Role In Castration-resistant Prostate Cancer Research Articles

Abstract 4369: CYP3A5 promotes aggressive and therapeutically resistant prostate cancer by modulating AR and Wnt signaling: Implications for racially disparate clinical outcomes

Abstract Prostate cancer (PC) is the second leading cause of cancer-related death in American men, which disproportionately affects Black/African American (AA) men. The androgen receptor (AR) signaling plays a pivotal role in PC development and is a primary target for intervention in patients with advanced disease. Aberrant activation of AR is also suggested to play a significant role in castration-resistant prostate cancer (CRPC). Despite advancements in anti-androgen therapies with new targeting agents, such as enzalutamide, metastatic CRPC (mCRPC) remains incurable. CYP3A5, a monooxygenase expressed in the prostate, liver and intestine, is involved in drug metabolism and steroid biosynthesis. We earlier reported that intratumoral CYP3A5 activated AR signaling by facilitating its nuclear translocation of AR. More notably, we found a higher CYP3A5 expression in AAs, attributed to the *1 CYP3A5 variant, as compared to non-Hispanic White Americans (NHWA) possessing the *3 variant. Analysis of RNA-seq data using patient tumor samples revealed that elevated CYP3A5 in AA PC patients was associated with Wnt-β catenin signaling activation via TCF4 overexpression. Here, we investigated the role of CYP3A5 in enzalutamide resistance and Wnt signaling by generating enzalutamide-resistant PC cell lines of NHWA (low CYP3A5-LNCaP) and AA-origin (high CYP3A5-MDAPCa2b). RT-qPCR assay was used to examine the changes in AR and CYP3A5 expression between the parental and the enzalutamide-resistant cell lines. The data show heightened CYP3A5 expression in enzalutamide-resistant cells of AA origin (MDAPCa2b/EnzR) but not of NHWA origin (LNCaP/EnzR). Interestingly, MDAPCa2b/EnzR exhibited no change in AR levels whereas it was increased in LNCaP/EnzR cells relative to parental lines. A loss of function assay was performed for siRNA-mediated silencing of CYP3A5 to study its effect on Wnt signaling. RNA sequencing results show that CYP3A5 inhibition in MDAPCa2b downregulated Wnt pathway genes (Wnt5A, Wnt10B, Wnt11, Fzd2, and Dvl3). These observations align with our previous RNA seq results using patient samples where we observed upregulated Wnt-β catenin signaling in high CYP3A5 expressing patient tumor samples. Western analysis post-CYP3A5 inhibition in LNCaP and MDAPCa2b cells revealed upregulated Axin and downregulated LRP6 and Dvl-3, signifying CYP3A5's regulation of the canonical Wnt-β catenin pathway. Additionally, MDAPCa2b saw downregulated Wnt 5A and Wnt 10B, known to influence the Wnt non-canonical pathway. Altogether, our data suggest that *1 CYP3A5 variant in AAs likely contributes to aggressive behavior and therapeutic resistance of PC and serves as a molecular determinant of disparate clinical outcomes. Citation Format: Jake McLean, Ajay P. Singh, Edwin Oh, Ranjana Mitra. CYP3A5 promotes aggressive and therapeutically resistant prostate cancer by modulating AR and Wnt signaling: Implications for racially disparate clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4369.

Melittin inhibits tumor cell migration and enhances cisplatin sensitivity by suppressing IL-17 signaling pathway gene LCN2 in castration-resistant prostate cancer.

