Aggressive Prostate Cancer Cells Research Articles

Stabilization of RRBP1 mRNA via an m6A-dependent manner in prostate cancer constitutes a therapeutic vulnerability amenable to small-peptide inhibition of METTL3

Mounting evidence has implicated the RNA m6A methylation catalyzed by METTL3 in a wide range of physiological and pathological processes, including tumorigenesis. The detailed m6A landscape and molecular mechanism of METTL3 in prostate cancer (PCa) remains ill-defined. We find that METTL3 is overexpressed in PCa and correlates with worse patient survival. Functional studies establish METTL3 as an oncoprotein dependent on its m6A enzymatic activity in both AR+ and AR− PCa cells. To dissect the regulatory network of m6A pathway in PCa, we map the m6A landscape in clinical tumor samples using m6A-seq and identify genome-wide METTL3-binding transcripts via RIP-seq. Mechanistically, we discover RRBP1 as a direct METTL3 target in which METTL3 stabilizes RRBP1 mRNA in an m6A-dependent manner. RRBP1 positively correlates with METTL3 expression in PCa cohorts and exerts an oncogenic role in aggressive PCa cells. Leveraging the 3D structural protein-protein interaction between METTL3 and METTL14, we successfully develop two potential METTL3 peptide inhibitors (RM3 and RSM3) that effectively suppress cancer cell proliferation in vitro and tumor growth in vivo. Collectively, our study reveals a novel METTL3/m6A/RRBP1 axis in enhancing aggressive traits of PCa, which can be therapeutically targeted by small-peptide METTL3 antagonists.

Overexpression of sialyl Lewisa carrying mucin-type glycoprotein in prostate cancer cell line contributes to aggressiveness and metastasis

Metastasis-promoting Lewis and sialyl Lewis antigens expressed on glycoproteins such as mucins are frequently displayed on the surface of prostate cancer cells and could thus be ideal candidates as measures of prostate cancer aggressiveness. The current study describes the altered expression of sialyl Lewisa (sLea) antigen attached to glycoproteins and key glycosyltransferases between normal prostate (RWPE-1) and cancerous cell lines (LNCaP and DU145). Our results suggest that the expression of sLea on different glycoproteins correlates with the aggressiveness of prostate cancer cells, as determined by flow cytometry and fluorescence microscopy. Blotting studies revealed that sLea-bearing glycoproteins, similar to mucins, are predominantly expressed in the more aggressive DU145 cells, followed by LNCaP cells. Immunohistochemistry technique showed a gradient of sLea expression, with low levels in low-grade prostate cancer (stage II/III) and increasing levels in high-grade cancer (stage IV), indicating its potential as a prognostic marker. Additionally, in qRT-PCR analysis significant upregulation of the glycosyltransferases GALNT5 and ST3GAL6 was observed, correlating with the increased sLea expression in LNCaP (3.2- and 14.5-fold) and DU145 (3.3- and 23.75-fold) cells. Our data indicates a correlation between sLea selectin ligand expression and prostate cancer aggressiveness. Furthermore, GALNT5 and ST3GAL6 could serve as benchmarks in PCa malignancy.

Abstract C097: PVT1 exon 9-dependent overexpression of RSAD2 is a characteristic of prostate cancer in males of African ancestry

Abstract Aggressive prostate cancer (PCa), including castrate-resistant and neuroendocrine subtypes, is projected to increase in incidence 5% each year. Aggressive PCa disproportionally impacts African American men, has limited effective treatment options and high mortality rates. Although racial disparities are established in PCa development, attributed to multiple contributory factors including systemic racism, differences in socioeconomic status and distrust in the medical system, work is still needed to understand the contribution of biological factors. We found the exon 9 region of plasmacytoma variant translocation 1 (PVT1) overexpressed in aggressive PCa cell lines derived from African American (AA) men. We found PVT1 exon 9 overexpression induced phenotypic changes to normal prostate epithelial cells in vitro and led to tumor formation in vivo. RNA-sequencing analysis revealed 156 significantly differentially expressed genes influenced by PVT1 exon 9 overexpression and radical S-adenosyl methionine domain containing 2 (RSAD2) was the top significantly overexpressed gene (log2Fold change 1.71, p-value < 0.05). We utilized the GSE223405 publicly available dataset to identify whether our identified PVT1 exon 9 downstream genes were enriched in AA prostate cancer cells. RSAD2 was significantly overexpressed in AA prostate cancer cells compared to prostate cancer cells from men of European ancestry (moEA) (log2Fold change 6.18, p-value < 0.05). Comparing normal cells to prostate cancer cells between these groups, we found that normal AA prostate cells had significantly higher expression of RSAD2 (log2fold change 4.25, p-value < 0.001) whereas moEA samples did not (log2fold change 0.837, p-value=0.150). From analysis of data obtained from cBioPortal, we found RSAD2 alteration significantly correlates (p-value < 0.001) with self-identified race and copy number overexpression of RSAD2 was found to be enriched in AA patients (18.16% moEA versus 24% AA). Finally, we found 88.6% of the significantly differentially expressed genes within our PVT1 exon 9 RNA-sequencing dataset were significantly differentially expressed in GSE223405 AA samples and of those, 51.7% of genes exhibited a similar differential expression pattern suggesting that the PVT1 exon 9/RSAD2 dependent pathway may be more prominent in AA men. Mechanistically, we found two differing pathways of RSAD2 overexpression in aggressive prostate cancer cell models, one dependent and one independent of PVT1 exon 9 overexpression, with similar but differing biological functions related to epigenetic processes and metabolism. Ongoing work is investigating the RSAD2 and PVT1 exon 9 expression axis in a diverse set of human prostate cancer tissues and investigating their utilization as a therapeutic target in prostate cancer. Citation Format: Rachel E. Sexton-Bonacci1, Murtaza Barkarar1, Chindeum Udekwu1, Olorunseun O. Ogunwobi1. PVT1 exon 9-dependent overexpression of RSAD2 is a characteristic of prostate cancer in males of African ancestry [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C097.

