the CYP inducers promoted increased AR nuclear migration and increased PSA production whereas CYP3A5 inhibitors blocked AR nuclear migration, decreasing PSA expression in both cell lines. A luminescence-based reporter assay using androgen response elements also supported that these CYP3A inducer and inhibitor drugs alter AR signaling. Conclusion: Our results indicate that concomitant medications that can alter CYP3A5 activity can modify AR signaling and PC growth. These results suggest that these CYP-modulating drugs can alter efficacy of ADT severely in AA men predominantly expressing wild-type CYP3A5 (*1/*1). We hypothesize that AR-directed therapeutic efficacy can be optimized by proper drug selection through minimizing the use of AR inducers while maximizing the use of AR inhibitors in patients undergoing ADT. Citation Format: Priyatham Gorjala, Oscar B. Goodman Jr., Ranjana Mitra. Effect of concomitant CYP3A5-modulating drugs on androgen-receptor signaling in African American men undergoing androgen deprivation therapy [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B48.

CYP17A1-independent production of the neurosteroid-derived 5α-pregnan-3β,6α-diol-20-one in androgen-responsive prostate cancer cell lines under serum starvation and inhibition by Abiraterone

CYP17A1-independent intratumoral steroid hormone synthesis is regarded as one possible explanation for resistance to treatment with the CYP17-inhibitor Abiraterone (Abi). The aim of our study was therefore to investigate the steroid metabolism of prostate cancer cells under serum starvation and the effects of Abi treatment.We assessed steroid metabolism in a panel of prostate cancer cells under serum starvation by radioactivity detector-coupled HPLC and HPLC-ESI-ToF-mass spectrometry after treatment with pregnenolone, progesterone and allopregnanolone. We further evaluated the effects of Abi on steroid metabolism of testosterone, dihydrotestosterone (DHT) and dehydroepiandrosterone (DHEA) by enzyme immunoassays (EIAs).Androgen-responsive cell lines metabolized pregnenolone primarily to mitogenic steroid 5α-pregnan-3β,6α-diol-20-one under serum starvation. Co-administration of Abi lead to detectable concentrations of the Abi metabolite Δ4-Abi (D4A), known to inhibit enzymes other than CYP17A1 in steroid metabolism. In addition, co-administration of Abi abrogated pregnenolone metabolism and resulted in a CYP17A1-independent significant increase of DHEA (13- to >100-fold) and DHT (2.5-fold) in androgen-responsive cells.Our results demonstrate the CYP17A1-independent formation of 5α-pregnan-3β,6α-diol-20-one by androgen-responsive prostate cancer cells under serum starvation and its inhibition by Abi. Its metabolism from pregnenolone suggests a major steroidogenesis shift in these cells, hinting at a neuroendocrine transdifferentiation phenomenon. The marked increase of DHEA levels by Abi resembles the steroidogenic pathways in nervous tissue, in a manner that precludes CYP17A1 activity. To which extent these processes are responsible or involved in the development of resistance to Abi, needs to be further elucidated.

Abstract 1578: Distinct expression and biological functions of PMEPA1 isoforms in prostate cancer

Abstract Introduction and Objectives: The androgen receptor (AR) and TGF-β dysfunctions contribute to prostate cancer (CaP) development and progression. Our laboratory since the discovery of PMEPA1, has established PMEPA1/NEDD4 and AR negative feedback loop and its role in AR regulation in prostate cancer. PMEPA1 gene has also identified as a TGF-β responsive gene and inhibits TGF-β signaling via a negative feedback loop. Five isoforms are transcribed from distinct promoters within the PMEPA1 locus. PMEPA1 isoforms were shown to have variations at the N-terminus of the protein. Our previous study has shown the differential correlations of 2 PMEPA isoforms (PMEPA1 and STAG1) in context of AR expression in CaP. This study focuses on understanding of the expression and biological functions of PMEPA1 isoforms in CaP. Methods: RNA-Seq data from LNCaP and VCaP cells, and from The Cancer Genome Atlas (TCGA) dataset, were used to evaluate the expression levels of PMEPA1 isoforms. LNCaP cells were treated with R1881 (0, 0.1, 1.0 nM) and DU-145 and PC-3 cells were treated with TGF-β (0, 5 and 25 ng/ml) for 24 hours. PMEPA1 isoform specific plasmids and siRNAs were transfected into LNCaP, DU-145 and PC-3 cells individually. Cell growth was analyzed by cell counting and BrdU incorporation assay. The protein levels of PMEPA1 isoforms and AR were detected by immunoblotting, and the transcript levels were evaluated by QRT-PCR. Results: Since the annotation of PMEPA1/TMEPA1 isoforms has been empirical in the literature, here we propose a new structure/expression based nomenclature: PMEPA1 (reading frame of 252 amino acids (aa)); PMEPA2 (344aa); PMEPA3 (287aa; STAG1); PMEPA4 (259aa); and PMEPA5 (237aa). Expression of PMEPA1 and PMEPA2 was restricted to androgen responsive prostate cancer cells in comparison to broader expression pattern of other isoforms (PMEPA3-5). The expression of PMEPA1-2 was androgen regulated, whereas expression of PMEPA 3-5 was regulated by TGF-β. Only PMEPA1 inhibited cell growth of LNCaP, DU-145 and PC-3 cells. In contrast, PMEPA2-4 promoted cell growth of DU-145 and PC-3 cells. Of all the isoforms only PMEPA1 mediated AR protein degradation in CaP. Conclusions: The PMEPA isforms appear to underscore distinct biological functions in the context of androgen and TGF-β signaling. Widely studied PMEPA1 was specific for AR degradation in prostate cancer cells and was consistent with previous observations of association of AR upregulation with loss of PMEPA1 in prostate cancer. The roles of PMEPA isoforms need to be better defined in prostate cancer and other cancers. Funding: This study was supported by CPDR, USUHS, HU0001-10-2-0002 to DGM. Citation Format: Shashwat Sharad, Hua Li, Allissa Dillman, Alagarsamy Srinivasan, Albert Dobi, Shiv Srivastava. Distinct expression and biological functions of PMEPA1 isoforms in 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 1578. doi:10.1158/1538-7445.AM2017-1578

