Enzalutamide Resistance Research Articles

Abstract 1445: TBX2 acts as a molecular switch to downregulate androgen receptor and upregulate glucocorticoid receptor signaling in castrate resistant prostate cancer

Abstract Background: A major obstacle in the treatment of metastatic castrate resistant prostate cancer (mCRPC) is acquired resistance to androgen deprivation therapy (ADT). It is now recognized that ADT, particularly the 2nd generation androgen receptor (AR) antagonists, such as enzalutamide, orchestrate plasticity changes/molecular alterations leading to therapy resistance. It is hypothesized in the field that the emergence of castrate resistance to 2nd generation ADT is driven by a switch/bypass from AR signaling to the glucocorticoid receptor (GR) signaling. Therefore, identifying the molecular mechanisms that concurrently drive the loss of AR and gain of GR signaling may be crucial in devising novel and effective therapeutic modalities against mCRPC. We have previously reported that TBX2, a T-box transcription factor (TF) with both repressor and activator functions, is over-expressed in CRPC and that TBX2 drives PCa bone metastatic progression. In agreement with our findings, a recent report showed that TBX2 is a key TF that drives plasticity associated with CRPC. In this study, we investigated the molecular mechanisms by which TBX2 drives plasticity associated with enzalutamide resistance. Methods: We genetically modulated TBX2 using the dominant negative (DN), sh-RNA, and overexpression (OE) approaches. RNA-seq was performed, and qRT-PCR, Western blot and immunohistochemical (IHC) analyses were used for validation. Further, we performed chromatin immunoprecipitation (ChIP) and site directed mutagenesis (SDM). Results: Using publicly available databases, we observed a negative correlation between TBX2 and AR; and a positive correlation between TBX2 and GR. Strikingly, blocking TBX2 expression in wildtype PC3 human PCa cells that do not express AR mRNA or protein led to a marked elevation of AR signature as assessed by RNA-seq analysis. These results were confirmed/validated at the protein level using additional human PCa cell lines. Further, these results were validated using all three approaches to genetically modulate TBX2, i.e. DN, shRNA, and OE. Further, ChIP and SDM analyses confirmed that TBX2 directly binds and transcriptionally represses AR. Conversely, genetic modulation of TBX2 was positively associated with GR expression, and TBX2 was found to directly bind to the GR promoter. Further, we found that knock-down of GR in LNCaP cells overexpressing TBX2 (LNCaPTBX2OE) reversed enzalutamide resistance that is associated with elevated TBX2 expression. Conclusions: Our studies suggest that TBX2 acts as the molecular switch that on one hand represses AR and on the other hand activates GR. Further our study paves the way for potential therapeutic strategies against the AR/GR switch/bypass in CRPC wherein AR and GR signaling could be co-inhibited through targeting TBX2. Citation Format: Sayanika Dutta, Girijesh Patel, Hamed Khedmatgozar, Daniel Latour, Manisha Tripathi, Srinivas Nandana. TBX2 acts as a molecular switch to downregulate androgen receptor and upregulate glucocorticoid receptor signaling in castrate resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1445.

Abstract 1455: The glucocorticoid receptor-LEDGFp75 interaction in prostate cancer therapy cross-resistance

Abstract Prostate cancer (PCa) is the second leading cause of cancer deaths in the U.S., disproportionally affecting African American (AA) men. PCa patients with recurrent disease develop therapy resistance and fail to respond to both anti-androgen receptor signaling (ARSI) therapy and taxane-based chemotherapy. Glucocorticoid receptor, a transcription factor, has been implicated in resistance to ARSI (via the GR bypass), and docetaxel (DTX) therapies. However, the mechanisms underlying this GR-mediated therapy cross-resistance are poorly understood. Previously, we demonstrated that glucocorticoids, which are co-administered with current PCa therapies and activate GR signaling, upregulate the chemoresistance-associated transcription coactivator LEDGFp75 in PCa cells. We also identified consensus GR binding sites in the promoter region of the gene encoding LEDGFp75, suggesting it is a GR target gene. We hypothesized that GR transcriptionally upregulates LEDGFp75 and then interacts with this protein in the nucleus to promote DTX resistance in PCa cells. Genetic silencing of GR in a panel of DTX-sensitive and -resistant PCa cell lines decreased the expression of LEDGFp75 at both the protein and transcript levels, confirming its status as a candidate GR target gene. However, genetic silencing of LEDGFp75 had no effects on GR protein expression. Pharmacological inhibition of GR also decreased LEDGFp75 in DTX-sensitive cells. The effects of Exicorilant and Relacorilant (Corcept Therapeutics) were evaluated on LEDGFp75 protein expression levels. Immunoprecipitation and confocal microscopy studies revealed that GR and LEDGFp75 interact in the nucleus of PCa cells. Interestingly, upregulation of GR in enzalutamide resistant LNCaP cells correlated with LEDGFp75 upregulation, and GR silencing in these cells decreased this upregulation. These results suggested a possible role for GR and LEDGFp75 in PCa therapy cross-resistance. Further studies are underway to determine if co-targeting these two proteins genetically and pharmacologically attenuates both enzalutamide resistance and DTX resistance and other aggressive properties of PCa cells. In addition, RNAseq studies have been initiated to determine the degree of transcriptional overlap between GR and LEDGFp75 in chemoresistant PCa cells. Our goal is to link mechanistically the GR-LEDGFp75 transcriptional network to ARSI/taxane cross-resistance in PCa and target this network to attenuate therapy resistance. Citation Format: Evelyn S. Sanchez-Hernandez, Pedro T. Ochoa, Greisha L. Ortiz-Hernandez, Shannalee Martinez, Carlos Casiano. The glucocorticoid receptor-LEDGFp75 interaction in prostate cancer therapy cross-resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1455.