Melittin is a small molecule polypeptide extracted from the abdominal cavity of bees, which is used to treat inflammatory diseases and relieve pain. However, the antitumor effect of melittin and its mechanisms remain unclear, especially in castration-resistant prostate cancer (CRPC). Through CCK-8 assay, colony formation assay, wound healing assay and Transwell migration assay, we explored the effect of melittin on CRPC cell lines. In addition, with microarray analysis, gene ontology analysis and kyoto encyclopedia of genes and genomes analysis, this study identified key genes and signaling pathways that influence the growth of PC-3 cells. Meanwhile, the effect of melittin on CRPC was also verified through subcutaneous tumor formation experiments. Finally, we also tested the relevant indicators of human prostate cancer (PCa) specimens through immunohistochemistry and H＆E stating. Here, melittin was verified to inhibit the cell proliferation and migration of CPRC. Moreover, RNA-sequence analysis demonstrated that Interleukin-17 (IL-17) signaling pathway gene Lipocalin-2 (LCN2) was downregulated by melittin treatment in CRPC. Further investigation revealed that overexpression of LCN2 was able to rescue tumor suppression and cisplatin sensitivity which melittin mediated. Interestingly, the expression of LCN2 is highly related to metastasis in PCa. In brief, our study indicates that LCN2 plays an oncogenic role in CRPC and melittin may be selected as an attractive candidate for CRPC therapy.

Switching mechanism from AR to EGFR signaling via 3-O-sulfated heparan sulfate in castration-resistant prostate cancer

Androgen deprivation therapy is given to suppress prostate cancer growth; however, some cells continue to grow hormone-independently as castration-resistant prostate cancer (CRPC). Sulfated glycosaminoglycans promote ligand binding to receptors as co-receptors, but their role in CRPC remains unknown. Using the human prostate cancer cell line C4-2, which can proliferate in hormone-dependent and hormone-independent conditions, we found that epidermal growth factor (EGF)-activated EGFR–ERK1/2 signaling via 3-O-sulfated heparan sulfate (HS) produced by HS 3-O-sulfotransferase 1 (HS3ST1) is activated in C4-2 cells under hormone depletion. Knockdown of HS3ST1 in C4-2 cells suppressed hormone-independent growth, and inhibited both EGF binding to the cell surface and activation of EGFR–ERK1/2 signaling. Gefitinib, an EGFR inhibitor, significantly suppressed C4-2 cell proliferation and growth of a xenografted C4-2 tumor in castrated mouse. Collectively, our study has revealed a mechanism by which cancer cells switch to hormone-independent growth and identified the key regulator as 3-O-sulfated HS.

Discovery of a Novel Bifunctional Steroid Analog, YXG-158, as an Androgen Receptor Degrader and CYP17A1 Inhibitor for the Treatment of Enzalutamide-Resistant Prostate Cancer.

The androgen/androgen receptor (AR) signaling pathway plays an important role in castration-resistant prostate cancer (CRPC). Bifunctional agents that simultaneously degrade AR and inhibit androgen synthesis are expected to block the androgen/AR signaling pathway more thoroughly, demonstrating the promising therapeutic potential for CRPC, even enzalutamide-resistant CRPC. Herein, a series of steroid analogs were designed, synthesized, and identified as selective AR degraders, among which YXG-158 (23-h) was the most potent antitumor compound with dual functions of AR degradation and CYP17A1 inhibition. In addition, 23-h abrogated the hERG inhibition and exhibited excellent PK profiles. In vivo, 23-h effectively inhibited the growth of hormone-sensitive organs in the Hershberger assay and exhibited robust antitumor efficacy both in enzalutamide-sensitive (LNCaP/AR) and enzalutamide-resistant (C4-2b-ENZ) xenograft models. Thus, 23-h was chosen as a preclinical candidate for the treatment of enzalutamide-resistant prostate cancer.

Investigation of the regulatory effects of synthesized antisense oligonucleotides on androgen receptor (AR) exon 3 splicing in prostate cancer cells

