Prostate Cells Research Articles

Smartphone-Based Fluorescent Profiling of Quaternary MicroRNAs in Urine for Rapid Diagnosis of Urological Cancers Using a Multiplexed Isothermal Exponential Amplification Reaction.

Urological cancers such as bladder or prostate cancer represent one of the most malignant tumors that accounts for an extremely high mortality. However, conventionally standard diagnostics for urological cancers are hardly available in low-resource settings. We developed herein a hand-held fluorescent imaging platform by integrating a multiplexed isothermal exponential amplification reaction (EXPAR) with a microgel-enriched methodology for sensitive profiling of quaternary microRNAs (miRNAs) in urine and quick diagnosis of urological cancers at the early stage. The target miRNA mixtures in the urine underwent four parallel EXPARs without cross-reactivity, followed by surface concentration and hybridization by the encoded polyacrylamide microgels. This mix-and-read strategy allowed for one-pot analysis of several key miRNAs simultaneously and provided 5-fold enhancement in fluorescent detection sensitivities compared to the individual EXPAR-based assays. Four urinary miRNAs (let-7a, miRNA-155, -223, and -143) could be quantitatively determined in a wide linear range from 50 fM to 30 nM, with the limits of detection at femtomolar levels. Using a smartphone-based imaging microreader, healthy and cancerous cohorts with prostate, bladder, and renal cell cancers could be discriminated in 30 min with the accuracy >83% using linear discriminant analysis. The developed detection platform has proven to be a portable, noninvasive, and useful complement to the toolbox for miRNA-based liquid biopsies, which holds immense potential and advantage for regular and large-scale applications in early cancer diagnosis.

Assessment of the in-vitro toxicity and in-vivo therapeutic capabilities of Juglans regia on human prostate cancer and prostatic hyperplasia in rats

Benign prostatic hyperplasia (BPH) and prostatic cancer are aging-associated urological conditions in men. While significant advances have been made in relation to treatment, poor response, treatment resistance and adverse side-effects limit currently available therapies. The possible antiproliferative and/or ameliorative potentials of Juglans regia have been reported, however, there is a dearth of scientific information on its effect on BPH or prostatic cancer. In this study, in-vivo and in-vitro studies were used to assess the possible benefits of fresh walnut fruits extract in BPH or prostate cancer. High-performance liquid chromatography coupled with electro spray ionization and quadrupole-time-of-flight mass spectrometry (HPLC-ESI-Q-TOF) was used to assess the chemical profile of the plant extract. The anti-proliferative activity of Juglans regia extract was tested against normal and cancerous human prostate cell lines by using the iCELLigence real-time and label-free cell analysis system. The ameliorative potential of fresh fruits Juglans regia extract was assessed by administering the extract at 50 and 100 mg/kg body weight to rats with testosterone induced BPH. Analysis of phytochemicals in Juglans regia revealed a high concentration of phenolic acids and flavonoids, Juglans regia also showing time- and dose-dependent anti-proliferative activity against prostate cancer cells, and reversed biochemical and histomorphological changes induced by testosterone-induced BPH.

Electrochemical Aptamer-Based Biosensor Developed to Monitor PCA3 and PSA Released by Prostate Cancer Cells