Vitamin K3 derivative inhibits androgen receptor signaling in targeting aggressive prostate cancer cells.

Prostate cancer (PCa) is the second critical cause of cancer-related deaths, with African Americans dying at higher rates in the U.S. The main reasons for the higher mortality rate are ethnic differences and lack of understanding of prostate cancer biology and affordable treatments, as well as the financial burden of African American men to obtain the most effective and safe treatments. The effect of micronutrients, including Vitamin K, on various cancer cell lines has been widely studied, but the potential anticancer effect of VK3-OCH3, an analog of vitamin K3 (Menadione), on African American prostate cancer has not been evaluated. In this study, we compared the anticancer effect of VK3-OCH3 on targeting African American derived PCa cell lines namely RC77-T and MDA-PCa-2b. Our results show that VK3-OCH3 significantly inhibits the proliferation of both RC77-T and MDA-PCa-2b African American PCa cells and promotes apoptosis, and the underlying mechanism of cell death appears to be similar in both the cell lines. Notably, VK3-OCH3 inhibits colony-forming ability and induces apoptosis by blocking the cell cycle at G0 in African American PCa cells. VK3-OCH3 also acts as an anti-metastatic agent by inhibiting the migration ability of the metastatic properties of African American PCa cells. The cell death of African American PCa cells mediated by VK3-OCH3 is associated with the production of free radicals, such as intracellular and mitochondrial reactive oxygen species (ROS). Interestingly, antioxidants such as N-Acetylcysteine (NAC) and Glutathione (GSH) effectively negated the oxidative stress induced by VK3-OCH3 on PCa cell lines derived from African American patients. Of note, VK3-OCH3 reduces androgen receptor and prostate-specific antigen expression in these PCa cells. Furthermore, molecular dynamic studies reiterated that VK3-OCH3 strongly binds to the androgen receptor, suggesting that the androgen receptor is the potential molecular target of VK3-OCH3. In addition, Western blot analysis showed that VK3-OCH3 reduces the expression of androgen receptor, TRX2, and anti-apoptotic signaling molecules such as Bcl-2 and TCTP in the MDA-PCa-2b metastatic PCa cellular model. In conclusion, our results suggested that VK3-OCH3 is a promising anticancer agent that could potentially reduce the mortality rates of African American PCa patients, warranting further preclinical and translational studies.

Targeting PLOD2 suppresses invasion and metastatic potential in radiorecurrent prostate cancer

BackgroundMetastatic relapse of prostate cancer after radiotherapy is a significant cause of prostate cancer-related morbidity and mortality. PLOD2 is a mediator of invasion and metastasis that we identified as being upregulated in our highly aggressive radiorecurrent prostate cancer cell line.MethodsPatient dataset analysis was conducted using a variety of prostate cancer cohorts. Prostate cancer cell lines were treated with siRNA, or the drug PX-478 prior to in vitro invasion, migration, or in vivo chick embryo (CAM) extravasation assay. Protein levels were detected by western blot. For RNA analysis, RNA sequencing was conducted on PLOD2 knockdown cells and validated by qRT-PCR.ResultsPLOD2 is a negative prognostic factor associated with biochemical relapse, driving invasion, migration, and extravasation in radiorecurrent prostate cancer. Mechanistically, HIF1α upregulation drives PLOD2 expression in our radiorecurrent prostate cancer cells, which is effectively inhibited by HIF1α inhibitor PX-478 to reduce invasion, migration, and extravasation. Finally, the long non-coding RNA LNCSRLR acts as a promoter of invasion downstream of PLOD2.ConclusionsTogether, our results demonstrate for the first time the role of PLOD2 in radiorecurrent prostate cancer invasiveness, and point towards its potential as a therapeutic target to reduce metastasis and improve survival outcomes in prostate cancer patients.

