CRPC Cells Research Articles

Abstract 5837: Aryl hydrocarbon receptor is critical for enzalutamide-resistant prostate cancer growth

Abstract Therapy resistance is a ubiquitous and major challenge for curing cancer including prostate cancer. As the most prevalent non-skin cancer for men, prostate cancer is responsible for more than 30,000 deaths annually in the U.S. Despite a favorable prognosis at early stages, prostate cancer progressively develop into an incurable castration-resistant stage (CRPC) where prostate cancer grows in a low-androgen environment. Enzalutamide (ENZ) is the first drug approved by FDA for treating CRPC by inhibiting the residual androgen signaling. While ENZ significantly improves overall survival and quality of life, resistance inevitably develops and greatly limits its benefit. To understand ENZ resistance and identify novel therapeutic targets with the hope of overcoming resistance, we performed RNA-seq to analyze mRNA expression profiles from three ENZ-resistant human CRPC cell lines (C4-2BENZR, CWR-R1ENZR and VCaPENZR) and identified the aryl hydrocarbon receptor (AHR) among the 114 commonly upregulated genes. AHR is a ligand-dependent transcription factor that has been shown to drive disease progression in multiple types of cancer. Stable shRNA-mediated knockdown of AHR substantially decreased growth of ENZ-resistant CRPC cells. Our pilot study also showed that AHR knockdown resulted in fewer and smaller CWR-R1ENZR tumors in a xenograft mouse model. To elucidate the underlying mechanism of AHR-mediated cancer growth, gene set enrichment analysis (GSEA) revealed a declining trend of enrichment of the 114 commonly ENZ-upregulated genes among differentially altered genes upon AHR knockdown in ENZ-resistant cells, suggesting that AHR may act as a master regulator of ENZ resistance. Moreover, we found that the Hallmark androgen response gene set is downregulated in AHR-silenced ENZ-resistant cells, implying a role of AHR in sustaining androgen response for ENZ-resistance. Consistently, we performed AHR ChIP-seq and confirmed that AHR bound to promoter regions of several androgen-regulated genes in CWR-R1ENZR cells. In addition, three peroxisome-related Hallmark gene sets are also negatively enriched in AHR-knockdown cells, which provides a rationale for further exploring a potential AHR-dependent connection of peroxisomal function to ferroptosis as a newly emerging mechanism of action for ENZ. In summary, our study suggests that AHR plays a critical role in maintaining ENZ-resistant CRPC growth with the likely involvement of modulating androgen signaling and ferroptosis susceptibility. Citation Format: Chia-Hui Chen, Boyang Jason Wu. Aryl hydrocarbon receptor is critical for enzalutamide-resistant prostate cancer growth [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 5837.

Cholinergic signaling via muscarinic M1 receptor confers resistance to docetaxel in prostate cancer

Docetaxel is the most commonly used chemotherapy for advanced prostate cancer (PC), including castration-resistant disease (CRPC), but the eventual development of docetaxel resistance constitutes a major clinical challenge. Here, we demonstrate activation of the cholinergic muscarinic M1 receptor (CHRM1) in CRPC cells upon acquiring resistance to docetaxel, which is manifested in tumor tissues from PC patients post- vs. pre-docetaxel. Genetic and pharmacological inactivation of CHRM1 restores the efficacy of docetaxel in resistant cells. Mechanistically, CHRM1, via its first and third extracellular loops, interacts with the SEMA domain of cMET and forms a heteroreceptor complex with cMET, stimulating a downstream mitogen-activated protein polykinase program to confer docetaxel resistance. Dicyclomine, a clinically available CHRM1-selective antagonist, reverts resistance and restricts the growth of multiple docetaxel-resistant CRPC cell lines and patient-derived xenografts. Our study reveals a CHRM1-dictated mechanism for docetaxel resistance and identifies a CHRM1-targeted combinatorial strategy for overcoming docetaxel resistance in PC.

DNA repair pathways-targeted cyclovirobuxine inhibits castration-resistant prostate cancer growth by promoting cell apoptosis and cycle arrest

BackgroundCastration-resistant prostate cancer (CRPC) is a deadly malignancy without effective therapeutics. Cyclovirobuxine (CVB) can play an anticancer role by inhibiting mitochondrial function, regulating tumor cell apoptosis, dysregulating autophagy, and other mechanisms. This study aimed to examine the function and mechanism of CVB in CRPC to provide new insights into CRPC treatment. MethodsThe effect of CVB on PC3 and C4-2 cell viability was determined using a CCK8 assay. Core therapeutic targets of CVB in CRPC cells were identified using RNA sequencing, online database, and PPI network analyses. Western blotting, RT–qPCR and molecular docking were performed to evaluate the regulation of core targets by CVB. Utilizing GO and KEGG enrichment analyses, the probable anti-CRPC mechanism of CVB was investigated. Immunofluorescence, flow cytometry and colony formation assays were used to verify the potential phenotypic regulatory role of CVB in CRPC. ResultsCVB inhibited CRPC cell activity in a concentration-dependent manner. Mechanistically, it primarily regulated BRCA1-, POLD1-, BLM-, MSH2-, MSH6- and PCNA-mediated mismatch repair, homologous recombination repair, base excision repair, Fanconi anemia repair, and nucleotide excision repair pathways. Immunofluorescence, Western blot, flow cytometry and colony formation experiments showed that CVB induced DNA damage accumulation, cell apoptosis, and cell cycle arrest and inhibited CRPC cell proliferation. ConclusionCVB can induce DNA damage accumulation in CRPC cells by targeting DNA repair pathways and then induce cell apoptosis and cell cycle arrest, eventually leading to inhibition of the long-term proliferation of CRPC cells.