1. Introduction Detection of prostate cancer (PCa) in the early stage of the disease is fundamental for the prevention of metastases and for the initiation of treatment, which may bring greater chance of patient cured; so several research groups have sought the manufacture of a simple and non-invasive methodology [1]. The early diagnosis relies on prostate specific antigen (PSA) serum test, even if it showed clear limits [2]. Recently, prostate cancer gene 3 (PCA3), a non-coding mRNA which is overexpressed in tumor cells when compared with noncancerous prostate tissue [3]. Here, we report a new method for detection of prostate cancer-specific antigen 3 (PCA3) and prostate specific antigen (PSA) released by cancer cells using an aptasensor. Electrochemical impedance spectroscopy (EIS), SEM and EDS were used to evaluate biosensor surface. 2. Experimental Part The aptamers were modified through Biosearch Technologies at 5′-terminus in C6-disulfide linker to have it bond to the gold electrode; and at 3′-end in methylene blue (MB) (as redox probe) to perform the electrochemical analysis, which was followed by the HPLC purification process to remove truncated-synthesis products and to enrich the purity of the aptamer sequences. Aptamer immobilization was done by incubation of 1 μM thiolated aptamer on the top of the gold-coated silicon electrode overnight, in the dark, at 4°C. Subsequently, the electrodes were washed with PBS and a backfilling consisted of 3 mM of MCH aqueous solution for 30 minutes was done to displace the nonspecifically adsorbed aptamer molecules and to passivate the electrode surface [26]. Finally, the electrodes were once more rinsed with PBS to remove MCH excesses. The electrochemical characterization was done in a potentiostat μAutolabIII (Metrohn, NL), using a three-electrode system, which consisted of Ag/AgCl (3M KCl), as reference electrode; Pt wire, as a counter electrode; and gold-coated silicon wafer, as a working electrode. The prostate cell lines used were normal human prostate cells (RWPE-1; ATCC® CRL-11™), androgen-sensitive human prostate cancer cells (LNCaP; ATCC® CRL-1740™), and androgen-independent human prostate cancer cells (PC-3; ATCC® CRL-1435™). The three lineages were chosen to better understand PCA3 and PSA production patterns by different cell types. 3. Results and Discussion The gold-coated silicon wafe electrode was analyzed by SEM and EDS, which acts as working electrode and substrate to anchor the thiolated aptamer on the surface. By using square wave voltammetry, the present aptasensor was able of monitoring the PCA3 and PSA released from prostate cells (normal and cancerous). The PSA and PCA3 detection limits were 1 ng/mL and 9.2 ng/mL, respectively, at linear range up to 150 ng/mL, and it was also able to detect both biomarker even in presence of several interfering compounds (UA, AA, BSA and MUC1). In addition, the herein developed aptasensor was able to verify, at accurate and robust response, the patterns of the PCA3 and PSA released by different cell lines (RWPE-1, LNCaP and PC3). The detection of PSA and PCA3 by aptasensor agree with the cell lineage type, the LNCaP is an androgen-dependent lineage. In this way, an overexpressed both biomarkres was release was verified [49,50] and it can be compared to early-stage patients. On the other hand, the PC3 cells PSA and PCA3 releasing was nearly background signal, demonstrating almost absent production of studied targets, the PC3 lineage is androgen-independent which can be compared to advanced-disease patients. Funding This work has been supported by a grant from FAPESP-Brazil, CNPq and Capes.

Claudins in genitourinary tract neoplasms: mechanisms, prognosis, and therapeutic prospects.

Genitourinary (GU) cancers are among the most prevalent neoplasms in the world, with bladder cancers constituting 3% of global cancer diagnoses. However, several pathogenetic mechanisms remain controversial and unclear. Claudins, for example, have been shown to play a significant role in several cancers of the human body. Their role in GU cancers has not been extensively studied. Aberrant expression of claudins -1, -2, -3, -4, -7, and -11 has been expressed in urothelial cell carcinomas. In prostate cancers, altered levels of claudins -1, -2, -3, -4, and -5 have been reported. Furthermore, the levels of claudins -1, -2, -3, -4, -6, -7, -8, and -10 have been studied in renal cell carcinomas. Specifically, claudins -7 and -8 have proven especially useful in differentiating between chromophobe renal cell carcinomas and oncocytomas. Several of these claudins also correlate with clinicopathologic parameters and prognosis in GU cancers. Although mechanisms underpinning aberrant expression of claudins in GU cancers are unclear, epigenetic changes, tumor necrosis factor-ɑ, and the p63 protein have been implicated. Claudins also provide therapeutic value through tailored immunotherapy via molecular subtyping and providing therapeutic targets, which have shown positive outcomes in preclinical studies. In this review, we aim to summarize the literature describing aberrant expression of claudins in urothelial, prostatic, and renal cell carcinomas. Then, we describe the mechanisms underlying these changes and the therapeutic value of claudins. Understanding the scope of claudins in GU cancers paves the way for several diagnostic, prognostic, and therapeutic innovations.

MYC-driven increases in mitochondrial DNA copy number occur early and persist throughout prostatic cancer progression.