The marine-derived HIF-1α inhibitor, Yardenone 2, reduces prostate cancer cell proliferation by targeting HIF-1 target genes

BackgroundProstate cancer (PCa) ranks as the second most prevalent cancer in men, with advanced stages posing significant treatment challenges. Given its solid tumor nature, PCa is highly susceptible to hypoxia, a condition associated with resistance to radiation and chemotherapy, metastasis, and unfavorable patient outcomes. Hypoxia-inducible factors (HIFs) play a pivotal role in cancer cell adaptation to hypoxic environments, contributing to treatment resistance. Consequently, inhibitors targeting HIFs hold promise for cancer therapy.MethodsIn this study, we aimed to characterize novel HIF-1α inhibitors including Sodwanones A (1), B (2), C (3), G (4) and Yardenone 2 (5) isolated from marine sponges belonging to the Axinella genus. Our investigation evaluated the impact of these compounds on various aspects of HIF-1α regulation, including stabilization, nuclear localization, expression of HIF-1 target genes (while sparing HIF-2 target genes), cellular metabolism, as well as cell proliferation and viability in prostate cells under hypoxic conditions.ResultsOur findings revealed that among the compounds tested, Yardenone 2 exhibited notable effects in hypoxia: it destabilized HIF-1α at the protein level, decreased its nuclear localization, selectively altered the expression of HIF-1 target genes, and restrained cell proliferation in aggressive PC3 prostate cancer cells as well as in an MSK-PCa3 patient-derived organoid line. Moreover, it affected the morphology of these organoid. Yardenone 2 was also compared to Docetaxel, a specific microtubule inhibitor and a drug used in the treatment of prostate cancer. The comparison between the two compounds revealed notable differences, such as a lack of specificity to hypoxic cells of Docetaxel.ConclusionThese results mark the first demonstration that Yardenone 2 functions as a cytostatic-like inhibitor impacting microtubules, specifically targeting hypoxic cancer cells. This discovery suggests a promising avenue for novel therapeutic interventions in prostate cancer.

Abstract 4619: Prolonged inhibition of androgen receptor signaling induces expression of Nuclear ErbB3 which renders prostate cancer cells susceptible to targeted inhibitors

Abstract Objective: Nuclear expression of the receptor tyrosine kinase (RTK) ErbB3/HER3 increases in highly aggressive prostate cancer cells, but its nuclear transport mechanism is currently unknown. Prostate tumors rely on the androgen receptor (AR), whose activation transcriptionally controls ErbB3 expression, but its role in ErbB3 nuclear localization had not previously been reported. Here, we investigated the mechanism by which subcellular localization of ErbB3 was altered in prostate cancer. Methods: ErbB3 localization was investigated in the human prostate cancer tumor progression model LNCaP, C4, C4-2 and C4-2B. ErbB3 was stimulated with heregulin-1β (HRG). Nuclear translocation was tested with a nucleocytoplasmic transport inhibitor panel (chlorpromazine, filipin III, amiloride and Leptomycin B) LNCaP and C4 cells were continuously cultured with the AR activation inhibitor abiraterone acetate (AbiAc) or ethanol (VEH) or treated with the AR inhibitors enzalutamide, darolutamide and apalutamide. ErbB3 activity and subcellular localization were analyzed using confocal microscopy/subcellular fractionation/immunoblot. Cell viability was determined by MTT assay. Proliferation and apoptosis were determined by flow cytometry. Invasive potential was investigated by crystal violet staining of cell colonies. ErbB3 expression was reduced by siRNA technology. Results: The ratio of nuclear to cytoplasmic ErbB3 increased in untreated cells from LNCaP<C4-2<C4<C4-2B. In all four lines, nuclear localization of ErbB3 peaked at 30 minutes after HRG treatment, with a rapid return to the cytoplasm in LNCaP cells (at 1 hour), slower in C4 cells (at 4 hours), and continued accumulation in the nucleus in C4-2 and C4-2B 8 hours following HRG treatment. Treatment with the transport inhibitor panel showed that nuclear accumulation of ErbB3 was prevented by the clathrin-dependent endocytosis inhibitor Chlorpromazine (CPZ) but not by the others. In LN-VEH and C4-VEH cells, HRG induced nuclear translocation of ErbB3 but not in LN-AbiAc or C4-AbiAc cells. Accordingly, HRG increased ErbB3 phosphorylation at Y1328 and nuclear Akt phosphorylation at S473 in VEH but not AbiAc cells. C4 cells, which had high baseline nuclear ErbB3, were more sensitive to the AR inhibitors and showed decreased viability and invasive potential compared to LNCaP cells. Conclusions: These results indicate that (1) ErbB3 nuclear localization required clathrin-dependent endocytosis (2) ligand binding of ErbB3 as well as the presence of an active AR is necessary for ErbB3 nuclear localization and (3) the presence of nuclear ErbB3 increases sensitivity to AR inhibitors. Citation Format: Maitreyee K. Jathal, Maria M. Mudryj, Paramita M. Ghosh. Prolonged inhibition of androgen receptor signaling induces expression of Nuclear ErbB3 which renders prostate cancer cells susceptible to targeted inhibitors [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 4619.