Abstract PR002: Loss of protein phosphatase PP2A activity drives AR addiction in prostate cancer

Abstract Reversible protein phosphorylation plays a fundamental role in numerous biological processes and is regulated by a dynamic interplay between protein kinases and phosphatases. Disruption of this balance due to aberrant activation of kinases and inactivation of phosphatases is a hallmark of cancer. The highly conserved protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase with a complex heterotrimeric composition involved in the regulation of numerous critical aspects of cellular function. PP2A is one of the most abundant enzymes, accounting for up to 1% of the total cellular protein in some tissues and represents the majority of serine/threonine phosphatase activities in many tissues. PP2A is comprised of a core dimer consisting of a scaffolding ‘‘A’’ subunit (PP2A-A), and a catalytic ‘‘C’’ subunit (PP2A-C), which form a heterotrimeric complex with one of at least 20 substrate-directing regulatory “B” subunits. PP2A heterotrimerization is regulated through Leucine309 methylation of catalytic C subunit, which influence the binding affinity of the B subunits to the AC dimer and thus the substrate specificity. Loss of the tumor suppressive activity of PP2A is associated with prostate cancer; however, the molecular basis for this loss is not well understood. Here, we report that the leucine carboxy methyltransferase (LCMT1), which methylates the L309 of the catalytic C subunit of the PP2A heterodimer, acts as a suppressor of androgen receptor (AR) addicted prostate cancer (PCa). In a series of tissue microarrays (TMAs) representing 17,747 localized PCa, low methyl-PP2A-C levels were negatively associated with critical parameters of disease progression including high Gleason score, and lymph node metastasis. Biologically, LCMT1 silencing leads to increased AR phosphorylation/activity and enhanced CRPC growth in vivo. Conversely, LCMT1 dependent methyl sensitive PP2A heterotrimers target AR and its critical coactivator MED1 for dephosphorylation through binding of the PP2A-B56α regulatory subunit to two conserved small linear motifs (SLiMs) on MED1, resulting in the eviction of the AR-MED1 complex from the chromatin and loss of target gene expression. Mechanistically, LCMT1 is regulated by PI3K-AKT-mTOR-S6K1-mediated phosphorylation-induced degradation requiring the β-TRCP E3 ubiquitin ligase. Targeted degradation of LCMT1 through this mechanism is associated with acquired resistance to second-generation anti-androgens such as enzalutamide, apalutamide and darolutamide resulting in restored AR-addiction in CRPC cells. Finally, feedforward stabilization of LCMT1 by a small molecule activator of phosphatase (SMAP) results in attenuation of AR signaling and tumor regression in enzalutamide refractory PCa xenografts. Collectively, these results demonstrate that decreased methyl-PP2A-C is a novel prognostic marker for PCa progression and that the loss of LCMT1 is a major determinant of AR-addicted advanced PCa, and support the therapeutic potential of selective small molecules modulators of PP2A for metastatic prostate cancer treatment. Citation Format: Irfan A. Asangani. Loss of protein phosphatase PP2A activity drives AR addiction in 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 PR002.

Targeting PHB1 to inhibit castration-resistant prostate cancer progression in vitro and in vivo

BackgroundCastration-resistant prostate cancer (CRPC) is currently the main challenge for prostate cancer (PCa) treatment, and there is an urgent need to find novel therapeutic targets and drugs. Prohibitin (PHB1) is a multifunctional chaperone/scaffold protein that is upregulated in various cancers and plays a pro-cancer role. FL3 is a synthetic flavagline drug that inhibits cancer cell proliferation by targeting PHB1. However, the biological functions of PHB1 in CRPC and the effect of FL3 on CRPC cells remain to be explored.MethodsSeveral public datasets were used to analyze the association between the expression level of PHB1 and PCa progression as well as outcome in PCa patients. The expression of PHB1 in human PCa specimens and PCa cell lines was examined by immunohistochemistry (IHC), qRT-PCR, and Western blot. The biological roles of PHB1 in castration resistance and underlying mechanisms were investigated by gain/loss-of-function analyses. Next, in vitro and in vivo experiments were conducted to investigate the anti-cancer effects of FL3 on CRPC cells as well as the underlying mechanisms.ResultsPHB1 expression was significantly upregulated in CRPC and was associated with poor prognosis. PHB1 promoted castration resistance of PCa cells under androgen deprivation condition. PHB1 is an androgen receptor (AR) suppressive gene, and androgen deprivation promoted the PHB1 expression and its nucleus-cytoplasmic translocation. FL3, alone or combined with the second-generation anti-androgen Enzalutamide (ENZ), suppressed CRPC cells especially ENZ-sensitive CRPC cells both in vitro and in vivo. Mechanically, we demonstrated that FL3 promoted trafficking of PHB1 from plasma membrane and mitochondria to nucleus, which in turn inhibited AR signaling as well as MAPK signaling, yet promoted apoptosis in CRPC cells.ConclusionOur data indicated that PHB1 is aberrantly upregulated in CRPC and is involved in castration resistance, as well as providing a novel rational approach for treating ENZ-sensitive CRPC.