Increased mitochondrial function may render some cancers vulnerable to mitochondrial inhibitors. Since mitochondrial function is regulated partly by mitochondrial DNA copy number (mtDNAcn), accurate measurements of mtDNAcn could help reveal which cancers are driven by increased mitochondrial function and may be candidates for mitochondrial inhibition. However, prior studies have employed bulk macrodissections that fail to account for cell type-specific or tumor cell heterogeneity in mtDNAcn. These studies have often produced unclear results, particularly in prostate cancer. Herein, we developed a multiplex in situ method to spatially quantify cell type-specific mtDNAcn. We show that mtDNAcn is increased in luminal cells of high-grade prostatic intraepithelial neoplasia (HGPIN), is increased in prostatic adenocarcinomas (PCa), and is further elevated in metastatic castration-resistant prostate cancer. Increased PCa mtDNAcn was validated by 2 orthogonal methods and is accompanied by increases in mtRNAs and enzymatic activity. Mechanistically, MYC inhibition in prostate cancer cells decreases mtDNA replication and expression of several mtDNA replication genes, and MYC activation in the mouse prostate leads to increased mtDNA levels in the neoplastic prostate cells. Our in situ approach also revealed elevated mtDNAcn in precancerous lesions of the pancreas and colon/rectum, demonstrating generalization across cancer types using clinical tissue samples.

Potential impacts of bisphenols on prostate cells: An overview of cytotoxicity, proliferation, oxidative stress, apoptosis, and ER-stress response activation

This study aimed to evaluate the toxic effects of Bisphenol A (BPA), Bisphenol F (BPF) and Bisphenol S (BPS) on PNT1A and PC-3 cells, focusing on their effects on endoplasmic reticulum (ER) stress and related pathways. PNT1A and PC-3 were treated with BPA, BPF and BPS at concentrations of 0.1, 1 and 10 μM for 48 h cytotoxicity, BrdU cell proliferation, ROS generation, apoptosis detection, gene expression analysis and Western blot analysis were performed. BPA induced proliferation and late apoptosis in PNT1A cells, whereas it induced both late apoptosis and early apoptosis in PC-3 cells. BPF and BPS induced late apoptosis in PC-3 cells. Increased ROS levels were observed in PNT1A cells exposed to 1–10 μM BPA. BPA, BPF and BPS increased the expression levels of ER stress-related genes in PNT1A cells. Furthermore, exposure to BPA increased the expression of ER stress-related CHOP/DDIT3 protein in PNT1A cells. These findings highlight the potential health risks associated with BPA, BPF and BPS exposure and emphasize the importance of investigating the underlying mechanisms by which these chemicals may affect human health. Further research is required to comprehensively understand the role of ER stress pathways in cellular responses to these substances.

Antioxidant, AChE inhibitory, and anticancer effects of Verbascum thapsus extract.

Verbascum thapsus (VT) is a medicinal plant that is used in folk medicine to treat a variety of ailments. For this study, the biological functions of VT methanol extract were determined in vitro. The plant's methanol extract was created through the maceration process. The phytochemical composition of plant extracts was investigated using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The antioxidant capacity of the extract was determined using the DPPH (2,2-diphenyl-1-picrylhydrazil) and ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) tests and its cytotoxicity was assessed using the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole)) assay on the Caco-2 (human colorectal adenocarcinoma cells), LNCaP (Lymph Node Carcinoma of the Prostate), and HEK293 cell lines (Human embryonic kidney 293 cells) used to model colon, prostate, and non-cancerous cells. VT extract showed low DPPH and ABTS radical scavenging activities compared to standard antioxidants at 30 mg/ml concentration. In addition, it was determined that VT extract inhibited acetylcholinesterase enzyme.

Receptor tyrosine kinase-like orphan receptor 1 inhibitor strictinin exhibits anti-cancer properties against highly aggressive androgen-independent prostate cancer