Abstract 5968: CDC7 is a targetable regulator of advanced prostate cancer

Abstract Prostate cancer is estimated to contribute to over 34,000 deaths in men residing in the United States, with the majority fatality due to metastatic disease. CDC7 is a kinase that regulates DNA replication and is an emerging cancer biomarker for poor prognosis in carcinomas such as Wilms tumor and hepatocellular carcinomas. In this study, we demonstrated that high level of CDC7 is associated with metastatic prostate cancer when compared to benign prostate tissues or localized prostate cancer. Furthermore, we found that CDC7 expression is highest in prostate cancer patient-derived xenografts (PDX) models of adenocarcinomas with neuroendocrine features. In vitro, we showed that inhibition of CDC7 with TAK-931, a CDC7 specific inhibitor, reduced the ability of aggressive prostate cancer cells to proliferate, migrate, and invade. Similarly, knock-down of CDC7 in prostate cancer cell lines inhibited cell growth in a colony formation assay. TAK-931 treated prostate cancer cell lines also showed an abnormal cell cycle profile, indicating that inhibiting CDC7 in aggressive prostate cancer could contribute to replication stress and promote apoptosis. Overall, this study demonstrates that CDC7 is a targetable protein that regulates advanced and aggressive prostate cancer growth. Citation Format: Alifiani Bonita Hartono, Sidharth Paparaju, Chung Lee, Shiqin Liu, Busola R. Alabi, En-Chi Hsu, James D. Brooks, Eva Corey, Tanya Stoyanova. CDC7 is a targetable regulator of advanced prostate cancer [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 5968.

Overexpression of REST Represses the Epithelial-Mesenchymal Transition Process and Decreases the Aggressiveness of Prostate Cancer Cells.

The RE-1 silencing transcription factor (REST) is a repressor factor related to neuroendocrine prostate cancer (PCa) (NEPC), a poor prognostic stage mainly associated with castration-resistant PCa (CRPC). NEPC is associated with cell transdifferentiation and the epithelial-mesenchymal transition (EMT) in cells undergoing androgen deprivation therapy (ADT) and enzalutamide (ENZ). The effect of REST overexpression in the 22rv1 cell line (xenograft-derived prostate cancer) on EMT, migration, invasion, and the viability for ENZ was evaluated. EMT genes, Twist and Zeb1, and the androgen receptor (AR) were evaluated through an RT-qPCR and Western blot in nuclear and cytosolic fractions of REST-overexpressing 22rv1 cells (22rv1-REST). The migratory and invasive capacities of 22rv1-REST cells were evaluated via Transwell® assays with and without Matrigel, respectively, and their viability for enzalutamide via MTT assays. The 22rv1-REST cells showed decreased nuclear levels of Twist, Zeb1, and AR, and a decreased migration and invasion and a lower viability for ENZ compared to the control. Results were expressed as the mean + SD of three independent experiments (Mann-Whitney U test, Kruskal-Wallis, Tukey test). REST behaves like a tumor suppressor, decreasing the aggressiveness of 22rv1 cells, probably through the repression of EMT and the neuroendocrine phenotype. Furthermore, REST could represent a response marker to ENZ in PCa patients.

Intracellular Fate of Sub-Toxic Concentration of Functionalized Selenium Nanoparticles in Aggressive Prostate Cancer Cells

Selenium 0 (Se0) is a powerful anti-proliferative agent in cancer research. We investigated the impact of sub-toxic concentrations of Se0 functionalized nanoparticles (SeNPs) on prostate cancer PC-3 cells and determined their intracellular localization and fate. An in-depth characterization of functionalized selenium nanoparticles composition is proposed to certify that no chemical bias relative to synthesis issues might have impacted the study. Selenium is an extremely diluted element in the biological environment and therefore requires high-performance techniques with a very low detection limit and high spatial resolution for intracellular imaging. This was explored with state-of-the-art techniques, but also with cryopreparation to preserve the chemical and structural integrity of the cells for spatially resolved and speciation techniques. Monodisperse solutions of SeNPs capped with bovine serum albumin (BSA) were shown to slow down the migration capacity of aggressive prostate cancer cells compared to polydisperse solutions of SeNPs capped with chitosan. BSA coating could prevent interactions between the reactive surface of the nanoparticles and the plasma membrane, mitigating the generation of reactive oxygen species. The intracellular localization showed interaction with mitochondria and also a localization in the lysosome-related organelle. The SeNPs-BSA localization in mitochondria constitute a possible explanation for our result showing a very significant dampening of the PC-3 cell proliferation capabilities. The purpose of the use of sublethal compound concentrations was to limit adverse effects resulting from high cell death to best evaluate some cellular changes and the fate of these SeNPs on PC-3. Our findings provide new insight to further study the various mechanisms of cytotoxicity of SeNPs.