Abstract 61: PRDM16 is key for promoting bone metastatic prostate cancer cell survival during entry into dormancy

Abstract Recurrent metastatic prostate cancer (PC) typically manifests in the skeleton. Despite our knowledge that DTCs give rise to incurable bone metastatic PC, the molecular mechanisms underpinning their entry into dormancy and exit, remain under-investigated. To address this, we developed a novel in vitro model of PC dormancy as determined by increased entry into G0/G1 state (flowcytometry) and increased p38/ERK activity ratio & p27 (immunoblotting). Interestingly, we also observed dormancy entry resulted in the formation of clusters that remained dormant for long periods (21 days) and could be reawakened via serum addition. RNASeq and bioinformatic analysis of control vs. dormant cells revealed PRDM16 to be significantly elevated during entry into dormancy across mouse (RM1) and human cell lines (LAPC4, 22RV1). In vitro, we observed that genetic silencing of PRDM16 leads to a significant reduction in the ability of PC cells to enter dormancy. Analyses show that PRDM16 increases the expression of anti-apoptotic proteins including BCL-2 while inhibiting pro-apoptotic proteins such as BIM and NOXA. Our preliminary data shows that PRDM16 expression in CRPC cells can be induced by BMP7 and conversely decreased by the BMP signaling antagonist, noggin, factors with known critical roles in regulating bone homeostasis. In vivo, we used intrailiac-artery technique to deliver actively growing RM1 cells or dormant RM1 clusters (D-RM1) to the hind limbs (n = 8) of syngeneic mice (C57BL/6). Bioluminescence shows that D-RM1 remains dormant for more than 30 days. Subsequent histological analyses show that D-RM1 home preferentially to the endosteal niche where, compared to RM1 controls, they remain largely negative for ki-67 and cleaved caspase 3 and exhibit high expression of PRDM16. In conclusion, our data points to PRDM16 as a key regulator of survival pathways during PC entry into dormancy. Citation Format: Mostafa Nasr, Tao Li, Ryan T. Bishop, Conor C. Lynch. PRDM16 is key for promoting bone metastatic prostate cancer cell survival during entry into dormancy [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 61.

Abstract 2716: Further development of microRNA-34a therapeutics to target castration-resistant prostate cancer

Abstract Prostate cancer (PCa) is heterogeneous and typically presents with multiple distinct cancer foci. Androgen receptor signaling inhibitors (ARSIs) are standard-of-care therapies for advanced PCa and can prolong patient survival by months. Treatment-naïve PCa harbors a population of stem-like cells (AR-/lo) functionally defined as PCa stem cells (PCSCs). During cancer progression or upon therapeutic treatment, non-CSCs may undergo cellular plasticity and be reprogrammed to PCSCs. As a result, PCSCs may become the predominant cell population in advanced (Gleason 9/10) and ARSI-resistant tumors, and the “root cause” for drug resistance. However, current ARSI-based treatment regimens primarily target AR+ CRPC cells or clones but largely ignore AR-/lo cells. microRNA-34a (miR-34a), a tumor-suppressive miRNA dysregulated in PCa, is a potent PCSC inhibitor by targeting cellular processes essential for CSC survival and functions. Here we aim to develop novel miR-34a therapeutics to target PCa cell heterogeneity and CRPC. miR-34a expression negatively correlates with the tumor stage in PCa samples. And miR-34a expression is significantly reduced in p53 mutated tumors. We explored two novel miR-34a delivery platforms. The first is packaging-free ligand-mediated conjugate utilizing 2- [3-(1,3-dicarboxy propyl) ureido] pentanedioic acid (DUPA) as a high-affinity binding ligand for prostate-specific membrane antigen (PSMA), which is upregulated in PCa. The second platform uses cationic lipid nanoparticles to package miR-34a (LNP-miR-34a). We show that during PCa progression and upon treatment, PSMA expression is upregulated and continues to be highly expressed in CRPC and PSMA expression level negatively correlates with overall survival in PCa. Moreover, DUPA-conjugate can specifically target PSMA-expressing PCa both in vitro and in vivo. DUPA-miR-34a significantly induces target gene silencing in vitro. On the other hand, LNP show good encapsulation efficiency for miR-34a, LNP-miR-34a significantly repressed cell proliferation by downregulating critical stemness factors in a dose-dependent manner. Meanwhile, we are pursuing chemical modifications of the miR-34a mimics aiming to improve serum stability and future in vivo applications. Both fully modified miR-34a (FM-miR-34a) and partially-modified-miR-34a (PM-miR-34a) exhibit improved serum stability without compromising the functionality. Our data suggest that DUPA-miR-34a represents a promising novel therapeutic for PCSC-enriched CRPC that continue to express PSMA, whereas LNP-miR-34a could be a systemic therapeutic for CRPC treatment in the combinatorial setting. miR-34a, as a direct target and a major mediator of p53 functions, may represent an effective replacement therapeutic in p53-mutated advanced PCa. Citation Format: Wen (Jess) Li, Xiaozhuo Liu, Ahmed M. Abdelaal, Sudarsan Kasireddy, Yafei Su, Yun Wu, Andrea L. Kasinski, Dean G. Tang. Further development of microRNA-34a therapeutics to target castration-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 2716.