The Androgen Receptor (AR) gene plays a key role in castration-resistant prostate cancer (CRPC). Controlling the progression of CRPC by inhibiting AR gene expression is one of the core directions for prostate cancer (Pca) drug development. A 23-amino acids retention, named exon 3a, into the DNA binding domain of the splice variant AR23 has been shown to prevent AR from entering the nucleus and restore the sensitivity of cancer cells to related therapies. In this study, we conducted a preliminary investigation of the splicing modulation of the AR gene in order to develop a splice-switching therapy for Pca by promoting exon 3a inclusion. Using mutagenesis-coupled RT-PCR with AR minigene and over-expression of certain splicing factors, we found that serine/arginine-rich (SR) proteins are key factors facilitating the recognition of the 3′ splice site of exon 3a (L-3′ SS), while the deletion or blocking of the polypyrimidine tract (PPT) region of the original 3′ splice site of exon 3 (S-3′ SS) could strongly enhance exon 3a splicing without affecting the function of any SR protein. Furthermore, we designed a series of antisense oligonucleotides (ASOs) to screen drug candidates, and ASOs targeting S-3′ SS and its PPT region or the exonic region of exon 3 turned out to be most effective in rescuing exon 3a splicing. A dose–response test indicated ASO12 as the lead candidate drug significantly promoting the inclusion of exon 3a to more than 85%. MTT assay confirmed that the cell proliferation was significantly inhibited after ASO treatment. Our results provide the first glance to AR splicing regulation. With several promising therapeutic ASO candidates obtained here, further development of ASO drugs to treat CRPC is strongly encouraged.

Molecular Mechanisms of Noncoding RNA in the Occurrence of Castration-Resistant Prostate Cancer.

Although several therapeutic options have been shown to improve survival of most patients with prostate cancer, progression to castration-refractory state continues to present challenges in clinics and scientific research. As a highly heterogeneous disease entity, the mechanisms of castration-resistant prostate cancer (CRPC) are complicated and arise from multiple factors. Among them, noncoding RNAs (ncRNAs), the untranslated part of the human transcriptome, are closely related to almost all biological regulation, including tumor metabolisms, epigenetic modifications and immune escape, which has encouraged scientists to investigate their role in CRPC. In clinical practice, ncRNAs, especially miRNAs and lncRNAs, may function as potential biomarkers for diagnosis and prognosis of CRPC. Therefore, understanding the molecular biology of CRPC will help boost a shift in the treatment of CRPC patients. In this review, we summarize the recent findings of miRNAs and lncRNAs, discuss their potential functional mechanisms and highlight their clinical application prospects in CRPC.

Hsa_circ_0092339 acts as a molecular sponge in castration-resistant prostate cancer via the hsa-mir-940/C-MYC axis.

Aims: We aimed to determine whether intronic circRNA acts as a molecular sponge in castration-resistant prostate cancer (CRPC). Materials & methods: A gene chip technique was used to conduct sequencing. A qPCR experiment was performed to verify the result. Radioimmunoprecipitation, RNA pull-down and dual-luciferase reporter assays were performed to particularly expound its function. Verification of downstream effects was carried out through qPCR and western blot, and a xenograft assay was performed in vivo for verification. Results: Intronic circRNA hsa_circ_0092339 in the nucleus was highly expressed in CRPC cell lines. hsa_circ_0092339 did not regulate the expression of its parental gene. hsa_circ_0092339 functions like a molecular sponge, preventing degradation of C-MYC mRNA by absorbing hsa-mir-940. Conclusion: hsa_circ_0092339 plays a critical role in CRPC through targeting C-MYC indirectly by absorbing hsa-mir-940.

Mechanisms of Resistance to Second-Generation Antiandrogen Therapy for Prostate Cancer: Actual Knowledge and Perspectives.

Prostate cancer therapy for locally advanced and metastatic diseases includes androgen deprivation therapy (ADT). Second-generation antiandrogens have a role in castration-resistant prostate cancer. Nevertheless, some patients do not respond to this therapy, and eventually all the patients became resistant. This is due to modifications to intracellular signaling pathways, genomic alteration, cytokines production, metabolic switches, constitutional receptor activation, overexpression of some proteins, and regulation of gene expression. The aim of this review is to define the most important mechanisms that drive this resistance and the newest discoveries in this field, specifically for enzalutamide and abiraterone, with potential implications for future therapeutic targets. Furthermore, apalutamide and darolutamide share some resistance mechanisms with abiraterone and enzalutamide and could be useful in some resistance settings.