Phenotype plasticity and altered sensitivity to chemotherapeutic agents in aggressive prostate cancer cells.

In 2023, approximately 288,300 new diagnoses of prostate cancer will occur, with 34,700 disease-related deaths. Death from prostate cancer is associated with metastasis, enabled by progression of tumor phenotypes and successful extracapsular extension to reach Batson's venous plexus, a specific route to the spine and brain. Using a mouse-human tumor xenograft model, we isolated an aggressive muscle invasive cell population of prostate cancer, called DU145J7 with a distinct biophysical phenotype, elevated histone H3K27, and increased matrix metalloproteinase 14 expression as compared to the non-aggressive parent cell population called DU145WT. Our goal was to determine the sensitivities to known chemotherapeutic agents of the aggressive cells as compared to the parent population. High-throughput screening was performed with 5,578 compounds, comprising of approved and investigational drugs for oncology. Eleven compounds were selected for additional testing, which revealed that vorinostat, 5-azacitidine, and fimepinostat (epigenetic inhibitors) showed 2.6-to-7.5-fold increases in lethality for the aggressive prostate cancer cell population as compared to the parent, as judged by the concentration of drug to inhibit 50% cell growth (IC50). On the other hand, the DU145J7 cells were 2.2-to-4.0-fold resistant to mitoxantrone, daunorubicin, and gimatecan (topoisomerase inhibitors) as compared to DU145WT. No differences in sensitivities between cell populations were found for docetaxel or pirarubicin. The increased sensitivity of DU145J7 prostate cancer cells to chromatin modifying agents suggests a therapeutic vulnerability occurs after tumor cells invade into and through muscle. Future work will determine which epigenetic modifiers and what combinations will be most effective to eradicate early aggressive tumor populations.

Mechanism of Tumor-Platelet Communications in Cancer.

Thrombosis is one of the main complications in cancer patients often leading to mortality. However, the mechanisms underlying platelet hyperactivation are poorly understood. Murine and human platelets were isolated and treated with small extracellular vesicles (sEVs) from various cancer cell lines. The effects of these cancer-sEVs on platelets were evaluated both in vitro and in vivo using various approaches, including the detection of cancer-sEV-specific markers in murine platelets and patient samples, measurement of platelet activation and thrombosis assays. Signaling events induced by cancer-sEVs and leading to platelet activation were identified, and the use of blocking antibodies to prevent thrombosis was demonstrated. We demonstrate that platelets very effectively take up sEVs from aggressive cancer cells. The process of uptake is fast, proceeds effectively in circulation in mice, and is mediated by the abundant sEV membrane protein-CD63. The uptake of cancer-sEVs leads to the accumulation of cancer cell-specific RNA in platelets in vitro and in vivo. The human prostate cancer-sEV-specific RNA marker PCA3 is detected in platelets of ~70% of prostate cancer patients. This was markedly reduced after prostatectomy. In vitro studies showed that platelet uptake of cancer-sEVs induces strong platelet activation in a CD63-RPTPα (receptor-like protein tyrosine phosphatase alpha)-dependent manner. In contrast to physiological agonists ADP and thrombin, cancer-sEVs activate platelets via a noncanonical mechanism. Intravital studies demonstrated accelerated thrombosis both in murine tumor models and in mice that received intravenous injections of cancer-sEVs. The prothrombotic effects of cancer-sEVs were rescued by blocking CD63. Tumors communicate with platelets by means of sEVs, which deliver cancer markers and activate platelets in a CD63-dependent manner leading to thrombosis. This emphasizes the diagnostic and prognostic value of platelet-associated cancer markers and identifies new pathways for intervention.

Abstract 1698: Novel role of Tribbles 2 driving treatment-emergent neuroendocrine differentiation in prostate cancer

