Normal Prostate Epithelial Cells Research Articles

Development of a Novel Risk Signature for Predicting the Prognosis and Immunotherapeutic Response of Prostate Cancer by Integrating Ferroptosis and Immune-Related Genes.

Ferroptosis and immune response correlation studies have not been reported in prostate cancer (PCa), and the main goal of this paper is to identify biomarkers that can be used for early diagnosis of prostate cancer. Data on PCa were retrieved from the TCGA and MSKCC2010 databases. Thereafter, the differentially expressed ferroptosis-related genes (DE-FRGs: ACSF2) and immune-related genes (DE-IRGs: ANGPT1, NPPC, and PTGDS) were identified using the "limma" package. Additionally, we used univariate and multivariate Cox regression analyses to obtain biochemical relapse (BCR)-free survival-related genes and construct a risk signature. Patients with high-risk scores were characterized by poor BCR-free survival, relatively low immune cell abundance, and comparably weak expression of immune checkpoint molecules. Moreover, gene set variation analysis (GSVA) was performed to explore the biological pathways related to the risk signature. Single sample gene set enrichment analysis (ssGESA) was applied to evaluate the status of immune cells in patients with PCa, which demonstrated that the risk score was intimately affiliated with immune response and cancer pathways. Ultimately, the connection between the risk score and response of PCa patients to immunotherapy was appraised using the TIDE algorithm. The TIDE algorithm implied that the high-risk score PCa population might benefit more from immunotherapy regimens. Finally, qRT-PCR were used to evaluate the expression of DE-FRGs and DE-IRGs in PCa cell and normal prostate epithelial cells. The result of qRT-PCR showed that the mRNA expression levels of ACSF2, ANGPT1, NPPC, and PTGDS in normal prostate epithelial cell were higher than that in PCa cells. Therefore, a risk score model was generated based on one DE-FRG and three DE-IRGs, which could predict the BCR-free survival and response of immunotherapy for patients with PCa.

Mode of action exploration for prostate epithelial cell injury caused by bisphenol A

Bisphenol A (BPA) is a typical food chemical contaminant with various detrimental effects, especially on reproductive system. Male prostate damage is also one of its major adverse health effects, of which mode of action (MOA) remains unclear. This study aims to explore the MOA for prostate toxicity of BPA using human normal prostate epithelial cell RWPE-1 for 28-day human-relevant-level exposure. A physiological based pharmacokinetic model was used to determine the concentration of BPA based on the actual oral exposure in China. The possible key events were identified by high-throughput transcriptome sequencing and validated by qPCR, Western blot and cell cycle assay, and the benchmark concentration analysis were conducted. The enriched KEGG pathways include the endocytosis, cell cycle, cellular senescence, MAPK and TNF signaling pathways. With increasing BPA concentrations, the increased mRNA and/or protein expressions of MAPKAPK2, c-JUN and c-fos in the MAPK signaling pathway, the increased mRNA expressions of CCND1 and CDKN1A, the decreased mRNA expression of CDC25C, the increased proportion of G0/G1 phase and S phase, as well as the decreased proportion of G2/M phase, were observed. The lowest value of benchmark concentration lower confidence limit (BMCL) was retrieved from G2/M phase ratio, with 110.580 and 175.862 nM for BMCL5 and BMCL10, respectively, much higher than the male gonad maximum concentration of 0.019 nM of BPA at the current exposure level of adult Chinese males. In conclusion, the MOA of BPA induced male prostatic toxicity at human-relevant levels may include: key event (KE)1-MAPK signaling pathway activation, KE2-disorder of cell cycle regulatory gene expression (increased expression of CCND1 and CDKN1A, decreased expression of CDC25C), and KE3-disturbance of cell cycle (increased proportion of G0/G1 and S phases, decreased proportion of G2/M phases). However, more studies are needed to validate and complete the MOA.

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.

Role of Vitamins in Therapeutic and Targeting Approaches for Prostate Cancer: An Overview.

Vitamins play a crucial role in cellular functions like cell cycling and proliferation, differentiation, and apoptosis. These also help in the induction of cell cycle arrest and/or apoptosis. They can inhibit normal prostatic epithelial cell growth and might be helpful for the prevention of prostate cancer (PCa). Many essential vitamins including the fat-soluble vitamins (vitamin A, vitamin D, vitamin E, and vitamin K) and the water-soluble vitamins (vitamin B complexes and vitamin C) have a huge impact on the inhibition of growth and progression of PCa. Vitamins show anticancer properties and are involved in regulatory processes like the DNA repairing process, which inhibit the growth of PCa. Consumption of multivitamins prevents methylation of cancer cells and possesses an enormous potential that can be applied for the prevention as well as in the management of PCa. They have a great role in the inhibition of different signalling pathways involved in PCa. Moreover, they have also displayed a significant role in targeting of PCa with various nanocarrier systems. This review encompasses the recent studies about the individual actions of different vitamins and vitamin analogs, the combination of vitamins, and their efficient functions in various therapeutic and targeting approaches for PCa.