Abstract 3141: Epiregulon infers single-cell transcription factor activity to dissect mechanisms of lineage plasticity and drug response

Abstract Single-cell multiomic technologies enable holistic reconstruction of cell states and hold the potential to identify drivers of tumor evolution and drug resistance. However, the data sparsity of single-cell assays can preclude accurate estimation of transcription factor activity. In addition, gene expression alone cannot capture transcription factor activity, especially in the context of drug treatment which alters transcription factor function without suppressing gene expression. To circumvent these challenges, we have developed epiregulon, a R package that constructs gene regulatory networks and infers transcription factor (TF) activity in single cells by integrating single cell gene expression, chromatin accessibility and bulk TF occupancy data. Epiregulon applies tests of independence to identify likely transcription factor - regulatory element - target gene relationships occurring at joint probabilities exceeding the expected probabilities of independent events. With epiregulon, we are able to detect lineage factor activity at enhanced sensitivity and predict perturbations more accurately than gene expression could. We further applied this tool to understand transcription factor activity to enzalutamide treatment and identify potential drivers of enzalutamide resistance. Finally, we generated a ground truth dataset using reprogram-seq, a technology that captures multi-omic profiles of single cells upon expression of defined transcription factors. Epiregulon correctly predicts NKX2-1 targets and demonstrates that NKX2-1 expression reprograms the epigenome towards a neuroendocrine state in LNCaP cells. Epiregulon is a useful tool that constructs gene regulatory networks to model the underlying gene regulation hierarchies that drive gene expression and cell states. Citation Format: Tomasz Wlodarczyk, Jenille Tan, Kerstin Seidel, Diana Wu, Shang-Yang Chen, Aleksander Chlebowski, Timothy Keyes, Yu Guo, Aaron Lun, Christopher Siebel, Shiqi Xie, Xiaosai Yao. Epiregulon infers single-cell transcription factor activity to dissect mechanisms of lineage plasticity and drug response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3141.

Abstract 6035: Glutaminase inhibitors block metabolic switch in prostate cancer and prevents anti-androgen treatment resistance

Abstract Background: All prostate cancers first manifest as androgen dependent tumors and most patients are initially treated with surgery or radiation. About 30% of these patients return to clinic with recurrent prostate cancer that initially responds to androgen ablation therapy (ADT). Unfortunately, almost all of them fail ADT and develop castrate resistant disease (CRPC) that responds poorly to most cancer therapies and progress to often lethal metastatic CRPC (mCRPC). There are accumulating evidence that at a certain state of progression of hormone sensitive prostate cancer (HSPC) to CRPC, prostate cancer cells adopt to androgen signaling axis targeted therapy (ASI) by switching their metabolism from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). High OXPHOS activity induces mitochondrial dysfunction, autophagy, cancer invasion, and metastasis. A mitochondrial enzyme glutaminase (GLS) produces L-glutamate from L-glutamine. L-glutamate is utilized in mitochondrial metabolism for energy production through TCA cycle. GLS inhibitors CB-839 and IACS-6274 are being clinically developed for treatment of various human malignancies. We have shown that growth of prostate cancer patient derived xenografts (PDX) is markedly inhibited by a combination treatment of a clinically used ASI enzalutamide (ENZA) followed by a GLS inhibitor. Methods: We followed the growth of one HSPC and two CRPC PDX tumors in mice and collected tumor tissues and circulating human tumor cells (CTC) from mouse blood samples at sacrifice. We used super resolution STED confocal microscopy to image mitochondria in the tissue samples and in the CTCs, desorption ionization mass spectrometry (DESI-MS) imaging of multiple metabolites in the tissue samples as well as hyperpolarized MRI (HP-MRI) of the xenografted tumors to follow the changes in cellular central carbon metabolism in vivo. Results: We observed that growth of PDXs developing ENZA resistance is markedly inhibited when ENZA is followed by adding GLS inhibitors with a concomitant reduction in OXPHOS activity. In addition, the ex vivo and in vivo methods introduced to monitor tumor metabolic activities can be easily translated into clinic to monitor the metabolic status of the tumors for early detection of resistance development and as a pharmacodynamic marker for metabolic inhibitor therapy. Conclusion: GLS inhibitors added to ASI could be effective against CRPC and mCRPC developing resistance to ASI treatment. Citation Format: Hirak Subhra Basu, Meredith Spradlin, Tian Weihua, Jose Enriquez, Pratip Bhattacharya, Livia Eberlin. Glutaminase inhibitors block metabolic switch in prostate cancer and prevents anti-androgen treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6035.