Spindle pole body component 25 in the androgen-induced regression of castration-resistant prostate cancer.

BackgroundAndrogen plays a critical role in the development and growth of prostate cancer (PCa) by binding to the androgen receptor, a steroid receptor for testosterone and dihydrotestosterone (DHT). Androgen deprivation therapy, a clinical endocrine therapy, has resulted in increases in the occurrence of castration-resistant prostate cancer (CRPC); however, the mechanisms of CRPC have not yet fully been determined. We previously showed that spindle pole body component 25 (SPC25), a component of the NDC80 complex that is critical in kinetochore formation and chromosome segregation during the cell cycle, plays a critical role in PCa tumorigenesis and cancer stemness. However, it is not yet known whether SPC25 plays a role in CRPC; thus, we sought to address this question in the current study.MethodsSPC25 levels were detected in androgen-insensitive PCa cells using the public database and bioinformatics tools. In vitro, SPC25 levels were determined in androgen-sensitive and androgen-insensitive PCa cells treated with or without DHT. The growth of the PCa cells was assessed by the Cell Counting Kit-8 assay. The invasiveness and migratory potential of the PCa cells were assessed by the transwell cell invasive assay and migratory assay, respectively. Gain-of-function and loss-of-function experiments examined the transfection of androgen-sensitive and androgen-insensitive PCa cells by plasmids carrying small-interfering ribonucleic acids for SPC25 or SPC25, respectively.ResultsSPC25 levels were significantly reduced in the androgen-insensitive PCa cells treated with DHT in the Public database. In vitro, PCa cell growth, invasion, and metastasis was reduced in androgen-insensitive PCa cells but increased in androgen-sensitive PCa cells treated with DHT, partially through DHT-regulated expression of SPC25 at transcriptional but not at translational levels.ConclusionsAndrogen treatment reduces CRPC growth, invasion, and metastasis partially through its regulation of SPC25. SPC25 represents a promising target in the treatment of CRPC.

CYP1B1-catalyzed 4-OHE2 promotes the castration resistance of prostate cancer stem cells by estrogen receptor \u03b1-mediated IL6 activation

BackgroundResistance to androgen deprivation therapy remains a major challenge for the clinical treatment of patients with castration-resistant prostate cancer (CRPC). CYP1B1, a critical enzyme that catalyzes the conversion of estradiol to 4-Hydroxy-17β-estradiol (4-OHE2), has been reported to promote the development and progression of hormone-related cancer, but its role in CRPC is unclear.MethodsTo explore the underlying mechanism which CYP1B1 promotes the prostate cancer stem cells (PCSCs) characteristics, bioinformatics analyses of human clinical prostate cancer (PCa) datasets were performed. CYP1B1, IL6, and estrogen receptor-α (ERα) expression levels were evaluated in PCa and CRPC tissues via immunohistochemistry. The high-performance liquid chromatography-mass spectrometry assay was carried out to examine intracellular 4-OHE2 levels. Serum-free suspension culture and flow cytometry assays were performed to evaluate PCSCs. Chromatin immunoprecipitation was used to validate that 4-OHE2 recruited ERα to the IL6 promoter.ResultsCYP1B1 expression was significantly increased in CRPC tissues and androgen-independent PCa cell lines. CYP1B1+ PCa cells were significantly enriched in bicalutamide-treated LNCaP cells, and CYP1B1 knockdown reduced the cell viability under bicalutamide treatment. In addition, CYP1B1 knockdown decreased the intracellular 4-OHE2 concentration, accompanied by reduced PCSC characteristics. In PCa cells, 4-OHE2 stimulated ERα transcriptional activity and upregulated the expression of IL6 and downstream genes of the IL6-STAT3 signaling. 4-OHE2 increased cell viability under bicalutamide treatment and promoted PCSC characteristics, while IL6 neutralizing antibody reversed these effects. Mechanistically, siERα and the ER antagonist ICI182780 significantly attenuated 4-OHE2-induced IL6 expression, and 4-OHE2 promoted the binding of ERα to the estrogen response element of the IL6 promoter.ConclusionsOur findings indicate that CYP1B1-catalyzed 4-OHE2 enhanced PCSC characteristics and attenuated bicalutamide sensitivity by ERα-mediated the IL6-STAT3 pathway activation. Our study further emphasizes the role of CYP1B1 in castration resistance and illustrates a novel mechanism of CRPC development.Graphical 54dyAth8NatsYXqV4_5FMeVideo .