Abstract Background: Prostate cancer initially responds to androgen receptor (AR) targeted therapies, such as enzalutamide. However, therapy-resistant, incurable disease invariably develops which remains as the major cause for mortality among prostate cancer patients. Molecular mechanism underlying emergence of resistant prostate cancer is yet to be fully characterized. Methods: We developed an in vitro cell culture model to mimic the clinical conditions in enzalutamide-resistant prostate cancer. These cells were transcriptionally profiled for gene expression analysis by Illumina Hi-Seq whole genome gene-expression array. Hits were validated by RT-qPCR and Western blot. For clinical correlation, enzalutamide treated prostate PDX models and surgical tumors (warm autopsy) from prostate cancer patients were comprehensively analyzed. Regulation and role of new hits were determined by treating cells and xenograft tumors with lentiviral-based shRNAs followed by analysis of cell viability, apoptosis, and effects on downstream targets. Pro-cancer effects were analyzed by cell proliferation, invasion, and soft-agar colony formation assays. Results: We identified that the Tribbles 2 (TRIB2) pseudokinase is overexpressed in enzalutamide-resistant prostate cancer (ERPC) cells. Elevated level of TRIB2 was also observed in enzalutamide treated PDX tumors, and in prostate tumors from patients who developed enzalutamide resistant metastatic disease. Interestingly, TRIB2 protein level is especially higher in prostate cancer cells and tumors, where the AR activity is low or absent due to genetic changes. Transfection and overexpression of TRIB2 results in aggressive prostate cancer cell growth and resistance to enzalutamide, apalutamide, darolutamide, and abiraterone. Furthermore, TRIB2 overexpression results in trans-differentiation in prostate cancer cells including neurite outgrowth and adeno-to-neuroendocrine lineage transition. Curiously, we found that TRIB2 downregulates luminal markers (AR, CK8), but upregulates the neuroendocrine markers (CHGA, NSE, SYP). Knockdown of TRIB2 triggers loss of neuroendocrine features, reduces viability of ERPC cells, and sensitize them back to enzalutamide, suggesting that TRIB2 plays a direct role in neuroendocrine differentiation and therapeutic resistance. We also found that TRIB2 upregulates the master neuronal transcription factor, BRN2, and the stemness factor, SOX2, and inhibition of either BRN2 or SOX2 re-sensitizes ERPC cells to enzalutamide. Conclusion: These findings reveal that TRIB2 is a novel driver for lineage plasticity and neuroendocrine differentiation in prostate cancer cells and greatly contributes to enzalutamide resistance. Citation Format: Jitender Monga, Craig Rogers, Shirish Gadgeel, Dhananjay Chitale, Jagadananda Ghosh. Novel role of Tribbles 2 driving treatment-emergent neuroendocrine differentiation in 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 1698.

Sonicated Extract from the Aril of Momordica Cochinchinensis Inhibits Cell Proliferation and Migration in Aggressive Prostate Cancer Cells.

Momordica cochinchinensis or gac fruit has been reported to have several biological activities, including antioxidation, anti-inflammatory, and anticancer activities. However, the effect on cancer cell metastasis has not been extensively studied. With this aim, the extract from the aril part was selected and investigated for prostate cancer cell migration. The aril extracts were prepared as boiled extract, sonicated extract, ethanol extract, and HAE (hexane:acetone:ethanol; 2 : 1 : 1) extract, while the prostate cancer cell models were PC-3 and LNCaP cells. An MTT assay was performed to compare the antiproliferative effect between prostate cancer cells and normal Vero cells. As a result, the sonicated extract had the highest efficiency in PC-3 cells, with IC50 values of 2 mg/mL and 0.59 mg/mL for 48 and 72 h, respectively, while it had less of an effect in LNCaP cells and was not toxic to normal cells. Cell damage was further confirmed using LDH and cell cycle analysis. As a result, the sonicated extract did not cause cell damage or death and only inhibited cell proliferation. The effect on cancer metastasis was further examined by wound healing, transwell migration assays, and western blotting. The results demonstrated that the sonicated extract inhibited PC-3 cell migration and decreased MMP-9 but increased TIMP-1 expression. All these results support that gac fruit is a valuable source for further development as an anticancer agent for prostate cancer patients.

PROTEIN KINASE C ALPHA IS A CENTRAL NODE FOR TUMORIGENIC TRANSCRIPTIONAL NETWORKS IN HUMAN PROSTATE CANCER.

Aberrant expression of protein kinase C (PKC) isozymes is a hallmark of cancer. The different members of the PKC family control cellular events associated with cancer development and progression. Whereas the classical/conventional PKCα isozyme has been linked to tumor suppression in most cancer types, here we demonstrate that this kinase is required for the mitogenic activity of aggressive human prostate cancer cells displaying aberrantly high PKCα expression. Immunohistochemical analysis showed abnormal up-regulation of PKCα in human primary prostate tumors. Interestingly, silencing PKCα expression from aggressive prostate cancer cells impairs cell cycle progression, proliferation and invasion, as well as their tumorigenic activity in a mouse xenograft model. Mechanistic analysis revealed that PKCα exerts a profound control of gene expression, particularly over genes and transcriptional networks associated with cell cycle progression and E2F transcription factors. PKCα RNAi depletion from PC3 prostate cancer cells led to a reduction in the expression of pro-inflammatory cytokine and epithelial-to-mesenchymal transition (EMT) genes, as well as a prominent down-regulation of the immune checkpoint ligand PD-L1. This PKCα-dependent gene expression profile was corroborated in silico using human prostate cancer databases. Our studies established PKCα as a multifunctional kinase that plays pleiotropic roles in prostate cancer, particularly by controlling genetic networks associated with tumor growth and progression. The identification of PKCα as a pro-tumorigenic kinase in human prostate cancer provides strong rationale for the development of therapeutic approaches towards targeting PKCα or its effectors.

The Differential Paracrine Role of the Endothelium in Prostate Cancer Cells.