PD04-11 MCT INHIBITION RESENSITIZES ENZALUTAMIDE-RESISTANT PROSTATE CANCER CELLS TO ENZALUTAMIDE

You have accessJournal of UrologyCME1 Apr 2023PD04-11 MCT INHIBITION RESENSITIZES ENZALUTAMIDE-RESISTANT PROSTATE CANCER CELLS TO ENZALUTAMIDE Sayani Bhattacharjee, Rebecca Wynn, Puneet Sindhwani, Firas Petros, and Nagalakshmi Nadiminty Sayani BhattacharjeeSayani Bhattacharjee More articles by this author , Rebecca WynnRebecca Wynn More articles by this author , Puneet SindhwaniPuneet Sindhwani More articles by this author , Firas PetrosFiras Petros More articles by this author , and Nagalakshmi NadimintyNagalakshmi Nadiminty More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003228.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate cancers (PCa) exhibit a unique metabolic profile with reliance on different forms of glucose metabolism at different stages of disease progression. Early stage PCa cells use the more efficient TCA cycle, while metastatic PCa cells switch to glycolysis (Warburg effect), leading to the accumulation of lactate. Monocarboxylate transporters (MCTs) play a major role in the export/import of lactate to maintain redox balance. Such metabolic reprogramming can lead to gain-of-function mutations and affect drug sensitivity. MCT expression levels are high in enzalutamide-resistant PCa cells. Hence, we hypothesized that MCT inhibition may be an attractive therapeutic strategy to overcome enzalutamide resistance in PCa cells. METHODS: We analyzed the expression levels of MCTs in PCa tissues using public datasets from Oncomine. We treated C4-2B and 22Rv1 parental and enzalutamide-resistant cells with varying concentrations of MCT inhibitors AR-C155858, AZD3965, or syrosingopine either singly or in combination with enzalutamide and assessed cell survival, cell viability, proliferation, clonogenic ability, survival in 3-D models, and glycolytic activity. We treated mouse xenografts of PCa cells with MCT inhibitors or enzalutamide either singly or in combination and assessed tumor growth. RESULTS: We found that the suppression of MCT activity using the MCT antagonists AR-C155858, AZD3965, or syrosingopine significantly diminished the proliferation, survival, and clonogenic ability of enzalutamide-resistant PCa cells compared with parental cells. MCT inhibition in combination with enzalutamide inhibited the metabolic adaptation of PCa cells. Tumor growth of enzalutamide-resistant PCa cell xenografts was suppressed significantly when treated with a combination of MCT inhibitors with enzalutamide. These findings imply that MCT inhibition may be an effective strategy to counter enzalutamide resistance in PCa. CONCLUSIONS: MCT activation may underlie the development of enzalutamide resistance in PCa and targeting it may represent a viable combinatorial option. Source of Funding: Department of Defense Prostate Prostate Cancer Research Program W81XWH2010794 (PC190332) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e146 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sayani Bhattacharjee More articles by this author Rebecca Wynn More articles by this author Puneet Sindhwani More articles by this author Firas Petros More articles by this author Nagalakshmi Nadiminty More articles by this author Expand All Advertisement PDF downloadLoading ...

