Signaling In Prostate Cancer Cells Research Articles

Role of KDM2B epigenetic factor in regulating calcium signaling in prostate cancer cells

KDM2B, a histone lysine demethylase, is expressed in a plethora of cancers. Earlier studies from our group, have showcased that overexpression of KDM2B in the human prostate cancer cell line DU-145 is associated with cell adhesion, actin reorganization, and improved cancer cell migration. In addition, we have previously examined changes of cytosolic Ca2+, regulated by the pore-forming proteins ORAI and the Ca2+ sensing stromal interaction molecules (STIM), via store-operated Ca2+ entry (SOCE) in wild-type DU-145. This study sought to evaluate the impact of KDM2B overexpression on the expression of key molecules (SGK1, Nhe1, Orai1, Stim1) and SOCE. Furthermore, this is the first study to evaluate KDM2B expression in circulating tumor cells (CTCs) from patients with prostate cancer. mRNA levels for SGK1, Nhe1, Orai1, and Stim1 were quantified by RT-PCR. Calcium signals were measured in KDM2B-overexpressing DU-145 cells, loaded with Fura-2. Blood samples from 22 prostate cancer cases were scrutinized for KDM2B expression using immunofluorescence staining and the VyCAP system. KDM2B overexpression in DU-145 cells increased Orai1, Stim1, and Nhe1 mRNA levels and significantly decreased Ca2+ release. KDM2B expression was examined in 22 prostate cancer patients. CTCs were identified in 45 % of these patients. 80 % of the cytokeratin (CK)-positive patients and 63 % of the total examined CTCs exhibited the (CK + KDM2B + CD45−) phenotype. To conclude, this study is the first to report increased expression of KDM2B in CTCs from patients with prostate cancer, bridging in vitro and preclinical assessments on the potentially crucial role of KDM2B on migration, invasiveness, and ultimately metastasis in prostate cancer.

EP300/CREBBP acetyltransferase inhibition limits steroid receptor and FOXA1 signaling in prostate cancer cells

The androgen receptor (AR) is a primary target for treating prostate cancer (PCa), forming the bedrock of its clinical management. Despite their efficacy, resistance often hampers AR-targeted therapies, necessitating new strategies against therapy-resistant PCa. These resistances involve various mechanisms, including AR splice variant overexpression and altered activities of transcription factors like the glucocorticoid receptor (GR) and FOXA1. These factors rely on common coregulators, such as EP300/CREBBP, suggesting a rationale for coregulator-targeted therapies. Our study explores EP300/CREBBP acetyltransferase inhibition’s impact on steroid receptor and FOXA1 signaling in PCa cells using genome-wide techniques. Results reveal that EP300/CREBBP inhibition significantly disrupts the AR-regulated transcriptome and receptor chromatin binding by reducing the AR-gene expression. Similarly, GR’s regulated transcriptome and receptor binding were hindered, not linked to reduced GR expression but to diminished FOXA1 chromatin binding, restricting GR signaling. Overall, our findings highlight how EP300/CREBBP inhibition distinctively curtails oncogenic transcription factors’ signaling, suggesting the potential of coregulatory-targeted therapies in PCa.

Abstract 2077: Exploring the multifaceted efficacy of de novo lipogenesis inhibitor in cancer therapy

Abstract Cancer cells frequently exhibit altered lipid metabolism to sustain their rapid growth and proliferation.Sterol Regulatory Element-Binding Proteins (SREBPs) are key transcription factors that orchestratelipid biosynthesis and uptake in cells. Dysregulation of SREBPs is a common feature of many cancertypes, making them attractive targets for therapeutic intervention. In this study, we present acomprehensive exploration of the potential and rationale for targeting lipid metabolism in cancerthrough the use of a small molecule inhibitor that targets activation of SREBP. We illustrate that oursmall molecule inhibitor (SMI) exhibits potent anticancer activity and effectively targets SREBPsignaling in a variety of prostate cancer models as well as in non-prostate cancer models that arelipogenesis driven. Our novel SMI induces apoptosis and reduces PCa cell proliferation, suppressescell invasion and migration. Furthermore, we illustrate that in addition to suppressing the transcriptionfactor activity of SREBPs, these novel SMIs are highly effective in suppressing both the full length andvariant androgen receptor and their signaling in prostate cancer cells. Notably, in cell and organoidmodels, these novel inhibitors exhibit synergistic anticancer activity in combination with the ARsignaling inhibitor, Enzalutamide. The anticancer activity of these inhibitors extend beyond ARdependent prostate cancer and are also effective in organoid models of neuroendocrine PCa, aparticularly drug-resistant and aggressive PC subtype. Lastly, we illustrate excellent pharmacokineticsand potent anti-tumor effects of these novel SREBP activation inhibitors using in vivo castration-resistant prostate cancer CDX tumors. Given the urgent need for novel targets in PCa, our first-in-class inhibitor represents a promising therapeutic strategy with the potential to effectively targetdysregulated lipogenesis in cancer patients. Citation Format: Dallin Lowder, Jin Na Shin, Maria E. Ruiz-Echartea, Jenny Deng, Aleksandra Rusin, Darlene Skapura, Michael Ittmann, Salma Kaochar. Exploring the multifaceted efficacy of de novo lipogenesis inhibitor in cancer therapy [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 2077.