Differential regulation of heparan sulfate biosynthesis in fibroblasts cocultured with normal vs. cancerous prostate cells.

Heparan sulfate proteoglycans (HSPGs) regulate a wide range of biological activities in both physiological and pathological conditions. Altered expression or deregulated function of HSPGs and their heparan sulfate (HS) chains significantly contribute to carcinogenesis as well and crucially depends on the functioning of the complex system of HS biosynthetic/modifying enzymes termed as "GAGosome". Here, we aimed at investigating the expression profile of the system in a cell culture model of stroma-epithelial crosstalk and searching for transcription factors potentially related to the regulation of expression of the genes involved. Coculture of BjTERT-fibroblasts with normal PNT2 human prostate epithelial cells resulted in significant downregulation (2-4-fold) of transcriptional activity of HS metabolism-involved genes (EXT1/2, NDST1/2, GLCE, HS2ST1, HS3ST1/2, HS6ST1/2, SULF1/2, HPSE) in both cell types, whereas coculture with prostate cancer cells (LNCaP, PC3, DU145) demonstrated no significant interchanges. Human Transcription Factor RT2 Profiler PCR array and manual RT-PCR verification supposed FOS, MYC, E2F, SRF, NR3C1 as potential candidates for regulation and/or coordination of HS biosynthesis. Taken together, transcriptional activity of HS biosynthetic system in normal fibroblasts and prostate epithelial cells during their coculture might be controlled by their intercellular communication, reflecting of adaptation of these cells to each other. The regulation is attenuated or abrogated if normal fibroblasts interact with prostate cancer cells making the cancer cells independent of the limiting effects of fibroblasts, thus contributing to possibility of unlimited growth and progression. Overall, these data demonstrate an ability of cell-cell interactions to affect transcriptional activity of HS biosynthesis-involved genes.

Brucine Suppresses Malignant Progression of Prostate Cancer by Decreasing Sarcosine Accumulation via Downregulation of GNMT in the Glycine/sarcosine Metabolic Pathway.

Prostate cancer (PCa) remains a leading cause of cancer-related incidence and mortality in men. Disruptions in amino acid (AA) metabolism contribute to the disease progression, with brucine, a glycine antagonist, exhibiting antitumor effects. This study explores the antitumor impact of brucine on PCa and investigates its mechanisms in regulating AA metabolic pathways. The study employed the PCa cell line DU-145, characterized by high sarcosine (Sar) levels, for various assays including Cell Counting Kit-8 (CCK8), wound healing, Transwell, 5-Ethynyl-2'-deoxyuridine (EDU), TdT mediated dUTP Nick End Labeling (TUNEL), flow cytometry, Western blot, and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Network pharmacological analysis determined the anticancer mechanisms of brucine. Sar levels in DU-145 cells were significantly higher than in normal prostatic epithelial cells RWPE-1. Treatment with brucine resulted in a marked decrease in cell viability, proliferation, invasion, and migration, while promoting apoptosis in a dose-dependent manner. Sar levels decreased with increasing brucine concentration. Network pharmacology analysis linked brucine's anticancer effect to the AA metabolism and glycine N-methyltransferase (GNMT) pathways. GNMT expression in prostate cancer tissues and The Cancer Genome Atlas database was significantly elevated compared to controls. Treatment with brucine led to downregulation of GNMT expression in DU-145 cells without significant effect on sarcosine dehydrogenase (SARDH). Addition of recombinant GNMT partially reversed the inhibitory effects of brucine on DU-145 cells. Treatment with brucine downregulates GNMT expression in DU-145 cells, reducing Sar accumulation and inhibiting tumor progression. These findings provide new insights into the antitumor mechanisms of brucine in PCa.