PD04-09 COMBINATION TREATMENT WITH HSP70/STUB1 MODULATOR AND ENZALUTAMIDE YIELDS SYNERGISTIC ACTIVITY IN ADVANCED PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023PD04-09 COMBINATION TREATMENT WITH HSP70/STUB1 MODULATOR AND ENZALUTAMIDE YIELDS SYNERGISTIC ACTIVITY IN ADVANCED PROSTATE CANCER Pengfei Xu, Joy C. Yang, Bo Chen, Christopher Nip, Shu Ning, Allen C. Gao, Hao Shao, Jason Gestwicki, Christopher P. Evans, and Chengfei Liu Pengfei XuPengfei Xu More articles by this author , Joy C. YangJoy C. Yang More articles by this author , Bo ChenBo Chen More articles by this author , Christopher NipChristopher Nip More articles by this author , Shu NingShu Ning More articles by this author , Allen C. GaoAllen C. Gao More articles by this author , Hao ShaoHao Shao More articles by this author , Jason GestwickiJason Gestwicki More articles by this author , Christopher P. EvansChristopher P. Evans More articles by this author , and Chengfei LiuChengfei Liu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003228.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Ubiquitin proteasome system is suppressed in enzalutamide resistant prostate cancer cells and that the HSP70/STUB1 machinery is involved in androgen receptor (AR) and AR variant protein stabilization. Targeting HSP70 could be a valuable strategy to overcome the resistance of androgen receptor signaling inhibitors (ARSI) in advanced prostate cancer. In this study, we tested novel HSP70/STUB1modulators in enzalutamide resistant cells and patient-derived prostate cancer cell models. METHODS: Expression of AR-V7, HSP70 and STUB1 were determined by qRT-PCR and western blot. Expression of HSP70 and STUB1 was down-regulated using specific siRNA. HSP70/STUB1 and AR-V7 interaction was determined by co-immunoprecipitation and dual immunofluorescence. Tumor organoids were established from the patient derived xenografts (PDX) and the viability was stained by LIVE/DEAD™ Cell Imaging Kit. The effects of HSP70 inhibitors on enzalutamide sensitivity were examined in cell line model and patient-derived cell models. RESULTS: A novel HSP70 inhibitor JG98 significantly suppressed drug resistant C4-2B MDVR and CWR22Rv1 cell growth and enhanced enzalutamide treatment (p<0.001). JG98 also suppressed the PDX derived organoid growth and induced organoid death in a dose dependent manner (p<0.001). Mechanistically, JG98 suppressed AR/AR-V7 expression in resistant cells and promoted STUB1 entering into the nucleus and bound to AR-V7. Knockdown of STUB1 diminished the anti-cancer effect and AR-V7 inhibition by JG98. A more potent HSP70 inhibitor JG231 was developed from JG98. JG231 significantly improved the drug solubility and showed better pharmacokinetic characteristics than JG98. Moreover, JG231 effectively suppressed the cell growth and enhanced enzalutamide treatment in resistant cells and PDX derived prostate cancer cells (p<0.001). CONCLUSIONS: The HSP70/STUB1 machinery involved in AR/AR-V7 regulation and enzalutamide resistance. Targeting HSP70 by novel HSP70/STUB1 modulators overcome the ARSI resistance and improve their therapy. Source of Funding: This work was supported in part by grants NIH/NCI R37CA249108 (C, Liu) and R01CA251253 (C, Liu) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e145 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Pengfei Xu More articles by this author Joy C. Yang More articles by this author Bo Chen More articles by this author Christopher Nip More articles by this author Shu Ning More articles by this author Allen C. Gao More articles by this author Hao Shao More articles by this author Jason Gestwicki More articles by this author Christopher P. Evans More articles by this author Chengfei Liu More articles by this author Expand All Advertisement PDF downloadLoading ...

A kinome-wide CRISPR screen identifies CK1α as a target to overcome enzalutamide resistance of prostate cancer

Enzalutamide (ENZA), a second-generation androgen receptor antagonist, has significantly increased progression-free and overall survival of patients with metastatic prostate cancer (PCa). However, resistance remains a prominent obstacle in treatment. Utilizing a kinome-wide CRISPR-Cas9 knockout screen, we identified casein kinase 1α (CK1α) as a therapeutic target to overcome ENZA resistance. Depletion or pharmacologic inhibition of CK1α enhanced ENZA efficacy in ENZA-resistant cells and patient-derived xenografts. Mechanistically, CK1α phosphorylates the serine residue S1270 and modulates the protein abundance of ataxia telangiectasia mutated (ATM), a primary initiator of DNA double-strand break (DSB)-response signaling, which is compromised in ENZA-resistant cells and patients. Inhibition of CK1α stabilizes ATM, resulting in the restoration of DSB signaling, and thus increases ENZA-induced cell death and growth arrest. Our study details a therapeutic approach for ENZA-resistant PCa and characterizes a particular perspective for the function of CK1α in the regulation of DNA-damage response.