Androgen receptor-mediated transcriptional repression targets cell plasticity in prostate cancer.

Androgen receptor (AR) signaling remains the key therapeutic target in the management of hormone‐naïve‐advanced prostate cancer (PCa) and castration‐resistant PCa (CRPC). Recently, landmark molecular features have been reported for CRPC, including the expression of constitutively active AR variants that lack the ligand‐binding domain. Besides their role in CRPC, AR variants lead to the expression of genes involved in tumor progression. However, little is known about the specificity of their mode of action compared with that of wild‐type AR (AR‐WT). We performed AR transcriptome analyses in an androgen‐dependent PCa cell line as well as cross‐analyses with publicly available RNA‐seq datasets and established that transcriptional repression capacity that was marked for AR‐WT was pathologically lost by AR variants. Functional enrichment analyses allowed us to associate AR‐WT repressive function to a panel of genes involved in cell adhesion and epithelial‐to‐mesenchymal transition. So, we postulate that a less documented AR‐WT normal function in prostate epithelial cells could be the repression of a panel of genes linked to cell plasticity and that this repressive function could be pathologically abrogated by AR variants in PCa.

The HeyL-Aromatase Axis Promotes Cancer Stem Cell Properties by Endogenous Estrogen-Induced Autophagy in Castration-Resistant Prostate Cancer.

Treatment of patients with castration-resistant prostate cancer (CRPC) remains a major clinical challenge. We previously showed that estrogenic effects contribute to CRPC progression and are primarily caused by the increased endogenous estradiol produced via highly expressed aromatase. However, the mechanism of aromatase upregulation and its role in CRPC are poorly described. In this study, we report that HeyL is aberrantly upregulated in CRPC tissues, and its expression is positively correlated with aromatase levels. HeyL overexpression increased endogenous estradiol levels and estrogen receptor-α (ERα) transcriptional activity by upregulating CYP19A1 expression, which encodes aromatase, enhancing prostate cancer stem cell (PCSC) properties in PC3 cells. Mechanistically, HeyL bound to the CYP19A1 promoter and activated its transcription. HeyL overexpression significantly promoted bicalutamide resistance in LNCaP cells, which was reversed by the aromatase inhibitor letrozole. In PC3 cells, the HeyL-aromatase axis promoted the PCSC phenotype by upregulating autophagy-related genes, while the autophagy inhibitor chloroquine (CQ) suppressed the aromatase-induced PCSC phenotype. The activated HeyL-aromatase axis promoted PCSC autophagy via ERα-mediated estrogenic effects. Taken together, our results indicated that the HeyL-aromatase axis could increase endogenous estradiol levels and activate ERα to suppress PCSC apoptosis by promoting autophagy, which enhances the understanding of how endogenous estrogenic effects influence CRPC development.

The role of JMJD6/U2AF65/AR-V7 axis in castration-resistant prostate cancer progression

Castration-resistant prostate cancer (CRPC) remains prostate cancer research and treatment bottleneck. Abnormal androgen receptor (AR) activation still has a pivotal role in CRPC. Multiple mechanisms involve the process, of which overabundant AR-V7 mRNA splicing production is currently focused and increasingly studied. However, factually, there is no definite conclusion about regulation of AR-V7 mRNA splicing. Recently developed knowledge has demonstrated that JMJD6 and U2AF65 as a hopeful approach in mRNA splicing regulation. The authors propose a novel possible mechanism elucidating AR mRNA splicing for CRPC progression using dual-function enzyme JMJD6 and its induced JMJD6/U2AF65/AR-V7 axis. In this hypothesis JMJD6 introduces to AR promoter to demethylate H3R or H4R and promotes AR mRNA transcription via its demethylase activity and interaction with U2AF65. It is expected that JMJD6 could further effectively perform U2AF65 hydroxylation to achieve AR-V7 mRNA splicing via its hydroxylase activity.