Castration-resistant prostate cancer cells are dependent on the high activity of CDK7

PurposeProstate cancer (PC) is successfully treated with anti-androgens; however, a significant proportion of patients develop resistance against this therapy. Anti-androgen-resistant disease (castration-resistant prostate cancer; CRPC) is currently incurable. Cyclin-dependent kinase 7 (CDK7) is positioned to positively regulate both cell cycle and transcription, the two features critical for the rapid proliferation of the CRPC cells. Here, we assess if CDK7 is a viable target to halt the proliferation of CRPC cells.MethodsWe use recently developed clinically relevant compounds targeting CDK7 and multiple cell proliferation assays to probe the importance of this kinase for the proliferation of normal, androgen-dependent, and CRPC cells. PC patient data were used to evaluate expression of CDK7 at different disease-stages. Finally, comprehensive glycoproteome-profiling was performed to evaluate CDK7 inhibitor effects on androgen-dependent and CRPC cells.ResultsWe show that CDK7 is overexpressed in PC patients with poor prognosis, and that CRPC cells are highly sensitive to compounds targeting CDK7. Inhibition of O-GlcNAc transferase sensitizes the CRPC, but not androgen-dependent PC cells, to CDK7 inhibitors. Glycoproteome-profiling revealed that CDK7 inhibition induces hyper-O-GlcNAcylation of the positive transcription elongation complex (pTEFB: CDK9 and CCNT1) in the CRPC cells. Accordingly, co-targeting of CDK7 and CDK9 synergistically blocks the proliferation of the CRPC cells but does not have anti-proliferative effects in the normal prostate cells.ConclusionWe show that CRPC cells, but not normal prostate cells, are addicted on the high activity of the key transcriptional kinases, CDK7 and CDK9.

Discovery of Highly Potent Daphnane Diterpenoids Uncovers Importin-β1 as a Druggable Vulnerability in Castration-Resistant Prostate Cancer.

Importins are overexpressed in many cancers and mediate the abnormal nuclear transport of oncogenic factors. The druggable potential of importins still remains unclear, largely because of the lack of potent inhibitors. Herein, the anti-castration-resistant prostate cancer (CRPC) screening of a Euphorbiaceae diterpenoid library followed by target fishing led to the identification of a highly potent importin-β1 inhibitor, daphnane diterpenoid DD1. DD1 selectively inhibited the growth and survival of CRPC cells at subnanomolar concentrations and completely blocked tumor growth in preclinical models at an extremely low dosage. Mechanistic studies revealed that targeting of importin-β1 by DD1 significantly reduced the nuclear accumulation of key CRPC drivers, shutting down their downstream oncogenic signaling. Disruption of the predicted binding sites of DD1 on importin-β1 abolished this anti-CRPC effect. These findings suggest that importin-β1 is an effective therapeutic target in CRPC and that DD1 as the most potent importin-β1 inhibitor to date can be developed as therapeutics for treatment of this disease.

Targeting Castration-Resistant Prostate Cancer Using Mesenchymal Stem Cell Exosomes for Therapeutic MicroRNA-let-7c Delivery.

Castration-resistant prostate cancer (PCa; CRPC) has a poor response to androgen deprivation therapy and is considered an incurable disease. MicroRNA (miR)-lethal 7c (let-7c) was implied to be a tumor suppressor in PCa, and treatment with exogenous let-7c targets both cancer cells and their associated mesenchymal stem cells (MSCs) to prevent CRPC progression and metastasis. Exosomes are nanometer-sized membrane-bound vesicles which have an absolute predominance in biocompatibility for drug delivery and gene therapy by mediating cell-to-cell communication. By utilizing the intrinsic tumor-targeting property of MSCs, this study aimed to investigate the feasibility of MSC-derived exosomes as an exogenous miR delivery system to target CRPC, using miR let-7c as an example. Bioinformatics analysis was performed to observe miR-let-7c expression in clinical samples by utilizing the GEO database. MSC-derived exosomes were collected from a human bone marrow-derived MSC cell line after cell transfection with either a pre-miR negative control or pre-miR-let-7c, and further characterized through nanoparticle tracking analysis and Western blotting. miR-let-7c expression was determined using RT-qPCR, and the phenotypic effects of both naked and MSC-exosome-encapsulated let-7c on CRPC cells (PC3 and CWR22Rv1) were determined by WST-1 cell proliferation assay and wound healing migration assay. miR-let-7c was downregulated in metastatic PCa and high grade group patients. miR-let-7c expression was confirmed to be downregulated in PCa cell lines, with massively decreased in most metastatic CRPC-like cells. Exogenous miR-let-7c can be successfully packaged into MSC exosomes. Treatment with either naked or MSC-exosome-encapsulated miR-let-7c resulted in significant reductions in cell proliferation and migration in CRPC-like PC3 and CWR22Rv1 cells. MSC-derived exosomes could serve as a therapeutic let-7c delivery system to target CRPC.