Diterpene glycosides from Fructus Rubi ameliorates benign prostatic hyperplasia in rats through the androgen and TGF-β/Smad signaling pathway

Fructus Rubi (FR), a food material with medicinal value, is used in traditional Chinese medicine (TCM) for treatment of various kidney-related problems, such as impotence, spermatorrhea, and frequent urination. It is also frequently used to produce diverse functional foods in China. The purpose of this research was to assess the therapeutic effects of FR diterpene glycosides on RWPE-1 epithelial cell (RWPE-1), a human normal prostatic epithelial cell, and benign prostate hyperplasia (BPH) rats, both of which had been exposed to dihydrotestosterone (DHT) and testosterone propionate (TP), respectively, and to investigate the mechanism of action. Target proteins that could stably bind to certain diterpene glycosides were screened through drug affinity responsive target stability combined with mass spectrometry (DARTS/MS). DHT-induced RWPE-1cells were used to detect drug activity. TP was subcutaneously injected to induce BPH in rats. The extract of diterpene glycosides from FR (FDS) was orally administered for 28 days. The DHT levels in the serum and prostate tissue of the rats were measured through enzyme-linked immunosorbent assay (ELISA), and to analyze cell proliferation and epithelial-mesenchymal transition (EMT), the protein expression of prostate-specific antigen (PSA), androgen receptor (AR), steroid 5α-reductase type 2 (SRD5A2), proliferating cell nuclear antigen (PCNA), S100 calcium-binding protein A2 (S100A2), transforming growth factor-β1 (TGF-β1), E-cadherin, vimentin, and Smad4 was determined through western blotting (WB), immunohistochemistry (IHC), or immunofluorescence (IF). FDS reduced the proliferation of DHT-induced RWPE-1cells. It also significantly inhibited rat prostate enlargement; decreased DHT levels in the serum and prostate tissue; inhibited the protein expression of AR, PSA, PCNA, S100A2, TGF-β1, E-cadherin, and Smad4; and increased the protein expression of E-cadherin. The present study is the first to report that diterpene glycosides isolated from FR inhibited BPH at the cellular level, regulated the proliferation of prostate cells through the androgen signaling pathway, and prevented EMT in the prostate through the S100A2-mediated TGF-β/Smad signaling pathway. These results indicate that FDS is a promising multitarget therapy for BPH.

Distinctive expression and cellular localisation of zinc homeostasis-related proteins in breast and prostate cancer cells

BackgroundZinc transport proteins (ZIP and ZnT), metallothioneins (MT) and protein kinase CK2 are involved in dysregulation of zinc homeostasis in breast and prostate cancer cells. Following up our previous research, we targeted ZIP12, ZnT1, MT2A and CK2 in this study by investigating their expression levels and protein localisation. MethodsQuantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence confocal microscopy were employed to quantify the expression of ZIP12, ZnT1, MT2A and CK2 subunits in a panel of breast and prostate cell lines without or with extracellular zinc exposure. The cellular localisations of these target proteins were also examined by immunofluorescence confocal microscopy. ResultsIn response to the extracellular zinc exposure, the gene expression was elevated for SLC39A12 (ZIP12), SLC30A1 (ZnT1) and MT2A (MT2A) in normal prostate epithelial cells (RWPE-1) in contrast to their cancerous counterparts (PC3 and DU145), whilst the gene expression was higher for SLC39A12 (ZIP12) and SLC30A1 (ZnT1) in both normal (MCF10A) and basal breast cancer cells (MDA-MB-231) compared to luminal breast cancer cells (MCF-7). At the protein level, the expression for both ZIP12 and ZnT1 was trending lower in the time course for the breast cancer cells whilst their expression was remained constant in the normal breast epithelial cells. The expression of ZIP12 in prostate cancer cells was higher than the normal prostate cells. The protein expression for CK2 α/αꞌ and CK2β was markedly higher in prostate cancer cells than the normal prostate cells. Upon extracellular zinc exposure, ZIP12 was, for the first time, conspicuously localised in the plasma membrane of breast cancer cells but not in normal breast epithelial cells and prostate cells. ZnT1 is only localised in the plasma membrane of breast cancer cells. MT2A is distinctively seen close to the plasma membrane in breast cancer cells. CK2 is also for the first time shown to be localised in proximity to the plasma membrane of breast cancer cells. ConclusionThe findings, particularly the localisation of ZIP12 and CK2, are novel and significant for our understanding of zinc homeostasis in breast and prostate cancer cells.

Pentagalloyl Glucose (PGG) Exhibits Anti-Cancer Activity against Aggressive Prostate Cancer by Modulating the ROR1 Mediated AKT-GSK3β Pathway.