Astragalus membranaceus Extract Induces Apoptosis via Generation of Reactive Oxygen Species and Inhibition of Heat Shock Protein 27 and Androgen Receptor in Prostate Cancers.

Although Astragalus membranaceus is known to have anti-inflammatory, anti-obesity, and anti-oxidant properties, the underlying apoptotic mechanism of Astragalus membranaceus extract has never been elucidated in prostate cancer. In this paper, the apoptotic mechanism of a water extract from the dried root of Astragalus membranaceus (WAM) was investigated in prostate cancer cells in association with heat shock protein 27 (HSP27)/androgen receptor (AR) signaling. WAM increased cytotoxicity and the sub-G1 population, cleaved poly (ADP-ribose) polymerase (PARP) and cysteine aspartyl-specific protease 3 (caspase 3), and attenuated the expression of B-cell lymphoma 2 (Bcl-2) in LNCaP cells after 24 h of exposure. Consistently, WAM significantly increased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive LNCaP cells. WAM decreased the phosphorylation of HSP27 on Ser82 and inhibited the expression of the AR and prostate-specific antigen (PSA), along with reducing the nuclear translocation of p-HSP27 and the AR via the disturbed binding of p-HSP27 with the AR in LNCaP cells. WAM consistently inhibited the expression of the AR and PSA in dihydrotestosterone (DHT)-treated LNCaP cells. WAM also suppressed AR stability, both in the presence and absence of cycloheximide, in LNCaP cells. Taken together, these findings provide evidence that WAM induces apoptosis via the inhibition of HSP27/AR signaling in prostate cancer cells and is a potent anticancer candidate for prostate cancer treatment.

Immunosuppressive role of BDNF in therapy-induced neuroendocrine prostate cancer.

Prostate stromal cells play a crucial role in the promotion of tumor growth and immune evasion in the tumor microenvironment (TME) through intricate molecular alterations in their interaction with prostate cancer (PCa) cells. While the impact of these cells on establishing an immunosuppressive response and influencing PCa aggressiveness remains incompletely understood. Our study shows that the activation of the leukemia inhibitory factor (LIF)/LIF receptor (LIFR) pathway in both prostate tumor and stromal cells, following androgen deprivation therapy (ADT), leads to the development of an immunosuppressive TME. Activation of LIF/LIFR signaling in PCa cells induces neuroendocrine differentiation (NED) and upregulates immune checkpoint expression. Inhibition of LIF/LIFR attenuates these effects, underscoring the crucial role of LIF/LIFR in linking NED to immunosuppression. Prostate stromal cells expressing LIFR contribute to NED and immunosuppressive marker abundance in PCa cells, while LIFR knockdown in prostate stromal cells reverses these effects. ADT-driven LIF/LIFR signaling induces brain-derived neurotrophic factor (BDNF) expression, which, in turn, promotes NED, aggressiveness, and immune evasion in PCa cells. Clinical analyses demonstrate elevated BDNF levels in metastatic castration-resistant PCa (CRPC) and a positive correlation with programmed death-ligand 1 (PDL1) and immunosuppressive signatures. This study shows that the crosstalk between PCa cells and prostate stromal cells enhances LIF/LIFR signaling, contributing to an immunosuppressive TME and NED in PCa cells through the upregulation of BDNF.

New mode of action of curcumin on prostate cancer cells: Modulation of endoplasmic reticulum-associated degradation mechanism and estrogenic signaling.

Prostate cancer is leading to cancer-related mortality in numerous men each year worldwide. While there are several treatment options, acquired drug resistance mostly limits the success of treatments. Therefore, there is a need for the development of innovative treatments. Curcumin is one of the bioactive polyphenolic ingredients identified in turmeric and has numerous biological activities, such as anti-inflammatory and anticancer. In the present study, we investigated the effect of curcumin on the ER-associated degradation (ERAD) and estrogenic signaling in prostate cancer cells. The antiproliferative effect of curcumin on human androgen-dependent prostate cancer cell lines LNCaP and VCaP was estimated by WST-1 assay. Morphological alterations were investigated with an inverted microscope. We investigated the effect of curcumin on ERAD and estrogen signaling proteins by immunoblotting assay. To evaluate the impact of curcumin on endoplasmic reticulum (ER) protein quality-related, the expression level of 32 genes was analyzed by quantitative reverse transcription polymerase chain reaction. The nuclear translocation of estrogen receptor was examined by nuclear fractionation and immunofluorescence microscopy. We found that curcumin effectively reduced the proliferation rates of LNCaP and VCaP cells. ERAD proteins; Hrd1, gp78, p97/VCP, Ufd1 and Npl4 were strongly induced by curcumin. Also, the steady-state level of polyubiquitin was increased in a dose-dependent manner in both cell lines. Curcumin administration remarkably decreased the protein levels of estrogen receptor-alfa (Erα), whereas estrogen receptor-beta unaffected. Additionally, curcumin strongly restricted the nuclear translocation of Erα. Present data suggest that curcumin may be effectively used in therapeutic approaches associated with the targeting ER protein quality control mechanism and modulation of estrogen signaling in prostate cancer.