Abstract P1-13-14: Increased androgen receptor expression as a mechanism of acquired anti-androgen resistance in androgen receptor-positive triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) represents the most aggressive breast cancer subtype, and despite recent treatment advances, clinical outcomes remain poor, particularly in the metastatic setting. TNBC is a biologically heterogeneous entity, and up to 50% of TNBC express the androgen receptor (AR). Gene expression subtyping has identified a subset of AR-TNBC with a luminal AR-driven (LAR) phenotype. Consequently, there has been great interest in translating androgen receptor signaling inhibitors (ARSIs) approved for use in prostate cancer such as enzalutamide and bicalutamide into the management of LAR-TNBC. However, early phase clinical trials of anti-androgens in metastatic AR-TNBC have had modest results. While multiple mechanisms of anti-androgen resistance have been identified in prostate cancer, including AR amplification and over-expression, emergence of AR splice variants (AR-Vs), and transition to AR independent growth, molecular determinants of anti-androgen resistance in TNBC remain poorly understood, and pre-clinical models to study acquired ARSI resistance in LAR-TNBC have been limited. Methods: A novel pre-clincial model of acquired ARSI resistance was derived from the LAR subtype MDA-MB-453 cell line through serial passaging with increasing concentrations of enzalutamide. Celltiter Blue viability assays were used to determine enzalutamide IC50 of parental and enzalutamide-resistant (EnzR) cell lines. RNA was extracted from the parental and resistant cells, reverse transcribed and expression of AR, splice-variants and canonical AR target genes were evaluated using RT-qPCR. Immunofluorescence was used to investigate the amount and nuclear localization of the AR protein within the parental and enzalutamide resistant cells. Results: EnzR MDA-MB-453 cells were derived through culture in increasing concentrations of enzalutamide for &amp;gt;6 months, and found to have a significant increase in enzalutamide IC50 compared to the parental cell line. EnzR MDA-MB-453 cells had a 4.5 fold increase in full-length AR expression at the transcriptional level compared to the parental cell line. AR protein expression and nuclear localization were also increased in EnzR cells compared to the parental cell line. Canonical AR targets including NKX3.1 were downregulated in response to enzalutamide in parental but not EnzR cells. While pathogenic AR splice variants (AR-Vs) implicated in ARSI resistance in prostate cancer were detected at low levels in MDA-MB-453 cells, no increase in AR-V expression was observed in the EnzR cell line. Conclusions: We have developed a novel pre-clinical model of anti-androgen/ARSI resistance in LAR-TNBC, and identified AR overexpression and persistent AR signaling associated with acquired enzalutamide resistance in LAR-TNBC. While pathogenic AR splice variants have been implicated in ARSI resistance in metastatic prostate cancer and are detected in the MDA-MB-453 cell line model, enzalutamide resistance was not associated with increased AR splice variant expression in this model. Future work will investigate mechanisms leading to AR overexpression as well as combination therapeutic strategies to overcome acquired ARSI resistance. Citation Format: Hannah Krause, Marina N. Sharifi, Serena K. Wolfe, Jamie M. Sperger, Kari B. Wisinski, Ruth O’Regan, Joshua Lang. Increased androgen receptor expression as a mechanism of acquired anti-androgen resistance in androgen receptor-positive triple negative breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-14.

Enzalutamide-induced signatures revealed by epigenetic plasticity using single-cell multi-omics sequencing in prostate cancer

Prostate cancer is morphologically and molecularly heterogeneous, which poses obstacles for early diagnosis and treatment. Advancements in understanding the heterogeneity of prostate cancer will help navigate through these challenges and ultimately benefit patients. In this study, we integrated single-cell sequencing for transposase-accessible chromatin and whole transcriptome in prostate cancer cell lines, aiming to decode the epigenetic plasticity upon enzalutamide (ENZ) treatment. By comparing the cell populations representing early-treatment response or resistance to the initial tumor cells, we identified seven signature gene sets; they present consistent trends of chromatin closing co-occurred with down-regulated genes during early response and chromatin opening with up-regulated genes upon maintaining drug resistance. In the molecular signatures, we found genes ZNF337, MAPK15, and ESRRG are favorable in progression-free prognosis during early response, while genes CCDC150, CCDC18, and POC1A marked poor prognosis underpinning the pre-existing drug resistance in The Cancer Genome Atlas prostate adenocarcinoma cohort. Ultimately, drug-target analyses nominated combinatory drug candidates to either enhance early-treatment response or potentially overcome ENZ resistance. Together, our integrative, single-cell multi-omics approach in pre-clinical models is effective in identifying informative signatures from complex molecular events, illustrating diverse drug responses in prostate cancer, and invoking novel combinatory drug strategies to inform clinical decision making.

Macrophages promote anti-androgen resistance in prostate cancer bone disease.

Metastatic castration-resistant prostate cancer (PC) is the final stage of PC that acquires resistance to androgen deprivation therapies (ADT). Despite progresses in understanding of disease mechanisms, the specific contribution of the metastatic microenvironment to ADT resistance remains largely unknown. The current study identified that the macrophage is the major microenvironmental component of bone-metastatic PC in patients. Using a novel in vivo model, we demonstrated that macrophages were critical for enzalutamide resistance through induction of a wound-healing-like response of ECM-receptor gene expression. Mechanistically, macrophages drove resistance through cytokine activin A that induced fibronectin (FN1)-integrin alpha 5 (ITGA5)-tyrosine kinase Src (SRC) signaling cascade in PC cells. This novel mechanism was strongly supported by bioinformatics analysis of patient transcriptomics datasets. Furthermore, macrophage depletion or SRC inhibition using a novel specific inhibitor significantly inhibited resistant growth. Together, our findings elucidated a novel mechanism of macrophage-induced anti-androgen resistance of metastatic PC and a promising therapeutic approach to treat this deadly disease.

Knockdown of RhoA Expression Reverts Enzalutamide Resistance via the p38 MAPK Pathway in Castration-resistant Prostate Cancer.