Androgen-receptor-negative, androgen-independent (ARneg-AI) prostate cancer aggressively proliferates and metastasizes, which makes treatment difficult. Hence, it is necessary to continue exploring cancer-associated markers, such as oncofetal Receptor Tyrosine Kinase like Orphan Receptor 1 (ROR1), which may serve as a form of targeted prostate cancer therapy. In this study, we identify that Penta-O-galloyl-β-D-glucose (PGG), a plant-derived gallotannin small molecule inhibitor, modulates ROR1-mediated oncogenic signaling and mitigates prostate cancer phenotypes. Results indicate that ROR1 protein levels were elevated in the highly aggressive ARneg-AI PC3 cancer cell line. PGG was selectively cytotoxic to PC3 cells and induced apoptosis of PC3 (IC50 of 31.64 µM) in comparison to normal prostate epithelial RWPE-1 cells (IC50 of 74.55 µM). PGG was found to suppress ROR1 and downstream oncogenic pathways in PC3 cells. These molecular phenomena were corroborated by reduced migration, invasion, and cell cycle progression of PC3 cells. PGG minimally and moderately affected RWPE-1 and ARneg-AI DU145, respectively, which may be due to these cells having lower levels of ROR1 expression in comparison to PC3 cells. Additionally, PGG acted synergistically with the standard chemotherapeutic agent docetaxel to lower the IC50 of both compounds about five-fold (combination index = 0.402) in PC3 cells. These results suggest that ROR1 is a key oncogenic driver and a promising target in aggressive prostate cancers that lack a targetable androgen receptor. Furthermore, PGG may be a selective and potent anti-cancer agent capable of treating ROR1-expressing prostate cancers.

Increased levels of thymidine kinase 1 in malignant cell-derived extracellular vesicles

Extracellular vesicles (EVs), whose main subtypes are exosomes, microparticles, and apoptotic bodies, are secreted by all cells and harbor biomolecules such as DNA, RNA, and proteins. They function as intercellular messengers and, depending on their cargo, may have multiple roles in cancer development. Thymidine kinase 1 (TK1) is a cell cycle-dependent enzyme used as a biomarker for cell proliferation. TK1 is usually elevated in cancer patients' serum, making the enzyme a valuable tumor proliferation biomarker that strongly correlates with cancer stage and metastatic capabilities. Here, we investigated the presence of TK1 in EVs derived from three prostate cancer cell lines with various p53 mutation statuses (LNCaP, PC3, and DU145), EVs from the normal prostate epithelial cell line RWPE-1 and EVs isolated from human seminal fluid (prostasomes). We measured the TK1 activity by a real-time assay for these EVs. We demonstrated that the TK1 enzyme activity is higher in EVs derived from the malignant cell lines, with the highest activity from cells deriving from the most aggressive cancer, compared to the prostasomes and RWPE-1 EVs. The measurement of TK1 activity in EVs may be essential in future prostate cancer studies.

FOXA2 rewires AP-1 for transcriptional reprogramming and lineage plasticity in prostate cancer

FOXA family proteins act as pioneer factors by remodeling compact chromatin structures. FOXA1 is crucial for the chromatin binding of the androgen receptor (AR) in both normal prostate epithelial cells and the luminal subtype of prostate cancer (PCa). Recent studies have highlighted the emergence of FOXA2 as an adaptive response to AR signaling inhibition treatments. However, the role of the FOXA1 to FOXA2 transition in regulating cancer lineage plasticity remains unclear. Our study demonstrates that FOXA2 binds to distinct classes of developmental enhancers in multiple AR-independent PCa subtypes, with its binding depending on LSD1. Moreover, we reveal that FOXA2 collaborates with JUN at chromatin and promotes transcriptional reprogramming of AP-1 in lineage-plastic cancer cells, thereby facilitating cell state transitions to multiple lineages. Overall, our findings underscore the pivotal role of FOXA2 as a pan-plasticity driver that rewires AP-1 to induce the differential transcriptional reprogramming necessary for cancer cell lineage plasticity.

Oncogenic Role of SATB2 In Vitro: Regulator of Pluripotency, Self-Renewal, and Epithelial-Mesenchymal Transition in Prostate Cancer.

Special AT-rich sequence binding protein-2 (SATB2) is a nuclear matrix protein that binds to nuclear attachment regions and is involved in chromatin remodeling and transcription regulation. In stem cells, it regulates the expression of genes required for maintaining pluripotency and self-renewal and epithelial-mesenchymal transition (EMT). In this study, we examined the oncogenic role of SATB2 in prostate cancer and assessed whether overexpression of SATB2 in human normal prostate epithelial cells (PrECs) induces properties of cancer stem cells (CSCs). The results demonstrate that SATB2 is highly expressed in prostate cancer cell lines and CSCs, but not in PrECs. Overexpression of SATB2 in PrECs induces cellular transformation which was evident by the formation of colonies in soft agar and spheroids in suspension. Overexpression of SATB2 in PrECs also resulted in induction of stem cell markers (CD44 and CD133), pluripotency-maintaining transcription factors (cMYC, OCT4, SOX2, KLF4, and NANOG), CADHERIN switch, and EMT-related transcription factors. Chromatin immunoprecipitation assay demonstrated that SATB2 can directly bind to promoters of BCL-2, BSP, NANOG, MYC, XIAP, KLF4, and HOXA2, suggesting SATB2 is capable of directly regulating pluripotency/self-renewal, cell survival, and proliferation. Since prostate CSCs play a crucial role in cancer initiation, progression, and metastasis, we also examined the effects of SATB2 knockdown on stemness. SATB2 knockdown in prostate CSCs inhibited spheroid formation, cell viability, colony formation, cell motility, migration, and invasion compared to their scrambled control groups. SATB2 knockdown in CSCs also upregulated the expression of E-CADHERIN and inhibited the expression of N-CADHERIN, SNAIL, SLUG, and ZEB1. The expression of SATB2 was significantly higher in prostate adenocarcinoma compared to normal tissues. Overall, our data suggest that SATB2 acts as an oncogenic factor where it is capable of inducing malignant changes in PrECs by inducing CSC characteristics.