Aim: It is important to identify anti-cancer compounds that can inhibit specific molecular targets to eradicate androgen-receptor negative (ARneg), androgen-independent (AI) prostate cancer, which is an aggressive form of prostate cancer with limited treatment options. The goal of this study was to selectively target prostate cancer cells that have high levels of oncogenic protein Receptor tyrosine kinase-like orphan receptor 1 (ROR1) by using strictinin, a small molecule ROR1 inhibitor. Methods: The methods performed in this study include western blots, methyl thiazolyl tetrazolium (MTT) proliferation assays, phosphatidylserine apoptosis assays, apoptosis flow cytometry (Annexin V, caspase 3/7), migration scratch assays, Boyden chamber invasion assays, and cell cycle flow cytometry. Results: Strictinin was most lethal against PC3 [half-maximal drug inhibitory concentration (IC50) of 277.2 µmol/L], an ARneg-AI cell type that expresses the highest levels of ROR1. Strictinin inhibited ROR1 expression, downstream phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-glycogen synthase kinase 3beta (GSK3β) pro-survival signaling, and epithelial-to-mesenchymal transition markers in PC3 cells. Additionally, strictinin decreased PC3 cell migration and invasion, while increasing S-phase cell cycle arrest. In ARneg-AI DU145 cells, strictinin inhibited ROR1 expression and modulated downstream AKT-GSK3β signaling. Furthermore, strictinin exhibited anti-migratory, anti-invasive, but minimal pro-apoptotic effects in DU145 cells likely due to DU145 having less ROR1 expression in comparison to PC3 cells. Throughout the study, strictinin minimally impacted the phenotype of normal prostatic epithelial cells RWPE-1 (IC50 of 658.5 µmol/L). Strictinin was further identified as synergistic with docetaxel [combination index (CI) = 0.311] and the combination therapy was found to reduce the IC50 of strictinin to 38.71 µmol/L in PC3 cells. Conclusions: ROR1 is an emerging molecular target that can be utilized for treating prostate cancer. The data from this study establishes strictinin as a potential therapeutic agent that targets ARneg-AI prostate cancer with elevated ROR1 expression to reduce the migration, invasion, cell cycle progression, and survival of prostate cancer.

JNK promotes the progression of castration-resistant prostate cancer.

Prostate cancer is one of the most common cancers in men worldwide. This study aims to elucidate the roles of c-Jun N-terminal kinase (JNK) in the progression of castration-resistant prostate cancer (CRPC). JNK overexpressing and knockdown cell lines were established on the PC-3 prostate cell line. qPCR and Western blotting were performed to determine the mRNA and protein levels of target genes in prostate tissues and cell lines. MTT and Matrigel invasion assays were conducted to evaluate the cell viability and invasive ability, respectively. The Kaplan-Meier estimator was performed to estimate the overall survival rate and second progression-free survival rate. Pearson's correlation coefficient was used to evaluate the relationship between JNK and prostate-specific antigen (PSA). Relative JNK expression was correlated with Gleason score and PSA value in patients with CRPC. Kaplan-Meier analysis revealed that patients with low JNK expression exhibited high overall survival and second progression-free survival rate. In vitro assays demonstrated that JNK overexpression promoted cell viability and invasion as well as the protein expressions of extracellular signal-regulated kinase (ERK) and matrix metalloproteinase 1 (MMP1) in PC-3 cell lines. JNK overexpression promotes the development of CRPC via the regulation of ERK and MMP1.

Prostate Cell Lineage Identities Are Governed by Distinct Metabolomes

Abstract Basal and luminal cell identities are regulated by distinct metabolisms in the prostate epithelium.

Hypoxia activates the hypoxia-inducible factor-1α/vascular endothelial growth factor pathway in a prostatic stromal cell line: A mechanism for the pathogenesis of benign prostatic hyperplasia.

The development of benign prostatic hyperplasia (BPH) is closely related to hypoxia in the prostatic stroma, and the hypoxia-inducible factor-1α/vascular endothelial growth factor (HIF-1α/VEGF) pathway has been shown to significantly activate in response to hypoxia. The underlying mechanism for activation of this pathway in the pathogenesis of BPH remains unclear. We constructed HIF-1α overexpression and knockdown BPH stromal (WPMY-1) and epithelial (BPH-1) cell lines, which were cultured under different oxygen conditions (hypoxia, normoxia, and hypoxia + HIF-1α inhibitor). Quantitative real-time polymerase chain reaction (qPCR) and Western blotting were applied to detect the expression of the HIF-1α/VEGF pathway. Cell proliferation and apoptosis were analyzed by Cell Counting Kit-8 and flow cytometry. We used the miRWalk 2.0 database and Western blotting to predict the potential miRNA that selectively targets the HIF-1α/VEGF pathway, and verified the prediction by qPCR and dual-luciferase assays. In a BPH stromal cell line (WPMY-1), the expression of VEGF was in accordance with HIF-1α levels, elevated in the overexpression cells and decreased in the knockdown cells. Hypoxia-induced HIF-1α overexpression, which could be reversed by a HIF-1α inhibitor. Moreover, the HIF-1α inhibitor significantly depressed cellular proliferation and promoted apoptosis in hypoxic conditions, assessed by Cell Counting Kit-8 and flow cytometry. However, in the BPH epithelial cell line (BPH-1), the expression level of HIF-1α did not influence the expression of VEGF. Finally, a potential miRNA, miR-17-5p, regulating the HIF-1α/VEGF pathway was predicted from the miRWalk 2.0 database and Western blotting, and verified by qPCR and dual-luciferase assay. In hypoxia, activation of the HIF-1α/VEGF pathway plays a crucial role in regulating cell proliferation in a BPH stromal cell line. Regulation by miR-17-5p may be the potential mechanism for the activation of this pathway. Regulation of this pathway may be involved in the pathogenesis of BPH.