Synthetic Dual Cysteine-ADP Ribosylated Peptides from the Androgen Receptor are Recognized by the DTX3L/PARP9 Complex.

Androgen signaling in prostate cancer cells involves multisite cysteine ADP-ribosylation of the androgen receptor (AR) by PARP7. The AR modification is read by ADP-ribosyl binding macrodomains in PARP9, but the reason that multiple cysteines are modified is unknown. Here, we use synthetic peptides to show that dual ADP-ribosylation of closely spaced cysteines mediates recognition by the DTX3L/PARP9 complex. Mono and dual ADP-ribosylated cysteine peptides were prepared using a novel solid-phase synthetic strategy utilizing a key, Boc-protected, ribofuranosylcysteine building block. This synthetic strategy allowed us to synthesize fluorescently labeled peptides containing a dual ADP-ribosylation motif. It was found that the DTX3L/PARP9 complex recognizes the dual ADP-ribosylated AR peptide (Kd = 80.5 nM) with significantly higher affinity than peptides with a single ADP-ribose. Moreover, oligomerization of the DTX3L/PARP9 complex proved crucial for ADP-ribosyl-peptide interaction since a deletion mutant of the complex that prevents its oligomer formation dramatically reduced peptide binding. Our data show that features of the substrate modification and the reader contribute to the efficiency of the interaction and imply that multivalent interactions are important for AR-DTX3L/PARP9 assembly.

THU573 Paxillin Regulates Androgen Receptor In Granulosa Cells

Abstract Disclosure: A. Weidner: None. A. Roy: None. K. Vann: None. O. Astapova: None. Paxillin is a ubiquitously-expressed adaptor protein that is integral in focal adhesions and plays an active role in the regulation of cell motility and apoptosis. Recently, paxillin was shown to mediate pro-tumorigenic androgen signaling in prostate cancer cells, where it is necessary for rapid cytoplasmic kinase signaling triggered by activation of membrane-bound androgen receptor. We sought to investigate the relationship between paxillin and androgen receptor (AR) in granulosa cells, where androgen actions, apoptosis and focal adhesions are of known importance, but the role of paxillin is understudied. When paxillin expression was silenced using siRNA in human granulosa-derived KGN cells, AR protein expression was significantly reduced, while its RNA expression was unaffected. Similarly, in granulosa cell-specific paxillin knockout mice, AR protein expression in granulosa cells was reduced. Further, we found that AR and paxillin co-localize in focal adhesions in mouse primary granulosa cells and in KGN cells, and their interaction requires the activity of focal adhesion kinase. Disruption of focal adhesion with a focal adhesion kinase inhibitor also leads to reduced AR protein levels. To further explore the mechanisms by which paxillin regulates AR expression, we created paxillin-null KGN cell lines using CRISPR-Cas9 gene editing. While paxillin loss resulted in reduced AR protein expression early (days) in the gene-editing process, this effect disappeared after weeks of clonal expansion from selected single cells. Expanded paxillin-null KGN clones showed normal AR protein expression, but increased AR mRNA expression, suggesting an adaptive increase in AR gene transcription to compensate for reduced AR protein levels in cells that survived paxillin deletion. Studies of AR protein degradation using cycloheximide to block protein synthesis showed that the half-life of AR was reduced from 6.7 to 4.5 hours (approximately 33%) by paxillin deletion, indicating accelerated AR protein degradation in cells lacking paxillin. Based on our results, we hypothesize that paxillin brings AR into focal adhesions in granulosa cells, thereby sequestering it from proteasomal degradation in the cytoplasm. Thus, paxillin may be a novel target in the management of androgen-related disorders in women, such as polycystic ovary syndrome. Presentation Date: Thursday, June 15, 2023

Inhibition of GLI Transcriptional Activity and Prostate Cancer Cell Growth and Proliferation by DAX1.