Glucose-Functionalized Silver Nanoparticles as a Potential New Therapy Agent Targeting Hormone-Resistant Prostate Cancer cells.

PurposeSilver nanoparticles (AgNPs) have shown great potential as anticancer agents, namely in therapies’ resistant forms of cancer. The progression of prostate cancer (PCa) to resistant forms of the disease (castration-resistant PCa, CRPC) is associated with poor prognosis and life quality, with current limited therapeutic options. CRPC is characterized by a high glucose consumption, which poses as an opportunity to direct AgNPs to these cancer cells. Thus, this study explores the effect of glucose functionalization of AgNPs in PCa and CRPC cell lines (LNCaP, Du-145 and PC-3).MethodsAgNPs were synthesized, further functionalized, and their physical and chemical composition was characterized both in water and in culture medium, through UV-visible spectrum, dynamic light scattering (DLS), transmission electron microscopy (TEM) and Fourier-transform infrared spectroscopy (FTIR). Their effect was assessed in the cell lines regarding AgNPs’ entering pathway, cellular proliferation capacity, ROS production, mitochondrial membrane depolarization, cell cycle analysis and apoptosis evaluation.ResultsAgNPs displayed an average size of 61nm and moderate monodispersity with a slight increase after functionalization, and a round shape. These characteristics remained stable when redispersed in culture medium. Both AgNPs and G-AgNPs were cytotoxic only to CRPC cells and not to hormone-sensitive ones and their effect was higher after functionalization showing the potential of glucose to favor AgNPs’ uptake by cancer cells. Entering through endocytosis and being encapsulated in lysosomes, the NPs increased the ROS, inducing mitochondrial damage, and arresting cell cycle in S Phase, therefore blocking proliferation, and inducing apoptosis.ConclusionThe nanoparticles synthesized in the present study revealed good characteristics and stability for administration to cancer cells. Their uptake through endocytosis leads to promising cytotoxic effects towards CRPC cells, revealing the potential of G-AgNPs as a future therapeutic approach to improve the management of patients with PCa resistant to hormone therapy or metastatic disease.

The androgen receptor couples promoter recruitment of RNA processing factors to regulation of alternative polyadenylation at the 3' end of transcripts.

Prostate cancer (PC) relies on androgen receptor (AR) signaling. While hormonal therapy (HT) is efficacious, most patients evolve to an incurable castration-resistant stage (CRPC). To date, most proposed mechanisms of acquired resistance to HT have focused on AR transcriptional activity. Herein, we uncover a new role for the AR in alternative cleavage and polyadenylation (APA). Inhibition of the AR by Enzalutamide globally regulates APA in PC cells, with specific enrichment in genes related to transcription and DNA topology, suggesting their involvement in transcriptome reprogramming. AR inhibition selects promoter-distal polyadenylation sites (pAs) enriched in cis-elements recognized by the cleavage and polyadenylation specificity factor (CPSF) complex. Conversely, promoter-proximal intronic pAs relying on the cleavage stimulation factor (CSTF) complex are repressed. Mechanistically, Enzalutamide induces rearrangement of APA subcomplexes and impairs the interaction between CPSF and CSTF. AR inhibition also induces co-transcriptional CPSF recruitment to gene promoters, predisposing the selection of pAs depending on this complex. Importantly, the scaffold CPSF160 protein is up-regulated in CRPC cells and its depletion represses HT-induced APA patterns. These findings uncover an unexpected role for the AR in APA regulation and suggest that APA-mediated transcriptome reprogramming represents an adaptive response of PC cells to HT.

Identification of the gossypol derivatives as androgen receptor inhibitor

Prostate cancer (PCa) is the most frequently diagnosed male malignant tumor and remains the second leading cause of male cancer mortality in western countries. The development of novel antiandrogens to circumvent the drug resistance in anti-PCa treatment is highly demanded. Herein, we identified that gossypol (GOS) specificly inhibited the AR signaling. Further optimization of GOS derivatives led to the discovery of compound XY-32. XY-32 efficiently inhibits AR signaling with the IC50 of 1.23 μM. XY-32 downregulates both the full-length AR and the AR variable splice AR-V7 via suppressing the mRNA expression. It inhibits the proliferation of CRPC cells such as the LNCaP cells, the PC-3 cells, and enzalutamide resistance 22Rv1 cells. The work demonstrates the GOS derivatives represent a novel series of anti-androgen to conquer the acquired AR mutations or AR splice variants induced drug resistance of mCRPC.