Enzalutamide has been approved clinically for the treatment of castrationresistant prostate cancer (CRPC) but is limited by the emergence of resistance. RhoA has been shown to play a vital role in carcinogenesis, invasion, and metastasis. However, the role of RhoA in enzalutamide-resistant prostate cancer (PCa) remains unclear. This study investigated the role of RhoA and the associated mechanisms of RhoA depletion in enzalutamide resistance in CRPC. Western blotting, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and colony formation assays were used to assess protein expression, survival, and proliferation of PCa cells, respectively. Xenograft experiments and hematoxylin and eosin (H&E) staining were used to detect further effects of RhoA on enzalutamide resistance in vivo. In the present study, the expression of RhoA, ROCK2, p38, p-p38, and AR was upregulated in enzalutamide-resistant PCa cells treated with enzalutamide, and silencing of RhoA or ROCK2 attenuated enzalutamide-resistant cell proliferation and colony formation. Furthermore, the deletion of RhoA dramatically increased the efficacy of enzalutamide in inhibiting 22RV1-derived xenograft tumor growth. Additionally, there was no significant change in ROCK1 expression in C4-2R cells treated with or without enzalutamide. Mechanistically, the knockdown of RhoA expression reverted the resistance to enzalutamide via RhoA/ROCK2/p38 rather than RhoA/ROCK1/p38. Our results suggested that RhoA is a promising therapeutic target. As the inhibition of RhoA reverted enzalutamide resistance, it may increase its effectiveness in CRPC.

VIM‑AS1 promotes proliferation and drives enzalutamide resistance in prostate cancer via IGF2BP2‑mediated HMGCS1 mRNA stabilization.

VIM‑AS1, a cancer‑specific long non‑coding RNA, has been recognized as a pivotal regulator in multiple types of cancer. However, the role of VIM‑AS1 in the proliferation and resistance to anti‑androgen therapy of LNCaP and C4‑2 prostate cancer cells remains to be determined. In the current study, gain‑and‑loss experiments were used to investigate the effects of VIM‑AS on the proliferation and anti‑androgen therapy of LNCaP and C4‑2 cells. RNA sequencing, RNA pulldown and RNA immunoprecipitation were used to elucidate the underlying mechanism of VIM‑AS1 driving prostate progression. It was demonstrated that VIM‑AS1 was upregulated in C4‑2 cells, an established castration‑resistant prostate cancer (CRPC) cell line, compared with in LNCaP cells, an established hormone‑sensitive prostate cancer cell line. The present study further demonstrated that VIM‑AS1 was positively associated with the clinical stage of prostate cancer. Functionally, overexpression of VIM‑AS1 decreased the sensitivity to enzalutamide treatment and enhanced the proliferation of LNCaP cells invitro, whereas knockdown of VIM‑AS1 increased the sensitivity to enzalutamide treatment and reduced the proliferation of C4‑2 cells invitro and invivo. Mechanistically, 3‑hydroxy‑3‑methylglutaryl‑CoA synthase 1 (HMGCS1) was identified as one of the direct downstream targets of VIM‑AS1, and VIM‑AS1 promoted HMGCS1 expression by enhancing HMGCS1 mRNA stability through a VIM‑AS1/insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2)/HMGCS1 RNA‑protein complex. Rescue assays indicated that knockdown of HMGCS1 expression ameliorated the increase in proliferation and enzalutamide resistance of prostate cancer cells induced by VIM‑AS1 overexpression. Overall, the present study determined the roles and mechanism of the VIM‑AS1/IGF2BP2/HMGCS1 axis in regulating proliferation and enzalutamide sensitivity of prostate cancer cells and suggested that VIM‑AS1 may serve as a novel therapeutic target for the treatment of patients with CRPC.

Daclatasvir, an Antiviral Drug, Downregulates Tribbles 2 Pseudokinase and Resensitizes Enzalutamide-Resistant Prostate Cancer Cells.

FDA-approved enzalutamide is commonly prescribed to reduce the growth of advanced prostate cancer by blocking androgen receptor function. However, enzalutamide-resistant prostate cancer (ERPC) invariably develops and progresses to metastatic, lethal disease. Management of ERPC poses a special problem not only because available therapeutic regimens cannot effectively kill ERPC cells but also due to their propensity to invade large bones. Moreover, molecular mechanism(s) behind enzalutamide resistance is not properly understood, which is delaying development of newer agents. We found that the pseudokinase, Tribbles 2 (TRIB2), is overexpressed in ERPC cells and plays a critical role in their survival. Forced overexpression of TRIB2 enhances prostate cancer cell growth and confers resistance to physiologic doses of enzalutamide, suggesting that TRIB2 plays an important role in the development and progression of ERPC. Though TRIB2 has emerged as an excellent molecular target for ERPC, suitable inhibitors are not commercially available for effective targeting. By designing a luciferase-tagged TRIB2 fusion protein-based assay system, we screened a library of about 1,600 compounds and found that daclatasvir (DCV), an antiviral drug, effectively inhibits TRIB2-luciferase. We also found that DCV degrades TRIB2 proteins by direct binding and resensitizes ERPC cells to enzalutamide treatment. Moreover, DCV at lower, sublethal doses synergizes with enzalutamide to decrease the viability and induce apoptosis in prostate cancer cells. Because DCV is already approved by the FDA and well tolerated in humans, based on our findings, it appears that DCV is a promising new agent for development of an effective therapy for advanced, enzalutamide-resistant, lethal prostate cancer.

CircROBO1 promotes prostate cancer growth and enzalutamide resistance via accelerating glycolysis.