Comparing the DNA‐Binding Specificity of Glucocorticoid and Androgen Receptors

Prostate cancer is the second most common cancer among men, with an estimated 175,000 diagnoses expected in 2019. Androgen hormones fuel prostate tumor growth through the actions of the androgen receptor (AR), a member of the nuclear hormone family of transcription factors. Therapies for prostate cancer include surgery and inhibition of AR with antagonists. Patients, however, often relapse with castration‐resistant prostate cancer (CRPC) in which the cancer grows in the absence of AR ligand action. In some cases, the need for androgens is subverted by overexpression of the AR‐related glucocorticoid receptor (GR). GR effectively replaces AR by regulating genes that drive prostate growth and survival. We have previously used the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method along with the SelexGLM computational algorithm to demonstrate that GR and AR have similar, but distinct DNA sequence specificities. Upon overexpression, GR is able to bind genomic loci once preferentially bound by AR and regulate associated genes. To better understand differences in AR and GR DNA recognition, we are now measuring DNA sequence preferences of both AR and GR using the No Read Left Behind (NRLB) method. NRLB yields high resolution models of specificity after just one round of selection, allowing rapid screening of both wildtype and hybrid AR and GR DNA binding domains. We have over‐expressed and purified both the AR and GR DNA binding domains, and are designing hybrid mutants. Exploring the similarities and differences between AR, GR and the hybrid molecules should help us understand how GR might be playing a role in CRPC.Support or Funding Information• Grant: NSF:CAREER (MCB‐1552862) to M.A. Pufall

Inhibitor 9 Combined With Androgen Deprivation Therapy or Chemotherapy Delays the Malignant Behavior of Castration-Resistant Prostate Cancer Through K-Ras/PLCε/PKCε Signaling Pathway.

Castration-resistant prostate cancer (CRPC) is a progressed stage of prostate cancer, which requires better understanding of the mechanisms and remains an unmet clinical need. As a common oncogene, K-Ras is associated with malignant behavior in different types of tumors but its role in CRPC is unknown. The present study aims to find the mechanism of K-Ras in CRPC and whether it can be used as a crucial molecule for the treatment of CRPC. For this purpose, tissue samples from primary prostate cancer (PPC) and CRPC patients were analyzed by immunohistochemistry and the data showed that K-Ras was elevated in CRPC. More importantly, higher K-Ras expression was related to a shorter recurrence-free survival time in patients with CRPC. In addition, K-Ras promoted the invasion, migration, and drug resistance of CRPC cells by activation of PLCε/PKCε signaling pathway. Meanwhile, the inhibitor of K-RasG12C mutants was able to inhibit malignant behavior of CRPC cells in vitro and in vivo. Inhibitors of K-RasG12C mutants have entered clinical trials. Taken together, the study shows that K-Ras may activate PKCε through PLCε, resulting in the alterations of malignant behavior of CRPC. Inhibitor 9, an inhibitor of the K-RasG12C mutant, has a strong anti-tumor effect in CRPC, which potentially suggests that inhibitors of this nature may serve as a promising treatment for CRPC.

Upregulated KDM4B promotes prostate cancer cell proliferation by activating autophagy.