The Hedgehog (Hh) signaling pathway plays an essential role in the initiation and progression of prostate cancer. This is mediated by transcriptional factors belonging to the GLI (glioma-associated oncogene) family, which regulate downstream targets to drive prostate cancer progression. The activity of GLI proteins is tightly controlled by a range of mechanisms, including molecular interactions and post-translational modifications. In particular, mitogenic and oncogenic signaling pathways have been shown to regulate GLI protein activity independently of upstream Hh pathway signaling. Identifying GLI protein regulators is critical for the development of targeted therapies that can improve patient outcomes. This study aimed to identify a novel protein that directly regulates the activity of GLI transcription factors in prostate cancer. We performed gene expression, cellular analyses, and reporter assays to demonstrate that DAX1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) interacts with GLI1 and GLI2, the master regulators of Hh signaling. Interestingly, DAX1 overexpression significantly inhibited Hh signaling by reducing GLI1 and GLI2 activity, prostate cancer cell proliferation, and viability. Our results shed light on a novel regulatory mechanism of Hh signaling in prostate cancer cells. The interaction between DAX1 and GLI transcription factors provides insight into the complex regulation of Hh signaling in prostate cancer. Given the importance of Hh signaling in prostate cancer progression, targeting DAX1-GLI interactions may represent a promising therapeutic approach against prostate cancer. Overall, this study provides new insights into the regulation of the Hh pathway and its role in prostate cancer progression. The findings suggest that DAX1 could serve as a potential therapeutic target for the treatment of prostate cancer.

Apoptotic and anti-Warburg effect of Morusin via ROS mediated inhibition of FOXM1/c-Myc signaling in prostate cancer cells.

Though Morusin is known to induce apoptotic, antiprolifertaive, and autophagic effects through several signaling pathways, the underlying molecular mechanisms of Morusin still remain unclear until now. To elucidate antitumor mechanism of Morusin, cytotoxicity assay, cell cycle analysis, Western blotting, TUNEL assay, RNA interference, immunofluorescense, immunoprecipitation, reactive oxygen species (ROS) measurement, and inhibitor study were applied in this study. Morusin enhanced cytotoxicity, increased the number of TUNEL positive cells, sub-G1 population and induced the cleavages of PARP and caspase3, attenuated the expression of HK2, PKM2, LDH, c-Myc, and Forkhead Box M1 (FOXM1) along with the reduction of glucose, lactate, and ATP in DU145 and PC3 cells. Furthermore, Morusin disrupted the binding of c-Myc and FOXM1 in PC-3 cells, which was supported by String and cBioportal database. Notably, Morusin induced c-Myc degradation mediated by FBW7 and suppressed c-Myc stability in PC3 cells exposed to MG132 and cycloheximide. Also, Morusin generated ROS, while NAC disrupted the capacity of Morusin to reduce the expression of FOXM1, c-Myc, pro-PARP, and pro-caspase3 in PC-3 cells. Taken together, these findings provide scientific evidence that ROS mediated inhibition of FOXM1/c-Myc signaling axis plays a critical role in Morusin induced apoptotic and anti-Warburg effect in prostate cancer cells. Our findings support scientific evidence that ROS mediated inhibition of FOXM1/c-Myc signaling axis is critically involved in apoptotic and anti-Warburg effect of Morusin in prostate cancer cells.

Triiodothyronine positively regulates endoplasmic reticulum-associated degradation (ERAD) and promotes androgenic signaling in androgen-dependent prostate cancer cells

Thyroid hormones (THs) play crucial roles in numerous physiological processes of nearly all mammalian tissues, including differentiation and metabolism. Deterioration of TH signaling has been associated with several pathologies, including cancer. The effect of highly active triiodothyronine (T3) has been investigated in many in vivo and in vitro cancer models. However, the role of T3 on cancerous prostate tissue is controversial today. Recent studies have focused on the characterization of the supportive roles of the endoplasmic reticulum-associated degradation (ERAD) and unfolded protein response (UPR) signaling in prostate cancer (PCa) and investigating new hormonal regulation patterns, including estrogen, progesterone and 1,25(OH)2D3. Additionally, androgenic signaling controlled by androgens, which are critical in PCa progression, has been shown to be regulated by other steroid hormones. Today, the effects of T3 on ERAD and UPR are unknown, the impact on androgenic signaling is also still not fully understood in PCa. Therefore, we aimed to investigate the molecular action of T3 on the ERAD mechanism and UPR signaling in PCa cells and also extensively examined the effect of T3 on androgenic signaling. Our data indicated that T3 tightly regulated ERAD and UPR signaling in androgen-dependent PCa cells. We also found that T3 hormone stimulated androgenic signaling by upregulating AR mRNA and protein levels and enhancing its nuclear translocation. Additionally, advanced computational studies supported the ligand binding effect of T3 on AR protein. Our data suggest that targeting thyroidal signaling should be considered in therapeutic approaches to be developed for prostate malignancy in addition to other steroidal regulations.