Abstract 3978: B7-H1/PD-L1 soluble expression in multiple cancer subtypes: High sensitivity measurement by immunoassay

Abstract Aberrant expression of the B7-H1/PD-L1 immunomodulatory molecule is evident in many forms of cancer. The evidence for increased protein levels of B7-H1/PD-L1 comes largely from immunohistochemistry analysis of surgical and biopsy specimens. While several studies attempt to correlate B7-H1/PD-L1 expression with poor prognosis, there are studies that conclude no correlation based on technical difficulties associated with detecting B7-H1/PD-L1 protein expression on the cell surface. We were able to quantifiably measure soluble B7-H1/PD-L1 in conditioned medium from tumor cell cultures using two new immunoassays, a Quantikine® ELISA and a Simple Plex™ Assay. We show that breast, prostate, glioma, and non-Hodgkin’s lymphoma cells express various levels of B7-H1/PD-L1 which correlate to their PTEN or proliferation status. This is in agreement with previous findings showing that increases in B7-H1/PD-L1 expression levels are frequently coincident with loss of the PTEN tumor suppressor or increased activation of the phosphoinositide 3-kinase (PI 3-K) pathway. The androgen-responsive LNCaP prostate cancer cell line does not exhibit measurable B7-H1/PD-L1 in any instance, while the androgen-independent prostate cancer cell line PC-3 expresses measurable B7-H1/PD-L1 that is further inducible with PMA treatment. In all cell lines included in our study, incubation with PMA increases the level of B7-H1/PD-L1 released into the culture media, while pretreatment with the PI 3-K inhibitor, LY294002, abrogates this increase. Importantly, the Simple Plex platform is able to quantify very low levels of B7-H1/PD-L1 that are below detection by ELISA. Our data suggest that combining the Quantikine ELISA and Simple Plex platforms is a powerful strategy for reliably and quantitatively assessing soluble B7-H1/PD-L1 levels. Citation Format: Justin Haworth, Rainer Grant, Samuel Thayer, Melissa Floren, Nathan Steere, Isabel O'Brien, Paul Younge, Michael Anderson, Greta Wegner, Kathryn Brumbaugh. B7-H1/PD-L1 soluble expression in multiple cancer subtypes: High sensitivity measurement by immunoassay [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 3978. doi:10.1158/1538-7445.AM2017-3978

Abstract 853: C1orf116, a gene with unknown function, is a novel driver of epithelial phenotype in epithelial-to-mesenchymal transition in human cancer