Calcium Activation of the Androgen Receptor in Prostate Cells.

Although prostate cancer patients initially respond to androgen deprivation therapy, most patients progress to a resistant phenotype. Castration resistance is due, in part, to intratumoral and/or adrenal synthesis of androgens, overexpression or mutation of the androgen receptor (AR), stabilization of AR by chaperones, and ligand-independent activation of AR. Increasing evidence also links disruption of calcium homeostasis to progression of prostate cancer. Our previous study shows that heavy metal cadmium activates the AR through a ligand-independent mechanism. Cadmium mimics calcium in biological systems due to their similar ionic charge and radius. This study determines whether calcium activates AR and whether first- and second-generation antiandrogens block the ability of calcium to activate the receptor. The expression of androgen-responsive genes and calcium channels was measured in prostate cells using a quantitative real-time polymerase chain reaction assay. Cell growth was measured. To ask whether calcium activates AR, prostate cells were treated with calcium in the absence and presence of the first-generation antiandrogens hydroxyflutamide and bicalutamide and the second-generation antiandrogen enzalutamide, and the expression of androgen-responsive genes and cell growth was measured. In the normal PWR-1E cells and HEK293T cells transiently expressing AR, treatment with calcium increased the expression of androgen-responsive genes by approximately 3-fold. The increase was blocked by enzalutamide but was not consistently blocked by the first-generation antiandrogens. In LNCaP cells which contain a mutant AR, treatment with calcium also increased the expression of androgen-responsive genes by approximately 3-fold, and the increase was more effectively blocked by enzalutamide than by hydroxyflutamide or bicalutamide. Treatment with calcium also increased cell growth that was blocked by enzalutamide. To ask whether dysregulation of calcium channels is associated with castration resistance, calcium channels were measured in the normal PWR-1E prostate cells, the hormone-responsive LNCaP cells, and the castration-resistant VCaP and 22RV1 cells. Compared to normal prostate cells, the hormone-responsive and hormone-resistant cells overexpressed several calcium channels. The results of this study show that calcium activates AR and increases cell growth and that calcium channels are overexpressed in hormone-responsive and hormone-resistant prostate cancer cells. Taken together, the results suggest a novel role of calcium in the castration-resistant phenotype.

Correction: Metformin inhibits the proliferation of benign prostatic epithelial cells

Correction: Metformin inhibits the proliferation of benign prostatic epithelial cells

Carrier-Tumor Cell Membrane Interactions for Optimized Delivery of a Promising Drug, 4(RS)-4-F4t-Neuroprostane.

Nanomedicines engineered to deliver molecules with therapeutic potentials, overcoming drawbacks such as poor solubility, toxicity or a short half-life, are targeted towards their cellular destination either passively or through various elements of cell membranes. The differences in the physicochemical properties of the cell membrane between tumor and nontumor cells have been reported, but they are not systematically used for drug delivery purposes. Thus, in this study, a new approach based on a match between the liposome compositions, i.e., membrane fluidity, to selectively interact with the targeted cell membrane was used. Lipid-based carriers of two different fluidities were designed and used to deliver 4(RS)-4-F4t-Neuroprostane (F4t-NeuroP), a potential antitumor molecule derived from docosahexaenoic acid (DHA). Based on its hydrophobic character, F4t-NeuroP was added to the lipid mixture prior to liposome formation, a protocol that yielded over 80% encapsulation efficiency in both rigid and fluid liposomes. The presence of the active molecule did not modify the liposome size but increased the liposome negative charge and the liposome membrane fluidity, which suggested that the active molecule was accommodated in the lipid membrane. F4t-NeuroP integration in liposomes with a fluid character allowed for the selective targeting of the metastatic prostate cell line PC-3 vs. fibroblast controls. A significant decrease in viability (40%) was observed for the PC-3 cancer line in the presence of F4t-NeuroP fluid liposomes, whereas rigid F4t-NeuroP liposomes did not alter the PC-3 cell viability. These findings demonstrate that liposomes encapsulating F4t-NeuroP or other related molecules may be an interesting model of drug carriers based on membrane fluidity.