Functional analysis of GCNT3 for cell migration and EMT of castration-resistant prostate cancer cells.

Castration-resistant prostate cancer (CRPC) is a malignant tumor that is resistant to androgen deprivation therapy. Treatments for CRPC are limited, and no diagnostic markers are currently available. O-glycans are known to play an important role in cell proliferation, migration, invasion, and metastasis of cancer cells. However, the differences in the O-glycan expression profiles for normal prostate cancer (PCa) cells compared with CRPC cells have not yet been investigated. In this study, the saccharide primer method was employed to analyze the O-glycans expressed in CRPC cells. Expression levels of core 4-type O-glycans were significantly increased in CRPC cells. Furthermore, the expression level of N-Acetylglucosaminyltransferase 3 (GCNT3), a core 4-type O-glycan synthase gene, was increased in CRPC cells. The expression of core 4-type O-glycans and GCNT3 was presumed to be regulated by androgen deprivation. GCNT3 knockdown induced cell migration and epithelial-mesenchymal transition. These observations elucidate the mechanism of acquisition of castration resistance in PCa and offer new possibilities for the development of diagnostic markers and therapeutic targets in the treatment of PCa.

Combination of the NRF2 Inhibitor and Autophagy Inhibitor Significantly Inhibited Tumorigenicity of Castration-Resistant Prostate Cancer.

Prostate cancer (PCa) is the most frequent cancer in men. Developing new treatment methods for CRPC will be a significant challenge in the clinical treatment of PCa. In conclusion, the results of this study show that NRF2 is downregulated in untreated PCa samples compared to normal PCa samples; however, it was upregulated in mCRPC samples compared to HSPC samples. These results demonstrated that NRF2 may serve as a tumor suppressor in tumorigenesis but promote PCa androgen-independent transferring after ADT treatment. Bioinformatics analysis showed that NRF2 was related to multiple signaling, such as the AGE-RAGE pathway, MAPK pathway, NF-kappa B signaling, PI3K-Akt signaling pathway, and VEGF signaling pathway. Moreover, we revealed that the NRF2 inhibitor significantly inhibited tumorigenicity of CRPC cells in vitro. Of note, combination of the NRF2 inhibitor and autophagy inhibitor had a more significantly suppressive role than either ML385 or CQ, indicating that combination of CQ (autophagy inhibitor) and ML385 (NRF2 inhibitor) is a potential treatment of CRPC. Finally, we conformed that high levels of autophagy regulators LC3B, ULK1, and beclin1 significantly correlated to longer PSA recurrence-free survival time. We think that this study could provide more evidence to confirm that NRF2 is a crucial regulator and targeting NRF2 and autophagy is a potential therapy option for CRPC.

Abstract 1742: Molecular characterization of CRPC pathogenesis and development of a novel anti-PSMA-drug conjugated hybrid nanoparticle therapy with reduced cellular toxicity

Abstract Background: Metastatic castration-resistant prostate cancer (mCRPC) is responsible for most of PCa deaths. Androgen deprivation therapy (ADT), taxanes and enzalutamide are the standard care for CRPC and mCRPC. Unfortunately, these approaches often fail to prevent tumor relapse due to at least in part, the inherently low oxygen levels within the prostate tumor environment. In addition to this, lower drug stability and fast drug clearance among ADT patients are the major therapeutic limitations in drug delivery and drug efficacy. Therefore, it is clinically warranted to develop an increased drug stability system to enhance the drug efficacy. Recent developments in drug delivery system including our earlier report, nanoparticle loaded drugs shows greater stability and better drug efficacy with sub-optimal drug concentrations in vitro. It is an essential to develop an ideal CRPC preclinical model system to test the anti-PSMA-drug conjugated hybrid nanoparticle efficacy with high drug stability with limited cellular toxicity. Materials and Methods: CRPC lines were developed from PSMA proficient LNCaP, LAPC4, C4-2, 22Rv1, and PSMA deficient PC3-ML cells with intermittent hypoxia. CRPC cells were analyzed for proliferation, RB status, AR signaling, migration invasion and drug resistance in both in vitro and in vivo. Further, CRPC bone metastases clinical samples from Thomas Jefferson University Hospital were compared with preclinical CRPC model. Efficient anti-PSMA-drug conjugated hybrid nanoparticle therapy was established. Results: Hypoxia resistance models show CRPC, RB loss, hormone therapy resistance in vitro and in vivo. Anti-PSMA-drug conjugated hybrid nanoparticle shows enhanced drug stability and drug efficacy in vitro and in vivo. Interestingly we observed fibrosis, immune impairment and inactive RB in bone metastatic CRPC samples correlated with our preclinical studies. Further, our clinical bone metastases samples present osteoblastic, osteolytic and osteoblastic & osteolytic nature. Anti-PSMA conjugated-drug loaded hybrid nanoparticles localized to PSMA specific cells in vitro & in vivo, causing cell cycle arrest. Drug loaded anti-PSMA conjugated hybrid nanoparticles did not cause major toxicities in mice. Conclusion: Hypoxia resistant mCRPC models are ideal and are comparable with CRPC induced bone metastases in patient’s samples. Drug-loaded anti-PSMA conjugated hybrid nanoparticles targets PSMA specific prostate cancer cells and show enhanced drug stability and drug efficacy in CRPC preclinical model. Citation Format: Thangavel Chellappagounder. Molecular characterization of CRPC pathogenesis and development of a novel anti-PSMA-drug conjugated hybrid nanoparticle therapy with reduced cellular toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1742.