Simple SummaryA growing body of literature supports the concept that a tumor mass is under the strict control of the microvascular endothelium and that the perfusion of oxygen and nutrients by capillary vessels to the tumor mass is reinforced by potent paracrine activity from the vascular endothelial cells. In our study, we investigate the biological and molecular implications of the paracrine crosstalk between vascular endothelial cells and prostate cancer cells. Our results indicate that the endothelial cells were able to secrete molecular signals that promote the proliferation and growth of low and highly aggressive prostate cancer cells and selectively increased the migration, invasion and metastatic potential of highly aggressive prostate cancer cells. The molecular analyses indicated that endothelial cells induced a differential effect on gene expression profile when comparing low versus highly aggressive prostate cancer cells, causing an enrichment of epigenetic changes in migratory pathways in highly aggressive prostate cancer cells. In conclusion, our results indicate that endothelial cells release signals that favor tumor growth and aggressiveness and that this interaction may play an important role in the progression of prostate cancer.The survival of patients with solid tumors, such as prostate cancer (PCa), has been limited and fleeting with anti-angiogenic therapies. It was previously thought that the mechanism by which the vasculature regulates tumor growth was driven by a passive movement of oxygen and nutrients to the tumor tissue. However, previous evidence suggests that endothelial cells have an alternative role in changing the behavior of tumor cells and contributing to cancer progression. Determining the impact of molecular signals/growth factors released by endothelial cells (ECs) on established PCa cell lines in vitro and in vivo could help to explain the mechanism by which ECs regulate tumor growth. Using cell-conditioned media collected from HUVEC (HUVEC-CM), our data show the stimulated proliferation of all the PCa cell lines tested. However, in more aggressive PCa cell lines, HUVEC-CM selectively promoted migration and invasion in vitro and in vivo. Using a PCa-cell-line-derived xenograft model co-injected with HUVEC or preincubated with HUVEC-CM, our results are consistent with the in vitro data, showing enhanced tumor growth, increased tumor microvasculature and promoted metastasis. Gene set enrichment analyses from RNA-Seq gene expression profiles showed that HUVEC-CM induced a differential effect on gene expression when comparing low versus highly aggressive PCa cell lines, demonstrating epigenetic and migratory pathway enrichments in highly aggressive PCa cells. In summary, paracrine stimulation by HUVEC increased PCa cell proliferation and tumor growth and selectively promoted migration and metastatic potential in more aggressive PCa cell lines.

Prostate cancer cells of increasing metastatic potential exhibit diverse contractile forces, cell stiffness, and motility in a microenvironment stiffness-dependent manner.

During metastasis, all cancer types must migrate through crowded multicellular environments. Simultaneously, cancers appear to change their biophysical properties. Indeed, cell softening and increased contractility are emerging as seemingly ubiquitous biomarkers of metastatic progression which may facilitate metastasis. Cell stiffness and contractility are also influenced by the microenvironment. Stiffer matrices resembling the tumor microenvironment cause metastatic cells to contract more strongly, further promoting contractile tumorigenic phenotypes. Prostate cancer (PCa), however, appears to deviate from these common cancer biophysics trends; aggressive metastatic PCa cells appear stiffer, rather than softer, to their lowly metastatic PCa counterparts. Although metastatic PCa cells have been reported to be more contractile than healthy cells, how cell contractility changes with increasing PCa metastatic potential has remained unknown. Here, we characterize the biophysical changes of PCa cells of various metastatic potential as a function of microenvironment stiffness. Using a panel of progressively increasing metastatic potential cell lines (22RV1, LNCaP, DU145, and PC3), we quantified their contractility using traction force microscopy (TFM), and measured their cortical stiffness using optical magnetic twisting cytometry (OMTC) and their motility using time-lapse microscopy. We found that PCa contractility, cell stiffness, and motility do not universally scale with metastatic potential. Rather, PCa cells of various metastatic efficiencies exhibit unique biophysical responses that are differentially influenced by substrate stiffness. Despite this biophysical diversity, this work concludes that mechanical microenvironment is a key determinant in the biophysical response of PCa with variable metastatic potentials. The mechanics-oriented focus and methodology of the study is unique and complementary to conventional biochemical and genetic strategies typically used to understand this disease, and thus may usher in new perspectives and approaches.

Seleno-aspirin compound AS-10 promotes histone acetylation ahead of suppressing androgen receptor transcription, G1 arrest, and apoptosis of prostate cancer cells.