Abstract B066: Genome-wide CRISPR screens identify PTGES3 as a druggable AR modulator

Abstract BACKGROUND The androgen receptor (AR) is the central therapeutic target in advanced prostate cancer (PCa). While treatments that directly target AR are effective in prolonging patient survival, aggressive prostate cancers invariably find a way to evade these therapies. Frequently, evasion mechanisms reestablish AR expression and therefore AR-driven oncogenic pathways are restored. Comprehensively understanding the protein regulation machinery of AR and identifying alternative therapeutic targets are crucial for effective treatment of this disease. METHODS To study regulators of AR, we utilized CRISPR-Cas9 technology to knock-in a split neon green fluorescent protein onto AR in prostate cancer cells. We evaluated the AR expression and activity in these AR reporter cell lines by multiple approaches including ARE-luciferase assay, ChIP, and RNA-seq. We further built a unique loss-of-function CRISPRi screening system in this AR reporter cell line where we performed a genome-wide fluorescence-activated cell sorting based screen and functionally interrogated hits that modulate AR protein levels. RESULTS We established the first prostate cancer cell line models harboring an endogenous AR reporter. Extensive genotypic and phenotypic characterization of this system demonstrated that the reporter is effective and does not alter AR activity. These models have enabled us to precisely measure the dynamic endogenous AR protein levels by its fluorescence signal. By utilizing a genome-wide CRISPRi screening method in this system, we identified classic AR regulators such as HOXB13, GATA2 and GRHL2, as well as many novel genes that have not been characterized in the context of prostate cancer. Among these novel hits, Prostaglandin E synthase 3 (PTGES3) is the highest ranked druggable target. We validated this finding by establishing PTGES3 knockdown cell lines and demonstrated that loss of PTGES3 downregulated AR and its downstream genes. Follow-up studies show that beyond the canonical chaperone function, PTGES3 physically interacts with AR in the nucleus, where it promotes AR activity. PTGES3 is required for cell proliferation in vitro and in vivo in multiple AR-driven PCa models but not in AR-independent PCa models, such as PC3 cells. From our preliminary data, this unique interaction further suggests that high tumor PTGES3 expression is associated with resistance to first-line androgen deprivation therapies in a retrospective clinical cohort. These results collectively confirm that PTGES3 regulates AR activity and is a potential therapeutic target. Lastly, using a disulfide tethering fragment screen, we developed a PTGES3 inhibitor that blocks the PTGES3/AR interaction and represses AR signaling in PCa cells. We aim to further optimize this PTGES3-specific inhibitor as a therapeutic alternative for patients with aggressive prostate cancer. CONCLUSIONS Together, these genetic, biochemical and chemical biology experiments point to a new class of AR-directed PTGES3 inhibitor therapies that may overcome known mechanisms of ARSI resistance in mCRPC. Citation Format: Haolong Li, James E. Melnyk, Becky Xu Hua Fu, Siyu Feng, Martin Sjöström, Raunak Shrestha, Meng Zhang, Lisa N. Chesner, Hyun Jin Shin, Marsha Calvert, Jonathan Chou, Rajdeep Das, Emily A. Egusa, Aidan Winters, Jun Zhu, Ashutosh Maheshwari, Junjie T. Hua, Mohammed Alshalalfa, William S. Chen, Bradley A. Stohr, Javed Siddiqui, Bo Huang, Eric J. Small, David A. Quigley, Kevan M. Shokat, Luke A. Gilbert, Felix Y. Feng. Genome-wide CRISPR screens identify PTGES3 as a druggable AR modulator [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 B066.

Anti-Warburg effect via generation of ROS and inhibition of PKM2/β-catenin mediates apoptosis of lambertianic acid in prostate cancer cells.

To elucidate the underlying antitumor mechanism of lambertianic acid (LA) derived from Pinus koraiensis, the role of cancer metabolism related molecules was investigated in the apoptotic effect of LA in DU145 and PC3 prostate cancer cells. MTT assay for cytotoxicity, RNA interference, cell cycle analysis for sub G1 population, nuclear and cytoplasmic extraction, lactate, Glucose and ATP assay by ELISA, Measurement of reactive oxygen species (ROS) generation, Western blotting, and immunoprecipitation assay were conducted in DU145 and PC3 prostate cancer cells. Herein LA exerted cytotoxicity, increased sub G1 population and attenuated the expression of pro-Caspase3 and pro-poly (ADP-ribose) polymerase (pro-PARP) in DU145 and PC3 cells. Also, LA reduced the expression of lactate dehydrogenase A (LDHA), glycolytic enzymes such as hexokinase 2 and pyruvate kinase M2 (PKM2) with reduced production of lactate in DU145 and PC3 cells. Notably, LA decreased phosphorylation of PKM2 on Tyr105 and inhibited the expression of p-STAT3, cyclin D1, C-Myc, β-catenin, and p-GSK3β with the decrease of nuclear translocation of p-PKM2. Furthermore, LA disturbed the binding of p-PKM2 and β-catenin in DU145 cells, which was supported by Spearman coefficient (0.0463) of cBioportal database. Furthermore, LA generated ROS in DU145 and PC3 cells, while ROS scavenger NAC (N-acetyl L-cysteine) blocked the ability of LA to reduce p-PKM2, PKM2, β-catenin, LDHA, and pro-caspase3 in DU145 cells. Taken together, these findings provide evidence that LA induces apoptosis via ROS generation and inhibition of PKM2/β-catenin signaling in prostate cancer cells.