Brevilin A Inhibits Prostate Cancer Progression by Decreasing PAX5-Activated SOX4.

Brevilin A possesses inhibitory effects on the development of prostate cancer (PCa); however, the underlying mechanism remains unclear. The present work aims to analyze how Brevilin A regulates PCa cell malignancy. RNA expression of paired box 5 (PAX5) and SRY-box transcription factor 4 (SOX4) was analyzed by quantitative real-time polymerase chain reaction. Protein expression of PAX5, SOX4, and nuclear proliferation marker (Ki67) was detected by western blotting or immunohistochemistry assay. The viability, proliferation, apoptosis, and migratory and invasive abilities of PCa cells were investigated by cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), flow cytometry, and transwell assays, respectively. The association between PAX5 and SOX4 was identified by dual-luciferase reporter assay and chromatin immunoprecipitation assay. Xenograft mouse model assay was used to reveal the effect of Brevilin A on tumor tumorigenesis in vivo. PAX5 and SOX4 expression were upregulated in PCa tissues and cells relative to normal prostate tissues and human prostate epithelial cells. Brevilin A treatment inhibited PAX5 protein expression in PCa cells. Additionally, Brevilin A inhibited proliferation, migration and invasion and induced apoptosis of PCa cells, whereas these effects were attenuated after PAX5 overexpression. SOX4 was transcriptionally activated by PAX5, and its introduction partially relieved the inhibitory effects of PAX5 knockdown on PCa cell malignancy. Moreover, Brevilin A delayed tumor formation in vivo. Brevilin A inhibited PCa progression by regulating SOX4 expression in a PAX5-dependent manner, providing a promising anti-tumor drug for PCa.

DSCAM-AS1 promotes the development of prostate cancer

PurposeThe purpose of this study was to investigate the role of lncRNA DSCAM-AS1 in prostate cancer to find new therapeutic targets and promote the research progress of prostate cancer.MethodsRT-qPCR was used to detect DSCAM-AS1 expression in prostate cancer tissues, normal tissues, human normal prostate epithelial cells (RWPE), and four prostate cancer cell lines. The clinical and prognostic role of DSCAM-AS1 was evaluated by the Kaplan–Meier curve and chi-square test. Secondly, a dual luciferase reporter gene assay was used to study the regulatory mechanism between miR-338-3p and DSCAM-AS1. Finally, the roles of DSCAM-AS1 and miR-338-3p in prostate cancer cell proliferation and metastasis were explored by CCK-8 and Transwell assays.ResultsIt was found that DSCAM-AS1 upregulation could serve as a warning of deterioration and poor prognosis in prostate cancer patients, and that knockdown of DSCAM-AS1 expression inhibited the progression of prostate cancer cells. In addition, miR-338-3p, a target of DSCAM-AS1, was found to be down-regulated in prostate cancer cells and miR-338-3p knockdown could reverse the inhibitory effect of DSCAM-AS1 silencing on prostate cancer.ConclusionDSCAM-AS1 is up-regulated in prostate cancer and regulates the progression of prostate cancer cells by targeting miR-338-3p.

Abstract LB324: STING protein expression is increased in intermediate luminal cells in proliferative inflammatory atrophy of the prostate (PIA) and is absent in the majority of primary prostatic adenocarcinomas