Abstract 6125: ADAM9 secreted by cancer-associated stromal cells promotes metastasis of prostate cancer

Abstract Introduction: Cancer-associated fibroblasts (CAFs), the main cellular component of the tumor microenvironment (TME), contribute to tumor metastasis. Prostate cancer (PCa) is the most common malignancy among the elderly male population, and it is typically fatal when the disease progresses to metastasis and castration-resistant stage (CRPC). We have previously demonstrated that ADAM9 increased during PCa progression, and ROS is the critical mediator regulating ADAM9 expression in CRPC cells to promote tumor growth. In addition to membrane-anchored ADAM9, a secreted variant of ADAM9 (sADAM9) was recently identified in activated liver stromal cells that facilitated colon cancer liver metastasis. However, it is not clear whether sADAM9 is expressed by prostatic CAFs and, if so, whether it contributes to aggressive tumor phenotypes through tumor-stromal interactions. Objective: To investigate the sADAM9 variants in CAFs and to elucidate the effects of CAF-derived sADAM9 on the aggressiveness of PCa cells. Methods: Immunohistochemistry staining and ELISA of clinical samples were used to assess the expression of ADAM9 during PCa tumor progression. Laser microdissected tissues, primary cell lines, and experimental cell model of paired normal fibroblasts and CAF cells were analyzed. The effects of sADAM9 on CRPC progression were conducted in PC3 cells by WST-1 cell proliferation assay, transwell migration assay, and μ-slide chemotaxis assay. Immunoblot analyses of integrin β1 and migration-related kinases were used to verify the possible downstream signaling pathway of sADAM9. Results: Increased overall intensity of ADAM9 protein expression in PCa patients with metastasis compared. Elevated ADAM9 expression was observed in both cancerous epithelial cells and the tumor stroma. The average serum ADAM9 concentration measured by ELISA was 6.253 ± 0.448 ng/mL in BPH patient samples versus 10.89 ± 0.953 ng/mL in PCa patient samples. A smaller molecular weight (~50-kDa) of ADAM9 was detected in CM from cultured CAFs but not normal fibroblasts or PCa cell lines except for DU145 cells. Treatment of PC3 cells with sADAM9 induced a significant chemotactic migration while did not affect cell proliferation. Furthermore, a stark increase in activated integrin β1, phosphorylated focal adhesion kinase (p-FAK), and p-AKT, but not p-mTOR in PCa cells by sADAM9 suggested the induction of cell migration occurs through activation of the integrin β1/FAK signaling pathways. Conclusion: Herein, we have provided in vitro evidence to demonstrate the pro-metastatic function of sADAM9 from CAFs that facilitate the migration of CRPC cells through integrin receptor signaling, which could be partly responsible for the promotion of CRPC metastasis. Further molecular identification of controlling spliced ADAM9 expression in CAFs remains to investigate. Citation Format: Weichieh Chen, Le Thi Huynh Trang, Chia-Ling Hsieh, Katsumi Shigemura, Masato Fujisawa, Leland W.K. Chung, Shian-Ying Sung. ADAM9 secreted by cancer-associated stromal cells promotes metastasis of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6125.

Abstract 1542: MALAT1 ablation dismantles homologous recombination repair machinery and sensitizes castrate resistant prostate cancer cells to PARP inhibitor

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as a promising target of intervention for metastatic castration-resistant prostate cancer (mCRPC), as approximately one-third of mCRPC patients harbor mutations in the genes associated with the homologous recombination (HR) pathway. Nonetheless, most patients inevitably develop resistance to PARP inhibition (PARPi), due to the induction of reversion mutations in the HR pathway. These mutations restore the function of HR genes and revert to the HR-proficient stage, which in turn decreases the vulnerability of cancer cells to PARP inhibitors. Here, we report that the clinical utility of PARPi could be efficaciously extended by targeting Metastasis associated lung adenocarcinoma transcript (MALAT1), a long non-coding RNA (lncRNA) often elevated in advanced-stage prostate cancer (PCa). In support of this, we show that mCRPC patients exhibit higher expression of genes involved in HR, which positively correlate with MALAT1 levels. Furthermore, RNA interference (RNAi)-mediated depletion of MALAT1 in CRPC cells perturb the expression of key HR genes, namely BRCA1/2, RAD51, EXO1, CHEK1/2, subsequently resulting in HR deficiency. This, in turn, escalates accumulation of DNA lesions in the MALAT1 ablated cells as noticed by abundance in γH2AX, a marker for DNA damage. As a consequence, MALAT1 ablated cells instigates G1/S phase arrest to fetch additional time to repair the damaged DNA or to induce apoptosis. Furthermore, we also provide evidence that the HR deficiency induced by MALAT1 depletion phenocopies “BRCAness” and exhibits synergy with clinically approved DNA repair inhibitors such as Olaparib. In particular, co-inhibition of MALAT1 and PARP1 exhibits a significant anti-proliferative effect reduced clonogenic survival and delays the resolution of γH2AX foci in CRPC cell lines. Taken together, our results establish that MALAT1 plays a central role in regulating the DNA damage response and provides a mechanistic rationale for dual targeting of MALAT1 and PARP in mCRPC patients. Citation Format: Anjali Yadav, Tanay Biswas, Ayush Praveen, Bushra Ateeq. MALAT1 ablation dismantles homologous recombination repair machinery and sensitizes castrate resistant prostate cancer cells to PARP inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1542.