Palmitoyl acyltransferase ZDHHC7 inhibits androgen receptor and suppresses prostate cancer.

The hormonal transcription factor androgen receptor (AR) is a master regulator of prostate cancer (PCa). Protein palmitoylation, which attaches a palmitate fatty acid to a substrate protein, is mediated by a class of 23 ZDHHC (Zinc-Finger DHHC motif)-family palmitoyltransferases. Although palmitoylation has been shown to modify many proteins and regulate diverse cellular processes, little is known about ZDHHC genes in cancer. Here we examined ZDHHC family gene expression in human tissue panels and identified ZDHHC7 as a PCa-relevant member. RNA-seq analyses of PCa cells with ZDHHC7 de-regulation revealed global alterations in androgen response and cell cycle pathways. Mechanistically, ZDHHC7 inhibits AR gene transcription and therefore reduces AR protein levels and abolishes AR signaling in PCa cells. Accordingly, ZDHHC7 depletion increased the oncogenic properties of PCa cells, whereas restoring ZDHHC7 is sufficient to suppress PCa cell proliferation and invasion in vitro and mitigate xenograft tumor growth in vivo. Lastly, we demonstrated that ZDHHC7 is downregulated in human PCa compared to benign-adjacent tissues, and its loss is associated with worse clinical outcomes. In summary, our study reveals a global role of ZDHHC7 in inhibiting androgen response and suppressing PCa progression and identifies ZDHHC7 loss as a biomarker for aggressive PCa and a target for therapeutic intervention.

Caffeic acid phenethyl ester suppresses EGFR/FAK/Akt signaling, migration, and tumor growth of prostate cancer cells

Epidermal growth factor receptor (EGFR) is upregulated in prostate cancer (PCa). However, suppression of EGFR did not improve the patient outcome, possibly due to the activation of PI3K/Akt signaling in PCa. Compounds able to suppress both PI3K/Akt and EGFR signaling may be effective for treating advanced PCa. We examined if caffeic acid phenethyl ester (CAPE) simultaneously suppresses the EGFR and Akt signaling, migration and tumor growth in PCa cells. Wound healing assay, transwell migration assay and xenograft mice model were used to determine the effects of CAPE on migration and proliferation of PCa cells. Western blot, immunoprecipitation, and immunohistochemistry staining were performed to determine the effects of CAPE on EGFR and Akt signaling. CAPE treatment decreased the gene expression of HRAS, RAF1, AKT2, GSK3A, and EGF and the protein expression of phospho-EGFR (Y845, Y1069, Y1148, Y1173), phospho-FAK, Akt, and ERK1/2 in PCa cells. CAPE treatment inhibited the EGF-induced migration of PCa cells. Combined treatment of CAPE with EGFR inhibitor gefitinib showed additive inhibition on migration and proliferation of PCa cells. Injection of CAPE (15mg/kg/3 days) for 14 days suppressed the tumor growth of prostate xenografts in nude mice as well as suppressed the levels of Ki67, phospho-EGFR Y845, MMP-9, phospho-Akt S473, phospho-Akt T308, Ras, and Raf-1 in prostate xenografts. Our study suggested that CAPE can simultaneously suppress the EGFR and Akt signaling in PCa cells and is a potential therapeutic agent for advanced PCa.

Abstract 2547: YAP1 associates with the EPHA3 receptor tyrosine kinase to promote prostate cancer progression