The PENGUIN approach to reconstruct protein interactions at enhancer-promoter regions and its application to prostate cancer

We introduce Promoter-Enhancer-Guided Interaction Networks (PENGUIN), a method for studying protein-protein interaction (PPI) networks within enhancer-promoter interactions. PENGUIN integrates H3K27ac-HiChIP data with tissue-specific PPIs to define enhancer-promoter PPI networks (EPINs). We validated PENGUIN using cancer (LNCaP) and benign (LHSAR) prostate cell lines. Our analysis detected EPIN clusters enriched with the architectural protein CTCF, a regulator of enhancer-promoter interactions. CTCF presence was coupled with the prevalence of prostate cancer (PrCa) single nucleotide polymorphisms (SNPs) within the same EPIN clusters, suggesting functional implications in PrCa. Within the EPINs displaying enrichments in both CTCF and PrCa SNPs, we also show enrichment in oncogenes. We substantiated our identified SNPs through CRISPR/Cas9 knockout and RNAi screens experiments. Here we show that PENGUIN provides insights into the intricate interplay between enhancer-promoter interactions and PPI networks, which are crucial for identifying key genes and potential intervention targets. A dedicated server is available at https://penguin.life.bsc.es/.

YAP1 Recognizes Inflammatory and Mechanical Cues to Exacerbate Benign Prostatic Hyperplasia via Promoting Cell Survival and Fibrosis.

Chronic prostatic inflammation promotes cell survival and fibrosis, leading to benign prostatic hyperplasia (BPH) with aggravated urinary symptoms. It is investigated whether yes-associated protein 1 (YAP1), an organ size controller and mechanical transductor, is implicated in inflammation-induced BPH. The correlation between YAP1 expression and fibrosis in human and rat BPH specimens is analyzed. Furthermore, the effects of YAP1 activation on prostatic cell survival and fibrosis, as well as the underlying mechanism, are also studied. As a result, total and nuclear YAP1 expression, along with downstream genes are significantly upregulated in inflammation-associated human and rat specimens. There is a significant positive correlation between YAP1 expression and the severity of fibrosis or clinical performance. YAP1 silencing suppresses cell survival by decreasing cell proliferation and increasing apoptosis, and alleviates fibrosis by reversing epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition in prostatic BPH-1 and WPMY-1 cells. Mechanistically, inflammatory stimulus and rigid matrix stiffness synergistically activate the RhoA/ROCK1 pathway to provoke cytoskeleton remodeling, thereby promoting YAP1 activation to exacerbate BPH development. Overall, inflammation-triggered mechanical stiffness reinforcement activates the RhoA/ROCK1/F-actin/YAP1 axis, thereby promoting prostatic cell survival and fibrosis to accelerate BPH progression.

Thyroid Nodule Correlation with Serum Prolactin and TPO Antibody Levels: A Case-Control Study

Introduction: The prevalence of thyroid nodules poses a grave clinical concern as they may lead to malignancy. Prolactin has been found to have anti-cell death properties and stimulates the growth of breast and prostate cells, as has been discovered in exploratory examinations. In light of this, the current research was designed to investigate whether a possible relationship exists between thyroid nodules and serum prolactin levels. Material and Methods: Ninety participants were enrolled in this study, of which 60 presented with thyroid nodules (30 antithyroid peroxidase antibody (TPO-Ab) positive and 30 TPO-Ab negative) and 30 without thyroid nodules (control group). Patients' serum prolactin, TPO-Ab, thyroid-stimulating hormone (TSH), T4 values, and thyroid ultrasound reports were scrutinized. Statistically significant outcomes were determined by a P-value less than 0.05. Results: Serum TSH, prolactin, and TPO-Ab levels were significantly higher in the case group than in the control group (P=0.041, 0.050, and 0.000, respectively). The case group had more instances of hyperprolactinemia than the control group (35% vs. 13.3%) (OR=3.5, CI 95%=1.08-11.38, P=0.031). Furthermore, the TPO-Ab positive group demonstrated greater solidity than the TPO-Ab negative group (93.3% vs. 73.3%, P=0.038). Conclusion: The present study confirmed the presence of a positive correlation between thyroid nodules and increased serum TSH, prolactin, and TPO-Ab levels. Furthermore, TPO-Ab positive nodules exhibited greater solidity and elevated TSH levels.