Background and aim: As non-coding RNAs, circular RNAs (circRNAs) contribute to the progression of malignancies by regulating various biological processes. In prostate cancer, however, there is still a lack of understanding regarding the potential molecular pathways and roles of circRNAs. Methods: Loss-off function experiments were performed to investigate the potential biological function of circRNA in the progression of prostate cancer. Western blot, qRT-PCR, and IHC assay were used to examine the expression level of different genes or circRNAs. Further molecular biology experiments were conducted to uncover the molecular mechanism underlying circRNA in prostate cancer using dual luciferase reporter and RNA immunoprecipitation (RIP) assays. Results: A novel circRNA (hsa_circ_0124696, named circROBO1) was identified as a significantly upregulated circRNA in both prostate cancer cells and tissues. Suppression of circROBO1 significantly attenuated the proliferation of prostate cancer cells. In addition, we found that the knockdown of circROBO1 remarkably increased the sensitivity of prostate cancer to enzalutamide treatment. A deceleration in glycolysis rate was observed after inhibition of circROBO1, which could suppress prostate cancer growth and overcome resistance to enzalutamide. Our results revealed that circROBO1 promotes prostate cancer growth and enzalutamide resistance via accelerating glycolysis. Conclusion: Our study identified the biological role of the circROBO1-miR-556-5p-PGK1 axis in the growth and enzalutamide resistance of prostate cancer, which is the potential therapeutic target of prostate cancer.

A synthetic lethal screen for Snail-induced enzalutamide resistance identifies JAK/STAT signaling as a therapeutic vulnerability in prostate cancer

A synthetic lethal screen for Snail-induced enzalutamide resistance identifies JAK/STAT signaling as a therapeutic vulnerability in prostate cancer

Overexpression of RACGAP1 by E2F1 Promotes Neuroendocrine Differentiation of Prostate Cancer by Stabilizing EZH2 Expression.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer. It is characterized by the loss of androgen receptor (AR) signaling in neuroendocrine transdifferentiation, and finally, resistance to AR-targeted therapy. With the application of a new generation of potent AR inhibitors, the incidence of NEPC is gradually increasing. The molecular mechanism of neuroendocrine differentiation (NED) after androgen deprivation therapy (ADT) remains largely unclear. In this study, using NEPC-related genome sequencing database analyses, we screened RACGAP1, a common differentially expressed gene. We investigated RACGAP1 expression in clinical prostate cancer specimens by IHC. Regulated pathways were examined by Western blotting, qRT-PCR, luciferase reporter, chromatin immunoprecipitation, and immunoprecipitation assays. The corresponding function of RACGAP1 in prostate cancer was analyzed by CCK-8 and Transwell assays. The changes of neuroendocrine markers and AR expression in C4-2-R and C4-2B-R cells were detected in vitro. We confirmed that RACGAP1 contributed to NE transdifferentiation of prostate cancer. Patients with high tumor RACGAP1 expression had shorter relapse-free survival time. The expression of RACGAP1 was induced by E2F1. RACGAP1 promoted neuroendocrine transdifferentiation of prostate cancer by stabilizing EZH2 expression in the ubiquitin-proteasome pathway. Moreover, overexpression of RACGAP1 promoted enzalutamide resistance of castration-resistant prostate cancer (CRPC) cells. Our results showed that the upregulation of RACGAP1 by E2F1 increased EZH2 expression, which drove NEPC progression. This study explored the molecular mechanism of NED and may provide novel methods and ideas for targeted therapy of NEPC.

Idarubicin combats abiraterone and enzalutamide resistance in prostate cells via targeting XPA protein

Although second-generation therapies like abiraterone (ABI) and enzalutamide (ENZ) benefit patients with castration-resistant prostate cancer (CRPC), drug resistance frequently occurs, eventually resulting in therapy failure. In this study, we used two libraries, FDA-approved drug library and CRISP/Cas9 knockout (GeCKO) library to screen for drugs that overcome treatment resistance and to identify the potential drug-resistant genes involved in treatment resistance. Our screening results showed that the DNA-damaging agent idarubicin (IDA) overcame abiraterone and enzalutamide resistance in prostate cancer cells. IDA treatment inhibited the DNA repair protein XPA expression in a transcription-independent manner. Consistently, XPA knockout sensitized prostate cancer cells to abiraterone and enzalutamide treatment. In conclusion, IDA combats abiraterone and enzalutamide resistance by reducing XPA protein level in prostate cancer.

A novel inhibitor of ARfl and ARv7 induces protein degradation to overcome enzalutamide resistance in advanced prostate cancer.