Abstract Metastatic castration-resistant prostate cancer is lethal, and the mechanisms contributing to this deadly disease are largely unknown. The disparity in our knowledge poses a significant barrier to developing effective therapeutics against advanced prostate cancer in humans. Evidence suggests that the ephrin family of receptor tyrosine kinase signaling may play a critical role in the pathobiology of prostate cancer progression. However, the mechanism is elusive. Our initial study showed that YAP1, in concert with the TEAD1 transcription factor, regulated EPHA3 expression. However, the significance of EPHA3 signaling in prostate cancer remains elusive. Here we investigated the role of EPHA3 signaling in prostate cancer cells. We showed that the expression of EPHA3 correlated with YAP1 in prostate cancer clinical samples. We also showed that EPHA3 knockout reduced cell growth in 2D and 3D cultures. In addition, EPHA3 loss sensitized castration-resistant prostate cancer cells to enzalutamide. Moreover, EPHA3 knockout reduced the GTPase activity of RHOA compared to cells with EPHA3-WT, which coincided with reduced phospho-ERK1/2 levels. Consistently, EPHA3 knockout reduces phosphor-ERK1/2, similar to the inhibition of phospho-ERK1/2 by an inhibitor of ROCK1 (Rho-associated coiled-coil-containing protein kinase 1). Besides, EPHA3 knockout reduced the expression of EMT markers ZEB1/2 and the cancer stem cell marker CD44 compared to the wild type. Furthermore, the levels of EPAH3 transcripts were higher in high-grade PC of AA patients compared to other races. Finally, AA and Caucasian Americans (CA) PC cell lines differentially express the member of ephrin-A and ephrin-B receptors. These observations suggest that the YAP1-EPHA3 signaling is a viable therapeutic target for patients with advanced prostate cancer. Citation Format: Marwah M. Al-Mathkour, Abdulrahman M. Dwead, Kezhan Khazaw, Bekir Cinar. YAP1 associates with the EPHA3 receptor tyrosine kinase to promote prostate cancer progression [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 2547.

Targeting androgen receptor and the variants by an orally bioavailable Proteolysis Targeting Chimeras compound in castration resistant prostate cancer.

Despite the advent of improved therapeutic options for advanced prostate cancer, the durability of clinical benefits is limited due to inevitable development of resistance. By constitutively sustaining androgen receptor (AR) signaling, expression of ligand-binding domain truncated AR variants (AR-V(ΔLBD)) accounts for the major mechanism underlying the resistance to anti-androgen drugs. Strategies to target AR and its LBD truncated variants are needed to prevent the emergence or overcome drug resistance. We utilize Proteolysis Targeting Chimeras (PROTAC) technology to achieve induced degradation of both full-length AR (AR-FL) and AR-V(ΔLBD) proteins. In the ITRI-PROTAC design, an AR N-terminal domain (NTD) binding moiety is appended to von-Hippel-Lindau (VHL) or Cereblon (CRBN) E3 ligase binding ligand with linker. Invitro studies demonstrate that ITRI-PROTAC compounds mechanistically degrade AR-FL and AR-V(ΔLBD) proteins via ubiquitin-proteasome system, leading to impaired AR transactivation on target gene expression, and inhibited cell proliferation accompanied by apoptosis activation. The compounds also significantly inhibit enzalutamide-resistant growth of castration resistant prostate cancer (CRPC) cells. In castration-, enzalutamide-resistant CWR22Rv1 xenograft model without hormone ablation, ITRI-90 displays a pharmacokinetic profile with decent oral bioavailability and strong antitumor efficacy. AR NTD that governs the transcriptional activities of all active variants has been considered attractive therapeutic target to block AR signaling in prostate cancer cells. We demonstrated that utilizing PROTAC for induced AR protein degradation via NTD represents an efficient alternative therapeutic strategy for CRPC to overcome anti-androgen resistance. The funding detail can be found in the Acknowledgements section.

Bacterial RNA virus MS2 exposure increases the expression of cancer progression genes in the LNCaP prostate cancer cell line.

Bacteriophages effectively counteract diverse bacterial infections, and their ability to treat most types of cancer has been explored using phage engineering or phage-virus hybrid platforms. In the present study, it was demonstrated that the bacteriophage MS2 can affect the expression of genes associated with the proliferation and survival of LNCaP prostate epithelial cells. LNCaP cells were exposed to bacteriophage MS2 at a concentration of 1×107 plaque forming units/ml for 24-48 h. After exposure, various cellular parameters, including cell viability, morphology, and changes in gene expression, were examined. MS2 affected cell viability adversely, reducing viability by 25% in the first 4 h of treatment; however, cell viability recovered within 24-48 h. Similarly, the AKT, androgen receptor, integrin α5, integrin β1, MAPK1, MAPK3, STAT3, and peroxisome proliferator-activated receptor-γ coactivator 1α genes, which are involved in various normal cellular processes and tumor progression, were significantly upregulated, whereas the expression levels of HSP90, ITGB5, ITGB3, HSP27, ITGAV, and PI3K genes were unchanged. Therefore, based on viability and gene expression changes, bacteriophage MS2 severely impaired LNCaP cells by reducing anchorage-dependent survival and androgen signaling. A caveolin-mediated endocytosis mechanism for MS2-mediated signaling in prostate cancer cells was proposed based on reports involving bacteriophages T4, M13, and MS2, and their interactions with LNCaP and PC3 cell lines.