Abstract A056: Treatment with dual BRG1/BRM (SMARCA4/2) inhibitor FHD-286 ablates tumor-associated androgen response elements (AREs) in prostate cancer

Abstract The Androgen Receptor (AR) cistrome is critical in the development of prostate cell identity while its misregulation promotes prostate cancer development. The pioneer transcription factor Forkhead box A1 (FOXA1) has been shown to be essential for the recruitment of AR to androgen response elements (AREs) allowing for reprogramming of the AR cistrome resulting in prostate cell transformation. FHD-286 is a BRM/BRG1 dual ATPase inhibitor currently in clinical trials for AML. Here, we show that treatment of prostate cancer cell lines with FHD-286 results in ablation of FOXA1 mediated AREs at tumor-associated AR binding sites (T-ARBS). Dual ATPase treatment subsequently reduces the expression level of various oncogenic AR target genes, leading to a decrease in tumor cell viability. Both patient derived organoid and In vivo studies provide further validation by showing a decrease in tumor growth. Strikingly, inhibition of BAF complex activity bypasses AR resistance mechanisms commonly seen after castration and Enzalutamide treatment as cell lines containing AR-V7 splice variants and neuroendocrine organoids display sensitivity. Taken together, our data illustrates a novel mechanism of treating AR mediated prostate cancer though the inhibition tumor associated AREs by treatment with FHD-286. Citation Format: Gabriel J Sandoval, Richard C Centore, Sal Topal, Dave Lahr, GiNell Elliott, Heena Gandevia, Ammar Adam, Jessica Piel, Holly M Nguyen, Eva Corey, Yu Chen, Steven Bellon, Murphy Hentemann. Treatment with dual BRG1/BRM (SMARCA4/2) inhibitor FHD-286 ablates tumor-associated androgen response elements (AREs) in prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A056.

Abstract C128: PT-112, a novel immunogenic cell death inducer, causes ribosomal biogenesis inhibition and organelle stress in cancer cells

Abstract PT-112 is a novel immunogenic small molecule under Phase 2 development in metastatic castration-resistant prostate cancer and thoracic malignancies. PT-112 has been shown to release damage-associated molecular patterns, essential immune signaling components of immunogenic cell death (ICD), and to elicit anticancer immune responses. In addition, PT-112 causes mitochondrial and endoplasmic reticulum (ER) stress in vitro. Here, we sought to elucidate early molecular effects of PT-112 that culminate in cancer cell death. We assessed nucleolar protein phenotypes in human prostate cancer cells treated with PT-112 using antibody labeling, followed by confocal microscopy. We also conducted nascent RNA sequencing in human cancer cells (non-small cell lung, prostate, and renal cell carcinoma) following 1- and 6-hour PT-112 treatment. We then performed an analysis using Enrichr across multiple databases to identify biological pathways linked to clusters of differentially expressed genes (p ≤ 0.001) in PT-112-treated cells. Pathways affected by PT-112 (p ≤ 0.05) were sorted by statistical significance. In prostate cancer cells, PT-112 caused changes in nucleolar protein labeling. The most pronounced effect, observed after 8 or more hours of treatment, was rapid NPM1 relocation from nucleoli to nucleoplasm, a hallmark of nucleolar stress and disruption of ribosomal biogenesis (RiBi). PT-112 also induced gene expression changes in the treated cancer cell lines, resulting in statistically significant repression of pathways related to RiBi, ribosomal RNA (rRNA) processing and translation. Notably, a pattern across these pathways was the reduced expression of many ribosomal genes, consistent with RiBi inhibition. Additional analysis identified oncogenic pathways such as Myc and NF-κB significantly inhibited by PT-112. The degree of pathway suppression increased with treatment time. These results demonstrate that PT-112 rapidly causes nucleolar stress and RiBi inhibition, suggesting these are central to PT-112-induced cancer cell death. This mechanism could also explain the selective killing of cancer cells by PT-112, given that aberrant nucleolar size and number as well as ribosomal activity in cancer cells make them more sensitive to these drug effects. The broad consequences of impaired ribosome production and downstream protein translation may explain PT-112’s anticancer and immunogenic effects. Additionally, these disruptions have been reported to cause an increase in free ribosomal proteins and changes in nucleolar protein localization, which have been shown to induce ER stress and shuttle pro-apoptotic proteins to the mitochondria. These additional consequences of RiBi inhibition and nucleolar stress may contribute to or drive previously observed PT-112-induced cancer organelle stress and ICD. While further validation of these findings is needed, this work deepens our understanding of PT-112’s mechanism of action. Present findings, together with demonstrated clinical activity of PT-112, position it as a promising small molecule cancer immunotherapy. Citation Format: Christina Y. Yim, Maria T. Congenie, Hannah L. Johnson, Maureen G. Mancini, Fabio Stossi, Michael A. Mancini, Joey Azofeifa, Matthew R. Price, Johan Baeck, Tyler D. Ames. PT-112, a novel immunogenic cell death inducer, causes ribosomal biogenesis inhibition and organelle stress in cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C128.