The novel selenium-aspirin compound AS-10 was recently reported by us with a cancer cell killing potency three orders of magnitude greater than aspirin in pancreatic cancer cell lines with caspase-mediated apoptosis and a reasonable selectivity against malignant cells. Although we also observed its cytocidal activity against PC-3 and DU145 androgen receptor (AR)-negative and P53-null/mutant aggressive human prostate cancer (PCa) cell lines in NCI-60 screen, the potential involvement and targeting of AR and P53 pathways that are intact in early-stage prostate carcinogenesis has not been examined, nor its primary molecular signaling after exposure. Human LNCaP PCa cells with functional AR and intact P53 were used to examine their cell cycle and cell fate responses to AS-10 exposure and upstream molecular signaling events including histone acetylation as a known aspirin effect. The AR-positive 22Rv1 human PCa cells were used to validate key findings. In addition to confirming AS-10's superior cytocidal potency than aspirin against all four PCa cell lines, we report a rapid (within 5 min) promotion of histone acetylation several hours ahead of the suppression of AR and prostate-specific antigen (PSA, coded by KLK3 gene) in LNCaP and 22Rv1 cells. AS-10 decreased AR and KLK3 mRNA levels without impacting pre-existing AR protein degradation or nuclear translocation in LNCaP cells. Sustained exposure to AS-10 arrested cells predominantly in G1 , and induced caspase-mediated apoptosis without necrosis. The death induced by AS-10 in LNCaP cells was attenuated by nontranscriptional activation of P53 protein or Jun N-terminal Kinase cellular stress signaling and was mitigated modestly by glutathione-boosting antioxidant N-acetylcysteine. AS-10 synergized with histone deacetylase inhibitor SAHA to suppress AR/PSA abundance and kill LNCaP cells. RNA-seq confirmed AR suppression at the transcriptional level and suggested multiple oncogene, cyclin, and CDK/CKI transcriptional actions to contribute to the cellular consequences. AS-10 promotes histone acetylation as its probable primary mechanism of action to induce PCa cell-cycle arrest and apoptosis, regardless of AR and P53 status. Nevertheless, the inhibition of AR signaling through mechanisms distinct from canonical AR antagonists may hold promise for combinatorial use with androgen deprivation therapy regimens or AR-axis targeting drugs.

Adipocyte-Derived Extracellular Vesicles Promote Prostate Cancer Cell Aggressiveness by Enabling Multiple Phenotypic and Metabolic Changes.

Background: In recent decades, obesity has widely emerged as an important risk factor for prostate cancer (PCa). Adipose tissue and PCa cells have been shown to orchestrate a complex interaction network to support tumor growth and evolution; nonetheless, the study of this communication has only been focused on soluble factors, although increasing evidence highlights the key role of extracellular vesicles (EVs) in the modulation of tumor progression. Methods and Results: In the present study, we found that EVs derived from 3T3-L1 adipocytes could affect PC3 and DU145 PCa cell traits, inducing increased proliferation, migration and invasion. Furthermore, conditioning of both PCa cell lines with adipocyte-released EVs resulted in lower sensitivity to docetaxel, with reduced phosphatidylserine externalization and decreased caspase 3 and PARP cleavage. In particular, these alterations were paralleled by an Akt/HIF-1α axis-related Warburg effect, characterized by enhanced glucose consumption, lactate release and ATP production. Conclusions: Collectively, these findings demonstrate that EV-mediated crosstalk exists between adipocytes and PCa, driving tumor aggressiveness.

Abstract 127: Pre-exposure to stress-inducing agents increase the anticancer efficacy of focused ultrasound against aggressive prostate cancer cells

Abstract Despite the initial success in treatment of localized prostate cancer (PCa) using surgery, radiation or hormonal therapy, recurrence of aggressive tumors dictates morbidity and mortality. Focused ultrasound (FUS) is being tested as a targeted noninvasive approach to eliminate the localized PCa foci, and strategies to enhance the anticancer potential of FUS have a high translational value. Since aggressive cancer cells utilize oxidative stress (Ox-stress) and endoplasmic reticulum stress (ER-stress) pathways for their survival and recurrence, we hypothesized that pre-treatment with drugs that disrupt stress-signaling pathways in tumor cells may increase FUS efficacy. Using four different PCa cell lines, i.e. LNCaP, C4-2B, 22Rv1 and DU145, we tested the in vitro effects of FUS, alone and in combination with two clinically-tested drugs that increase Ox-stress (i.e. CDDO-me) or ER-stress (i.e. Nelfinavir). As compared to standalone FUS, significant (p<0.05) suppressions in both survival and recurrence of PCa cells were observed following pre-sensitization with low dose CDDO-me (100 nM) and/or nelfinavir (2 µM). In drug pre-sensitized cells, significant anticancer effects were evident at a FUS intensity of as low as 0.7 kW/cm2. This combined mechanochemical disruption (MCD) approach decreased cell proliferation, migration and clonogenic ability, and increased apoptosis/necrosis and reactive oxygen species (ROS) production. Furthermore, although activated in cells that survived standalone FUS, pre-sensitization with CDDO-me and/or nelfinavir suppressed both total and activated (phosphorylated) NF-κB and Akt protein levels in surviving cells. Thus, a combined MCD therapy may be an effective noninvasive approach towards targeted elimination of aggressive PCa cells. Citation Format: Hakm Y. Murad, Partha K. Chandra, Charles A. Kelly, Namrata Khurana, Heng Yu, Emma P. Bortz, Shirley N. Hong, Debasis Mondal, Damir B. Khismatullin. Pre-exposure to stress-inducing agents increase the anticancer efficacy of focused ultrasound against aggressive prostate cancer cells [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 127.