Abstract B039: Adipocyte regulation of ER stress and mTOR signaling in bone-metastatic PCa: The role of stearoyl-CoA desaturase

Abstract Bone metastatic disease is correlated with increased morbidity and mortality in prostate cancer (PCa) patients. Current treatments for bone-metastatic PCa are palliative, and new targets for therapy are desperately needed for this presently incurable disease. Various studies have determined that the supportive nature of the bone marrow (BM) niche and crosstalk between the bone-resident cells and the tumor lead to increased tumor cell survival and escape from therapy. Specifically, studies from our lab highlighted the critical role of bone marrow adipose tissue and its expansion due to obesity or age in PCa progression and chemoresistance in bone. Bone marrow adipocytes are metabolically active cells that, through secretion of lipids and adipokines, have the potential to affect the neighboring cells, including the tumor cells that have disseminated into the BM niche. We have shown previously that interaction with marrow adipocytes promotes lipid uptake by PCa cells, modulates their metabolic phenotype, and activates pro-survival signaling and ER/oxidative stress pathways; however, the molecular mechanisms behind the tumor-promoting effects of adipocytes are not understood. The main objective of this study was to investigate the molecular mechanisms underlying lipid-induced stress during tumor cell-adipocyte crosstalk and its functional relationship to tumor growth and progression in bone. Our preliminary data showed that adipocyte-tumor cell crosstalk increases lipid peroxidation in PCa cells, which coincides with augmented expression of ER stress markers and activation of mTOR signaling. We also observed that PCa cells under adipocyte exposure had augmented gene expression of stearoyl-CoA desaturase (SCD), an ER-resident enzyme responsible for the conversion of saturated fatty acids (SFA) to monounsaturated fatty acids (MUFA). We, therefore, hypothesized that SCD is a metabolic regulator that protects metastatic PCa cells against lipid-induced toxicity and ER stress and promotes tumor survival via activation of mTOR signaling. Here, we show that the inhibition of mTORC1 by Everolimus (EVO) decreases the gene expression of ER stress markers in PCa cells exposed to adipocytes. We also demonstrate that adipocyte-mediated induction of ER stress coincides with an increase in active mTOR signaling in PCa cells, indicating possible bidirectional crosstalk between ER stress and mTOR pathways. Notably, inhibition of SCD in PCa cells grown in Transwell co-culture with adipocytes by either siRNA or the small molecule inhibitor CAY10566 leads to reduced mTOR signaling and an increase in lipid peroxidation in PCa cells. SCD inhibition also increases the gene and protein expression of ER stress markers, specifically XBP1 (S) and ATF4, suggesting apoptosis induction. Collectively, our results place SCD as a mediator of ER stress-mTOR crosstalk and a promoter of tumor survival and suggest that targeting SCD activity might be a viable approach to rewire tumor metabolism, and inhibit metastatic progression in bone and sensitize PCa tumors to therapy. Citation Format: Alexis Wilson, Mackenzie Herroon, Shane Mecca, Laimar Garmo, Izabela Podgorski. Adipocyte regulation of ER stress and mTOR signaling in bone-metastatic PCa: The role of stearoyl-CoA desaturase [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B039.

Artepillin C overcomes apalutamide resistance through blocking androgen signaling in prostate cancer cells

Prostate cancer has a relatively good prognosis, but most cases develop resistance to hormone therapy, leading to castration-resistant prostate cancer (CRPC). Androgen receptor (AR) antagonists and a cytochrome P450 17A1 inhibitor have been used to treat CRPC, but cancer cells readily develop resistance to these drugs. In this study, to improve the therapy of CRPC, we searched for natural compounds which block androgen signaling. Among cinnamic acid derivatives contained in Brazilian green propolis, artepillin C (ArtC) suppressed expressions of androgen-induced prostate-specific antigen and transmembrane protease serine 2 in a dose-dependent manner. Reporter assays revealed that ArtC displayed AR antagonist activity, albeit weaker than an AR antagonist flutamide. In general, aberrant activation of the androgen signaling is involved in the resistance of prostate cancer cells to hormone therapy. Recently, apalutamide, a novel AR antagonist, has been in clinical use, but its drug-resistant cases have been already reported. To search for compounds which overcome the resistance to apalutamide, we established apalutamide-resistant prostate cancer 22Rv1 cells (22Rv1/APA). The 22Rv1/APA cells showed higher AR expression and androgen sensitivity than parental 22Rv1 cells. ArtC inhibited androgen-induced proliferation of 22Rv1/APA cells by suppressing the enhanced androgen signaling through blocking the nuclear translocation of AR. In addition, ArtC potently sensitized the resistant cells to apalutamide by inducing apoptotic cell death due to mitochondrial dysfunction. These results suggest that the intake of Brazilian green propolis containing ArtC improves prostate cancer therapy.