Abstract C061: The impact of the BRG1/BRM-associated factor (BAF) complex on the androgen receptor cistrome in African American prostate cancer

Abstract We aimed to establish in prostate cancer (PCa) how and why the androgen receptor displays differential functions in a manner driven by genomic ancestry, namely between African American (AA) and European American (EA) PCa, with the goal to assess how this may contribute to PCa health disparities. We utilized EA and AA non-malignant prostate and PCa cell models with confirmed genomic ancestry and defined the basal and dihydrotestosterone (DHT)-regulated AR and H3K27ac cistromes using CUT&RUN, and integrated these data with publicly available genomic counterparts. The DHT-dependent AR and H3K27ac cistromes significantly overlapped within AA cells and EA cells but there was significantly less overlap between AA and EA cells. For example, H3K27ac distributions were frequent (~15-20k sites) in each of non-malignant AA RC43N and EA HPr1AR, and malignant RC43T and LNCaP, and the significance of the overlaps were comparable for basal and DHT treated cells, but this was not significantly impacted by ancestry. By contrast the AR cistrome was highly DHT-dependent, with ~2k sites notably in LNCaP and RC43T, which very significantly overlapped with H3K27ac in the same cells but significantly less so across cells. Thus, although the H3K27ac cistrome was comparable across cells, the overlap with AR was driven by genomic ancestry. To investigate this, we focused on the BAF complexes as previously we identified reduced expression of a key component, SMARCA5, in AA PCa. These large multimeric complexes regulate nucleosome positioning during gene transcription and DNA repair. In PCa, the cBAF complex impacts different transcription factors, including ERG, and it is tempting to speculate that the corruption of cBAF identified in AA PCa phenocopies the impact of TMPRSS2-ETS impact observed in EA PCa. To test the possibility that the cBAF complex corrupts AR genomic functions in AA PCa we exploited scRNA-Seq data from PCa organoids from the Rb and Pten double knockout (PBCre4:Rb1f/f:Pten f/f ) to establish that expression of genes in the cBAF genes complex were collectively significantly altered in neuroendocrine PCa (NEPC) and Pouf23 androgen indifferent lineages. Secondly, knockdown of another cBAF component, SMARCC1, enhanced Enzalutamide sensitivity in same organoids. Finally, to test the impact of cBAF complex on AR signaling we have generated AA and EA PCa cell lines that stably express dCRISPR-activator (VP64) or repressor (KRAB) proteins and designed gRNAs to regulate expression of, for example, SMARCA5 and measure the impact on the AR cistrome and function. In summary, these findings identified that the shared AR and H3K27ac cistromes are driven by genomic ancestry, and that reduced expression of SMARCA5 and other components of the cBAF complex are significant in AA and advanced PCa, and impact AR signaling. Our future directions are focused on how SMARCA5 expression disrupts the balance between transcription factor functions, such as for the AR and other transcription factors, and effective DNA repair. Citation Format: Shahid S. Hussain, Debasis Nayak, David W. Goodrich, Clayton Yates, Moray Campbell. The impact of the BRG1/BRM-associated factor (BAF) complex on the androgen receptor cistrome in African American prostate cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C061.