Abstract Unresolved chronic inflammation has been postulated to drive prostate cancer development. We have proposed that proliferative inflammatory atrophy (PIA) can lead to the development of preneoplastic high grade prostatic intraepithelial neoplasia (PIN) lesions and/or directly to adenocarcinoma at times. Additionally, some PIA cells harbor molecular alterations seen in PIN and invasive carcinoma lesions, with a low frequency of PIA cells showing MYC over-expression, partial promoter hypermethylation and silencing of GSTP1, and formation of ETS gene fusions. PIA appears to result from inflammatory injury and regeneration in which proliferative and atrophic epithelial cells have an intermediate cell phenotype with both luminal and basal cell features, including basal cell markers, keratin 5 and BCL2, as well as varying levels of prostate luminal enriched genes such as androgen receptor, PSA and NKX3.1. Also, a number of gene products that are apparently stress induced, which are not expressed in normal prostate are induced in at least a subset of intermediate luminal cells in PIA, including GSTA1, PTGS2, LTF and PIGR (PMIDs:17384581, 37550827). Stimulator of interferon genes (STING), encoded by TMEM173, is a multipass transmembrane ER associated protein, and is a critical hub for innate responses directed against numerous bacterial, viral and parasitic pathogens, and is important for the induction and maintenance of adaptive immunity in a number of situations. Here, we examined STING expression by IHC, using a genetically validated assay, and show that the majority of atrophic prostatic luminal epithelial cells in all PIA lesions (N=20) consistently express high levels of STING, which is present in normal prostatic basal epithelial cells yet absent in normal-appearing prostatic luminal cells. In primary prostatic adenocarcinomas (> 260 tumors), STING protein was either completely negative , or extremely low in ~ 95% of cases. That STING in PIA is activate is supported by single cell RNA sequencing (scRNAseq) data from human prostatectomy tissues that show cells with the phenotype of intermediate luminal cells, indicative of PIA, express a number of interferon response genes that are not expressed in normal luminal cells (PMID 37905029). To our knowledge this is the first systematic report of STING protein staining in human prostate cancer and benign tissues. We postulate that upregulation of STING in human intermediate cells in PIA represents an inflammatory response that can drive interferon stimulated gene expression and inflammation in PIA. This innate immune response is absent, potentially by gene silencing, in prostatic adenocarcinoma, which may help neoplastic cells evade the immune response and could help contribute to the long standing finding that most prostate cancers are immunologically cold. Citation Format: Thomas M. Steele, Alan K. Meeker, Mindy K. Graham, Rulin Wang, Carolina Gomes-Alexandre, William G. Nelson, Fernanda Caramella Pereiria, Sushant Kachhap, Srinivasan Yegnasubramanian, Angelo Michael De Marzo. STING protein expression is increased in intermediate luminal cells in proliferative inflammatory atrophy of the prostate (PIA) and is absent in the majority of primary prostatic adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB324.

Abstract 3035: Bridging the gap: Exploring the roles of ETS- coactivator interactions in promoting prostate cancer

Abstract The ETS family of transcription factors is comprised of 28 members that all share a common C- terminal DNA-binding domain. ETS play essential roles in developmental processes, such as stem cell maintenance, blood vessel formation, and cellular fate. While absent in the normal prostate, four members of the ETS family become expressed in prostate tumors: ETV1, ETV4, ETV5 and ERG. Our lab has previously classified these proteins as oncogenic ETS for their ability to promote cellular migration in normal prostate epithelial RWPE1 cells. Transcription factors may need to interact with additional proteins to serve oncogenic roles. My research explores the interactions between 1) ETV1 and EWS, as well as 2) ERG and p53. Our lab has established that ETV1, ETV4 and ETV5 form essential interactions with EWS, a transcriptional coactivator that promotes ETS activity. In prostate tumors, chromosomal rearrangements result in the N-terminal truncations of ETV1, ETV4 and ETV5. I hypothesize that the EWS interaction domain is retained and that these truncated ETVs will remain oncogenic. Pulldown assays using purified full-length and truncated ETV1 suggests EWS interacts with both N-terminal and C-terminal portions ETV1. I am attempting to express truncated ETV1, ETV4 and ETV5 in RWPE1 cells. Upon stable expression, I will evaluate their interactions with EWS and their ability to promote migration in vitro. To identify putative proteins that bridge EWS to ETV1, immunoprecipitation mass spectrometry will be used. Mutations in p53, a tumor suppressor protein, promote oncogenesis and occur in nearly half of all cancer cases. We hypothesize that mutant p53 interacts with ETS to promote its recruitment to new genomic sites. Previous studies demonstrate that mutant p53 binds ETS2 at a higher affinity than wildtype p53 and we have expanded this list of interacting ETS to include ERG. We seek to explore whether ERG and mutant p53 cooperate to promote oncogenic phenotypes. I successfully cloned wildtype p53, mutant p53 (R248W) and double-mutant p53 (R248W, T81A) into a retroviral backbone. I then transduced RWPE1-Vector and RWPE1-ERG expressing cells to express these constructs. Although additional replicates are needed, it appears that mutant p53 promotes colony formation in both cell types relative to wildtype p53. I plan to validate these observations and generate an appropriate negative control cell line before extending into a mouse model. Through the continuation of this work, I will provide critical insight into the molecular mechanisms by which a subset of ETS factors achieve oncogenic function. We address the contributions of DNA-binding specificity and coactivator interactions in promoting the phenotypes associated with prostate cancer. Citation Format: Kaitlyn M. Mills, Nicholas Downing, Stephanie Metcalf, Peter Hollenhorst. Bridging the gap: Exploring the roles of ETS- coactivator interactions in promoting 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 3035.