Abstract Metastatic prostate cancer is an incurable lethal disease and is the cause of death for 28,000 men in the US annually. In order for a cancer cell to metastasize from the primary tumor, it must move. In a critical early event of metastasis, a rare population of cells in the primary tumor undergoes a phenotype switch from a proliferating epithelial cell to gaining the migratory capacity of a mesenchymal cell, a process, termed epithelial-to-mesenchymal transition (EMT). To identify previously overlooked regulators of EMT, we undertook a multi-study gene expression array analysis (15 published gene expression studies; 95 total samples; 6 cancer types) and identified C1orf116 as a candidate driver of the epithelial phenotype in EMT across multiple cancer types. Of note, C1orf116 is an unnamed gene of unknown function that is largely uncharacterized in any setting, including in cancer biology or in EMT. To substantiate its potential role in EMT, we queried the NCI-60 cell line collection gene expression dataset and found that C1orf116 shared a similar expression pattern with epithelial marker genes (ESRP1, ESRP2, OVOL1, OVOL2) and a converse expression pattern to mesenchymal marker genes (CDH2, SNAI2, VIM). Moreover, we found that C1orf116 expression is decreased in metastatic prostate cancer tumors compared to localized disease (Oncomine), suggesting the potential for clinical translation. To specifically interrogate the role of C1orf116 in EMT, we utilized the PC3 cell line, an in vitro model of prostate cancer EMT. We found that C1orf 116 expression was higher in an epithelial PC3 cell line (PC3-epi) compared to PC3 cells that had undergone a stable EMT (PC3-emt). Moreover, shRNA-mediated knockdown of C1orf116 expression in PC3-epi cells resulted in decreased gene expression of canonical epithelial markers (OVOL1, ESRP1, CDH1) and elevated expression of standard mesenchymal markers (CDH2, ZEB1, VIM), suggesting a role as a driver of the epithelial phenotype. Interestingly, the sole publication specifically studying C1orf116 describes it as Specifically Androgen Regulated Gene (SARG). The authors demonstrate the presence of an androgen response element upstream of the C1orf116 start site the in androgen-responsive prostate cancer cell line LNCaP. Our data indicate a role for C1orf116 in the androgen receptor-negative prostate cancer cell line PC3, suggesting a non-androgen-mediated role for C1orf116 in prostate cancer. In addition to elucidating its role in EMT, work is underway to characterize the RNA and protein products of C1orf116. Alternative splicing results in two transcripts of varying length that likely give rise to at least two protein product sizes. RNA sequencing and mass spectrometry will reveal transcript and protein identity and provide insight into potential isoform switching regulating EMT and other processes. Citation Format: Sarah R. Amend, James Hernandez, Princy Parsana, Kenneth J. Pienta. C1orf116, a gene with unknown function, is a novel driver of epithelial phenotype in epithelial-to-mesenchymal transition in human 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 853. doi:10.1158/1538-7445.AM2017-853

Inhibition of androgen receptor promotes CXC-chemokine receptor 7-mediated prostate cancer cell survival

The atypical C-X-C chemokine receptor 7 (CXCR7) has been implicated in supporting aggressive cancer phenotypes in several cancers including prostate cancer. However, the mechanisms driving overexpression of this receptor in cancer are poorly understood. This study investigates the role of androgen receptor (AR) in regulating CXCR7. Androgen deprivation or AR inhibition significantly increased CXCR7 expression in androgen-responsive prostate cancer cell lines, which was accompanied by enhanced epidermal growth factor receptor (EGFR)-mediated mitogenic signaling, promoting tumor cell survival through an androgen-independent signaling program. Using multiple approaches we demonstrate that AR directly binds to the CXCR7 promoter, suppressing transcription. Clustered regularly interspaced short palindromic repeats (CRISPR) directed Cas9 nuclease-mediated gene editing of CXCR7 revealed that prostate cancer cells depend on CXCR7 for proliferation, survival and clonogenic potential. Loss of CXCR7 expression by CRISPR-Cas9 gene editing resulted in a halt of cell proliferation, severely impaired EGFR signaling and the onset of cellular senescence. Characterization of a mutated CXCR7-expressing LNCaP cell clone showed altered intracellular signaling and reduced spheroid formation potential. Our results demonstrate that CXCR7 is a potential target for adjuvant therapy in combination with androgen deprivation therapy (ADT) to prevent androgen-independent tumor cell survival.

Proteasome-associated deubiquitinase ubiquitin-specific protease 14 regulates prostate cancer proliferation by deubiquitinating and stabilizing androgen receptor

Androgen receptor (AR) is frequently over-expressed and plays a critical role in the growth and progression of human prostate cancer. The therapy attempting to target AR signalling was established in decades ago but the treatment of prostate cancer is far from being satisfactory. The assignable cause is that our understanding of the mechanism of AR regulation and re-activation remains incomplete. Increasing evidence suggests that deubiquitinases are involved in the regulation of cancer development and progression but the specific underlying mechanism often is not elucidated. In the current study, we have identified ubiquitin-specific protease 14 (USP14) as a novel regulator of AR, inhibiting the degradation of AR via deubiquitinating this oncoprotein in the androgen-responsive prostate cancer cells. We found that (i) USP14 could bind to AR, and additionally, both genetic and pharmacological inhibition of USP14 accelerated the ubiquitination and degradation of AR; (ii) downregulation or inhibition of USP14 suppressed cell proliferation and colony formation of LNcap cells and, conversely, overexpression of USP14 promoted the proliferation; and (iii) reduction or inhibition of USP14 induced G0/G1 phase arrest in LNcap prostate cancer cells. Hence, we conclude that USP14 promotes prostate cancer progression likely through stabilization of AR, suggesting that USP14 could be a promising therapeutic target for prostate cancer.