Abstract 396: Therapeutic targeting of eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) in enzalutamide resistant (ENZR) prostate cancer

Abstract Objective: Deregulation of the translation initiation machinery is one of the critical steps for oncogenic mRNA translation in tumor cells. Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) is a crucial scaffold of translation initiation complex (EIF4F). Our recent studies suggest a role for EIF4G1 in multiple tumor types including prostate cancer progression. In the present study, we tested the unknown function of EIF4G1 in Enzalutamide resistant prostate cancer cells. Methods: We used enzalutamide resistant C4-2B (C4-2BENZR) and parental C4-2B cells for the current study. For the functional studies, we used shRNA targeting the EIF4G1 and pharmacological EIF4G-EIF4E complex inhibitor (4EGI-1). The functional assays such as clonogenic, cell proliferation, cell viability, trans-well migration/invasion, and prostasphere formation were performed. To further expand in vitro findings we employed, in-vivo tumor xenograft model derived from enzalutamide resistance C4-2B cells. Results: We found a higher level of EIF4G1 protein in ENZ resistance (C4-2B ENZR) cell lines as compared to parental C4-2B cell lines. Furthermore, knockdown of EIF4G1 by shRNA or treatment with 4EGI-1 inhibitor impaired the clonogenic potential, prostasphere formation, cell viability/proliferation, and cell migration/invasion in C4-2B ENZR cell line, suggesting a critical role of EIF4G1 in modulating enzalutamide resistance in CRPC cells. C4-2BENZR subcutaneous tumor xenograft treated with
combination of 4EGI-1 and ENZ were significantly reduced the tumor volume. Moreover 4EGI-1 sensitized ENZ Resistance C4-2B cells to Enzalutamide treatment in tumor xenograft model. Conclusions: Overall, the findings suggest that EIF4G1 could serve as a novel therapeutic target in overcoming therapeutic resistance to current treatments in PCa. Citation Format: Praveen K. Jaiswal, Sweaty Koul, Saikolappan Sankaralingam, Hari K. Koul. Therapeutic targeting of eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) in enzalutamide resistant (ENZR) prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 396.

Natural product myricetin is a pan-KDM4 inhibitor which with poly lactic-co-glycolic acid formulation effectively targets castration-resistant prostate cancer

BackgroundCastration-resistant prostate cancer (CRPC) with sustained androgen receptor (AR) signaling remains a critical clinical challenge, despite androgen depletion therapy. The Jumonji C-containing histone lysine demethylase family 4 (KDM4) members, KDM4A‒KDM4C, serve as critical coactivators of AR to promote tumor growth in prostate cancer and are candidate therapeutic targets to overcome AR mutations/alterations-mediated resistance in CRPC.MethodsIn this study, using a structure-based approach, we identified a natural product, myricetin, able to block the demethylation of histone 3 lysine 9 trimethylation by KDM4 members and evaluated its effects on CRPC. A structure-based screening was employed to search for a natural product that inhibited KDM4B. Inhibition kinetics of myricetin was determined. The cytotoxic effect of myricetin on various prostate cancer cells was evaluated. The combined effect of myricetin with enzalutamide, a second-generation AR inhibitor toward C4-2B, a CRPC cell line, was assessed. To improve bioavailability, myricetin encapsulated by poly lactic-co-glycolic acid (PLGA), the US food and drug administration (FDA)-approved material as drug carriers, was synthesized and its antitumor activity alone or with enzalutamide was evaluated using in vivo C4-2B xenografts.ResultsMyricetin was identified as a potent α-ketoglutarate-type inhibitor that blocks the demethylation activity by KDM4s and significantly reduced the proliferation of both androgen-dependent (LNCaP) and androgen-independent CRPC (CWR22Rv1 and C4-2B). A synergistic cytotoxic effect toward C4-2B was detected for the combination of myricetin and enzalutamide. PLGA-myricetin, enzalutamide, and the combined treatment showed significantly greater antitumor activity than that of the control group in the C4-2B xenograft model. Tumor growth was significantly lower for the combination treatment than for enzalutamide or myricetin treatment alone.ConclusionsThese results suggest that myricetin is a pan-KDM4 inhibitor and exhibited potent cell cytotoxicity toward CRPC cells. Importantly, the combination of PLGA-encapsulated myricetin with enzalutamide is potentially effective for CRPC.