Abstract 773: Overexpression of phosphoserine phosphatase (PSPH) in prostate cancer and in tumor microenvironment promotes tumor cell proliferation

Abstract Reprogrammed cellular metabolism is one of the key features of cancer and tumor microenvironment. L-3-phosphoserine phosphatase (PSPH) is one of the five rate-limiting enzymes in the biosynthesis of serine from glucose that supports cell proliferation. Here, we aim to study the role of PSPH in prostate cancer (PCa) and its tumor microenvironment. We find that PSPH not only is overexpressed in prostate cancer cell lines compared to normal prostate epithelial cells (PrEC) and benign prostate epithelial cells (BPH-1), but also exhibits a higher level in patient-derived carcinoma-associated fibroblasts (CAFs) compared to the matched benign associated fibroblasts (BAFs), which are derived from fresh radical prostatectomy specimens of African American (AA) and European American (EA) men. Notably, MDAPCa2b cells derived from an AA prostate cancer patient and AA CAFs have even higher expression of PSPH protein. The mRNA levels of PSPH are also significantly elevated in prostate cancer tissues in AA men compared to EA men and predict poor survival in PCa patients. Knock-down of PSPH expression significantly inhibits the proliferation and colony formation of prostate cancer cells. Growth of xenograft tumors derived from PSPH knockdown MDAPCa2b cells has also been suppressed markedly. Gene expression profiling revealed that suppression of PSPH expression by shRNAs in LNCaP and MDAPCa2b cells is associated with the regulation of gene expression related to metabolism, immunity, extracellular matrix remodeling, and Ion channels. Similarly, analysis of publicly available PCa RNA seq databases also demonstrates that PSPH amplification or over-expression is associated with alterations of gene expression related to metabolism and immunity. Our results suggest that PSPH is upregulated in prostate cancer cells and their surrounding fibroblasts to support tumor growth via reprogramming metabolic pathways and transcriptomes. Citation Format: Liankun Song, Noriko Yokoyama, Jun Xie, Yunjie Hu, Zhenyu (Arthur) Jia, Beverly Wang, Dan Mercola, Edward Uchio, Thomas Ahlering, Michael Lilly, Xiaolin Zi. Overexpression of phosphoserine phosphatase (PSPH) in prostate cancer and in tumor microenvironment promotes tumor cell proliferation [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 773.

Abstract 3033: Oncogenic ETS transcription factors avoid repression by EZH2 and FOXO1 in prostate cancer and Ewing sarcoma

Abstract ETS transcription factors play roles throughout development, aiding in hematopoiesis, blood vessel formation and cell fate. The ETS family is composed of 28 members, all of which share an ETS DNA-binding domain. Chromosomal rearrangements that lead to the overexpression of specific ETS promote oncogenic transformation and tumor development. More than half of prostate tumors are driven by aberrant expression of an ETS protein. Our lab has demonstrated that oncogenic ETS form essential interactions with a ubiquitous RNA-binding protein, EWS, to promote prostate tumorigenesis. A similar mechanism exists in Ewing sarcoma (ES). ES tumors are driven by the expression EWS/ETS fusion proteins. This project focuses on the similarities and differences between oncogenic ETS in prostate cancer and EWS/ETS fusions in Ewing sarcoma. We compared biological functions of the most common oncogenic ETS in prostate cancer, ERG, and the most common fusion in ES, EWS/FLI1. Both ERG and EWS/FLI1 promoted migration and clonogenic survival in normal prostate epithelial cell lines, RWPE1 and PNT2. These data suggest that the EWS-ERG complex and EWS/FLI1 fusion protein can function similarly in prostate cells. Knockdown of endogenous EWS/FLI1 in the ES cell line, A673, reduced anchorage-independent colony formation in soft agar. Rescue with exogenous EWS/FLI1 or EWS/ERG restored colony formation relative to control. Wildtype ERG was unable to rescue colony formation in A673. This suggests that ERG and EWS/FLI1 function differently in A673 cells. Pulldown assays from A673 lysates reveal that ERG interacts with EZH2 and FOXO1. Our lab has established that ERG acts as a transcriptional repressor through the formation of an ERG-EZH2-PRC2 complex. Others demonstrate that FOXO1 represses ERG activity by reducing its recruitment to the target sites. Both the N-terminal truncation of ERG and phosphorylation of ERG at S96 disrupted these interactions and rescued anchorage independent growth in A673 ES cells. These data suggest that EWS/ETS fusions avoid transcriptional repression in Ewing tumors due to loss of the N-terminus of the ETS protein. Citation Format: Nicholas F. Downing, Kaitlyn M. Mills, Peter C. Hollenhorst. Oncogenic ETS transcription factors avoid repression by EZH2 and FOXO1 in prostate cancer and Ewing sarcoma [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 3033.