Castration-resistant prostate cancer (CRPC) causes most of the deaths in patients with prostate cancer (PCa). The androgen receptor (AR) axis plays an important role in castration resistance. Emerging studies showed that the lysine demethylase KDM4B is a key molecule in AR signaling and turnover, and autophagy plays an important role in CRPC. However, little is known about whether KDM4B promotes CRPC progression by regulating autophagy. Here we used an androgen-independent LNCaP (LNCaP-AI) cell line to assay aberrant KDM4B expression using qPCR and western blot analysis and investigated the function of KDM4B in regulating cell proliferation. We found that KDM4B was markedly increased in LNCaP-AI cells compared with LNCaP cells. KDM4B level was significantly correlated with the Gleason score in PCa tissues. In vitro, KDM4B overexpression in CRPC cells promoted cell proliferation, whereas knockdown of KDM4B significantly inhibited cell proliferation. Upregulated KDM4B contributed to activate Wnt/β-catenin signaling and autophagy. Moreover, KDM4B activated autophagy by regulating the Wnt/β-catenin signaling. Finally, we demonstrated that autophagy inhibition attenuated KDM4B-induced CRPC cell proliferation. Our results provided novel insights into the function of KDM4B-driven CRPC development and indicated that KDM4B may be served as a potential target for CRPC therapy.

The small molecule WNT/\u03b2-catenin inhibitor CWP232291 blocks the growth of castration-resistant prostate cancer by activating the endoplasmic reticulum stress pathway

BackgroundAndrogen receptor (AR)-targeted treatments improve the survival of castration-resistant prostate cancer (CRPC) patients; however, secondary resistance to these agents ultimately occurs in virtually all patients. Therefore, alternative therapeutic targets are urgently needed. Since growing evidence demonstrates that WNT/β-catenin signaling plays an important role in CRPC, the antitumor activity and mechanism of action of CWP232291, a small molecule β-catenin inhibitor, were investigated in prostate cancer.MethodsWe assessed the antitumor activity of CWP232291 in prostate cancer cell lines and primary cells derived from CRPC patients. The effect of CWP232291 on apoptotic cell death, endoplasmic reticulum (ER) stress, cell viability, and WNT/β-catenin signaling was evaluated by flow cytometry, western blotting, luciferase reporter assay, and fluorescence microscopy. Antitumor efficacy was assessed in two CRPC xenograft mouse models.ResultsCWP232291 induced ER stress, resulting in upregulation of the proapoptotic protein CHOP and activation of caspase-3-dependent apoptosis. In addition, CWP232291 suppressed the expression of β-catenin by affecting WNT-dependent transcriptional activity, and downregulated AR and its splice variants in prostate cancer cells. Antitumor activity was observed in prostate cancer cells in vitro and ex vivo, and antitumor efficacy was observed in vivo.ConclusionsBeyond providing preclinical evidence of therapeutic efficacy for the novel small molecule β-catenin inhibitor CWP232291 in CRPC, our results show that inducing ER stress and targeting WNT/β-catenin signaling may be a novel strategy against CRPC.

Probing the Effects of Androgen and Glucocorticoid Receptor Sequences on DNA Binding Preferences

Prostate cancer is the second leading cause of cancer among men and although survival rates are high for regional tumors, it is estimated that almost 30,000 men will succumb to their diagnosis in 2018. Androgen hormones fuel prostate tumor growth by signaling through the androgen receptor (AR), a member of the nuclear receptor superfamily of transcription factors. Blocking androgen signaling through administration of antagonists (e.g bicalutamide, enzalutamide) or halting production of androgens through chemical or surgical castration arrests progression of the disease. Patients often relapse with castration resistant prostate cancer (CRPC) that continues to grow despite the lack of androgens. One mechanism of resistance is due to upregulation of a related nuclear receptor, the glucocorticoid receptor (GR). Because AR and GR have overlapping DNA binding specificities, GR is able to functionally substitute for AR at key disease genes. We have shown previously, using SELEX (Selective Evolution of Ligands by Exponential Enrichment) coupled with a novel computational algorithm (SelexGLM), that although AR and GR bind similar sequences, they recognize DNA differently. GR is less specific than AR, allowing it to accommodate predominantly AR sequences. To better understand the selectivity of AR and GR, and to identify the determinants of their specificity, we are measuring the sequence preference of AR and GR mutants using the No Read Left Behind (NRLB) method. As opposed to traditional SELEX, NRLB yields high resolution models of specificity with a single round of selection, which allows rapid screening of our mutants. We have over‐expressed and purified both the AR and GR DNA binding domains, and have designed hybrid mutants. Comparison of the DNA binding preferences of AR, GR and the hybrid molecules by NRLB should help uncover how GR might be playing a role in CRPC.Support or Funding InformationThis work was supported in part by the FUTURE program of the University of Iowa Carver College of Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MicroRNA-144-3p inhibits cell proliferation and promotes apoptosis in castration-resistant prostate cancer by targeting CEP55.