Enzalutamide (ENZ) is a second-generation androgen receptor (AR) antagonist used for the treatment of castration-resistant prostate cancer (CRPC) and reportedly prolongs survival time within a year of starting therapy. However, CRPC patients can develop ENZ resistance (ENZR), mainly driven by abnormal reactivation of AR signaling, involving increased expression of the full-length AR (ARfl) or dominantly active androgen receptor splice variant 7 (ARv7) and ARfl/ARv7 heterodimers. There is currently no efficient treatment for ENZR in CRPC. Herein, a small molecule LLU-206 was rationally designed based on the ENZ structure and exhibited potent inhibition of both ARfl and constitutively active ARv7 to inhibit PCa proliferation and suppress ENZR in CRPC. Mechanically, LLU-206 promoted ARfl/ARv7 protein degradation and decreased ARfl/ARv7 heterodimers through mouse double minute 2-mediated ubiquitination. Finally, LLU-206 exhibited favorable pharmacokinetic properties with poor permeability across the blood-brain barrier, leading to a lower prevalence of adverse effects, including seizure and neurotoxicity, than ENZ-based therapies. In a nutshell, our findings demonstrated that LLU-206 could effectively inhibit ARfl/ARv7-driven CRPC by dual-targeting of ARfl/ARv7 heterodimers and protein degradation, providing new insights for the design of new-generation AR inhibitors to overcome ARfl/ARv7-driven CRPC.

ODP440 Characterization of the Major Single Nucleotide Polymorphic Variants of Aldo-Keto Reductase 1c3 (Type 5 17β-Hydroxysteroid Dehydrogenase)

Abstract Background Aldo-keto-reductase (AKR) 1C3, also known as type 517β-hydroxysteroid dehydrogenase and prostaglandin F synthase,is a member of the AKR superfamily that reduces aldehydes and ketones to primary and secondary alcohols. It plays an essential role in the peripheral formation of androgens and is associated with several steroid hormone dependent diseases including prostate cancer (PC), breast cancer, and polycystic ovarian syndrome (PCOS). Furthermore, AKR1C3 is implicated in castration resistant prostate cancer (CRPC) drug resistance on the basis that it is upregulated in CRPC tumors and metastases and overexpressed in cell culture &amp; xenograft models of Abiraterone and Enzalutamide resistance, where resistance is surmounted by AKR1C3 knockdown or treatment with the AKR1C3 inhibitor indomethacin. AKR1C3 has 14 nonsynonymous single nucleotide polymorphisms (nsSNPs) with different global frequencies and ethnic distributions. Association studies support their role in PC and other diseases, but detailed functional genomic studies of these variants are lacking. One study examined5 AKR1C3 nsSNPs for their ability to reduce exemestane, an aromatase inhibitor used to treat breast cancer, to 17β-dihydroexemestane, and reported a 17-250-fold reduction in catalytic efficiency of H5Q, E77G, K104D, and R258C variants compared to WT. This observation provided the impetus to examine impact of these variants on AKR1C3 function in the context of PC. We purified AKR1C3 WT, and the top four most frequently occurring nsSNPs, H5Q, E77G, K104D, and R258C, from E. coli to expand upon their characterization and illuminate functional differences that could affect disease outcome and treatment. METHODS: Kinetic measurements were made using either a continuous spectrophotometric assay monitoring the appearance of the reduced cofactor NADH, or a discontinuous RP UV-HPLC assay based on chromatographic separation of substrate and product. Inhibitor sensitivity studies were conducted with a fluorescence-based plate-reader assay monitoring enzyme activity via oxidation of a standard substrate in the presence of increasing inhibitor. Stability was assessed by monitoring the percent of maximum activity remaining via oxidation of a standard substrate over a range of increasing temperatures for each variant. Results We report no major deviations in kinetics in the top four most frequently occurring AKR1C3 variants compared to WT. Additionally, variants did not display differences in inhibitor sensitivity to competitive inhibitor indomethacin. However, the K104D variant lost activity at a lower temperature than WT, suggesting that this mutation results in a decrease in stability. Conclusions Altogether, these findings contrast with previous claims that AKR1C3 variants, mainly H5Q, give rise to significant advantage or disadvantage over WT in PC and other diseases, and suggests that patients with AKR1C3 dependent diseases would likely be overall unaffected by variant status. Presentation: No date and time listed

Repression of SLC22A3 by the AR-V7/YAP1/TAZ axis in enzalutamide-resistant castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is an aggressive and fatal disease, with most patients succumbing within 1-2 years despite undergoing multiple treatments. Androgen-receptor (AR) inhibitors, including enzalutamide (ENZ), are used for the treatment of mCRPC; however, most patients develop resistance to ENZ. Herein, we propose that the repression of SLC22A3 by AR-V7/YAP1/TAZ conferred ENZ resistance in mCRPC. SLC22A3 expression is specifically downregulated in the ENZ-resistant C4-2B MDVR cells, and when YAP1/TAZ is hyperactivated by AR full-length or AR-V7, these proteins interact with DNMT1 to repress SLC22A3 expression. We observed low SLC22A3 expression and high levels of TAZ or YAP1 in mCRPC patient tissues harbouring AR-V7 and the opposite expression patterns in normal patient tissues. Our findings suggest a mechanism underlying ENZ resistance by providing evidence that the AR-V7/YAP1/TAZ axis represses SLC22A3, which could be a potential treatment target in prostate cancer.