Multifaceted impact of adipose conditioned media: Obesity-driven promotion of prostate cancer and cancer stem cell dynamics.

Obesity is an established risk factor for the development and progression of prostate cancer (PC). This study used adipose conditioned media (ACM) from differentiated adipocytes to assess its effect on PC development and aggressiveness. Due to limited research on ACM's impact on isolated PC stem cells (PCSCs), we also examined CD44+ PCSCs. ACM notably boosted interleukin-1β (IL-1β), IL-6, and IL-8 production in normal prostate epithelial cells and LNCaP cells. It also increased IL-6 and IL-8 production in PC3 and CD44+ LNCaP cells, and IL-1β and IL-6 production in CD44+ PC3 cells. This indicates that ACM induces the production of inflammatory cytokines in both cancer and prostate epithelial cells. Furthermore, ACM promoted proliferation in androgen receptor (AR)-negative PC3 cells, CD44+ PC3 PCSCs, and nonmalignant RWPE cells, without affecting AR-positive LNCaP cells. In addition, ACM-enhanced invasion and migration potential in both PC3 and CD44+ PC3 cells. Western blot analysis indicated the involvement of NF-κB and AKT pathways in ACM-induced proliferation in PC3 cells and NF-κB in PCSCs. In ACM-treated PC3 cells, E-cadherin was downregulated, while N-cadherin, Snail, vimentin, fibronectin, and Twist were upregulated, suggesting ACM-induced invasion via classical epithelial-to-mesenchymal transition (EMT) pathways. In response to ACM, PCSCs exhibited increased expression of E-cadherin, Snail, and vimentin, which are partial EMT markers promoting stemness and resistance to apoptosis. In addition, increased expressions of Nanog, Oct3/4, survivin, and Bcl-2 were observed. Although the molecules we studied have diverse effects on cellular regulation, our data emphasize obesity's multifaceted role in promoting and aggressing PC, notably affecting PCSC populations.

New 2-oxoindole derivatives as multiple PDGFRα/ß and VEGFR-2 tyrosine kinase inhibitors

Two new series of N-aryl acetamides 6a-o and benzyloxy benzylidenes 9a-p based 2-oxoindole derivatives were designed as potent antiproliferative multiple kinase inhibitors. The results of one-dose NCI antiproliferative screening for compounds 6a-o and 9a-p elucidated that the most promising antiproliferative scaffolds were 6f and 9f, which underwent five-dose testing. Notably, the amido congener 6f was the most potent derivative towards pancreatic ductal adenocarcinoma MDA-PATC53 and PL45 cell lines (IC50 = 1.73 µM and 2.40 µM, respectively), and the benzyloxy derivative 9f was the next potent one with IC50 values of 2.85 µM and 2.96 µM, respectively. Both compounds 6f and 9f demonstrated a favorable safety profile when tested against normal prostate epithelial cells (RWPE-1). Additionally, compound 6f displayed exceptional selectivity as a multiple kinase inhibitor, particularly targeting PDGFRα, PDGFRβ, and VEGFR-2 kinases, with IC50 values of 7.41 nM, 6.18 nM, and 7.49 nM, respectively. In contrast, the reference compound Sunitinib exhibited IC50 values of 43.88 nM, 2.13 nM, and 78.46 nM against the same kinases. The derivative 9f followed closely, with IC50 values of 9.9 nM, 6.62 nM, and 22.21 nM for the respective kinases. Both 6f and 9f disrupt the G2/M cell cycle transition by upregulating p21 and reducing CDK1 and cyclin B1 mRNA levels. The interplay between targeted kinases and these cell cycle regulators underpins the G2/M cell cycle arrest induced by our compounds. Also, compounds 6f and 9f fundamentally resulted in entering MDA-PATC53 cells into the early stage of apoptosis with good percentages compared to the positive control Sunitinib. The in silico molecular-docking outcomes of scaffolds 6a-o and 9a-p in VEGFR-2, PDGFRα, and PDGFRβ active sites depicted their ability to adopt essential binding interactions like the reference Sunitinib. Our designed analogs, specifically 6f and 9f, possess promising antiproliferative and kinase inhibitory properties, making them potential candidates for further therapeutic development.