MicroRNA-144-3p (miR-144-3p) has been implicated in the tumorigenesis of multiple types of cancer. However, its role in castration-resistant prostate cancer (CRPC) remains largely unknown. This study aimed to explore the biological role of miR-144-3p in the development of CRPC. RT-qPCR was performed to measure the expression levels of miR-144-3p in CRPC tissues. CRPC cells were transfected with miR-144-3p or NC. MTT and colony formation assays were used to determine cell growth; flow cytometry was used to measure apoptosis; a luciferase reporter assay was used to predict the target genes regulated by miR-144-3p. Finally, siCEP55 or siNC was transfected into DU145 cells, and the rates of cell proliferation and apoptosis were measured. Protein expression levels were confirmed by Western blot analysis. MiR-144-3p expression was significantly decreased in both CRPC tissues and cell lines compared with that from androgen-dependent prostate cancer (ADPC) tumors. Overexpression of miR-144-3p in CRPC cells effectively inhibited proliferation and colony formation and promoted apoptosis in these CRPC cells. Additionally, miR-144-3p directly targeted centrosomal protein 55 (CEP55) and suppressed CEP55 expression. Finally, CEP55 silencing remarkably suppressed proliferation and induced apoptosis of CRPC cells, indicating that miR-144-3p affects CRPC cell survival and proliferation by downregulating CEP55. MiR-144-3p serves as a tumor suppressor in CRPC cells by directly targeting CEP55, which appears to be a novel therapeutic target for CRPC.

Abstract 1088: Defining bone metastatic potential of Abi1 CRISPR prostate cancer cell lines

Abstract Prostate cancer is the second most common cancer in American men, and it is the third leading cause of cancer-related deaths in American men. Distant stage and metastatic disease is often treated with androgen-deprivation therapy (ADT); however, this treatment ultimately fails, leading to castration-resistant prostate cancer (CRPC). This further emphasizes the need for better therapeutic targets. Our work focuses on understanding the role of the Abi1 gene in prostate cancer tumor progression. Our current in vitro data demonstrate an invasive phenotype with our CRISPR-mediated Abi1 knockout (KO) cell lines. In total, using CRISPR-mediated technology we have generated four Abi1 gene KO cell lines, PC-3, DU145, LnCaP, and RWPE-1. Importantly, the androgen-dependent cell line, LNCaP, demonstrated upregulation of Abi1 to the androgen receptor stimulation and the opposite response to the receptor inhibition upon treatment with enzalutamide. These data indicated that low Abi1 level could be induced by ADT therapies and are consistent with our previous observation of low Abi1/WAVE complex in prostate tumor samples. To learn more about this mechanism, we set out to investigate the role of Abi1 in castrated mice. The overall goal of the experiment is to compare the ability of cells lacking the Abi1 gene to engraft in bone under androgen deprivation condition and compare it to the control cells and examine any change in the engraftment phenotype. It is hoped that this research will lead to better understanding of the Abi1/WAVE complex role in CRPC. Supported by NCI R01 CA16101. Citation Format: Alexander Nappi, Dawn Post, Disharee Das, Ishita Joshi, Megan Oest, Gennady Bratslavsky, Leszek Kotula. Defining bone metastatic potential of Abi1 CRISPR prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1088.