Prostate Cancer Tissue Samples Research Articles

Characterization of Hormone-Dependent Pathways in Six Human Prostate-Cancer Cell Lines: A Gene-Expression Study.

Prostate cancer (PCa), the most incident cancer in men, is tightly regulated by endocrine signals. A number of different PCa cell lines are commonly used for in vitro experiments, but these are of diverse origin, and have very different cell-proliferation rates and hormone-response capacities. By analyzing the gene-expression pattern of main hormone pathways, we systematically compared six PCa cell lines and parental primary cells. We compared these cell lines (i) with each other and (ii) with PCa tissue samples from 11 patients. We found major differences in the gene-expression levels of androgen, insulin, estrogen, and oxysterol signaling between PCa tissue and cell lines, and between different cell lines. Our systematic characterization gives researchers a solid basis to choose the appropriate PCa cell model for the hormone pathway of interest.

Long noncoding RNA LINC00261 suppresses prostate cancer tumorigenesis through upregulation of GATA6-mediated DKK3

BackgroundProstate cancer is one of the leading causes of cancer death in males. Recent studies have reported aberrant expression of lncRNAs in prostate cancer. This study explores the role of LINC00261 in prostate cancer progression.MethodsThe differentially expressed genes, transcription factors, and lncRNAs related to prostate cancer were predicted by bioinformatics analysis. Prostate cancer tissue samples and cell lines were collected for the determination of the expression of LINC00261 by reverse transcription quantitative polymerase chain reaction. The binding capacity of LINC00261 to the transcription factor GATA6 was detected by RIP, and GATA6 binding to the DKK3 promoter region was assessed by ChIP. In addition, luciferase reporter system was used to verify whether LINC00261 was present at the DKK3 promoter. After gain- and loss-of function approaches, the effect of LINC00261 on prostate cancer in vitro and in vivo was assessed by the determination of cell proliferation, invasion and migration as well as angiogenesis.ResultsLINC00261, GATA6, and DKK3 were poorly expressed in prostate cancer. LINC00261 could inhibit transcriptional expression of DKK3 by recruiting GATA6. Overexpression of LINC00261 inhibited prostate cancer cells proliferation, migration, and invasion as well as angiogenesis, which could be reversed by silencing DKK3. Furthermore, LINC00261 could also suppress the tumorigenicity of cancer cells in vivo.ConclusionsOur study demonstrates the inhibitory role of LINC00261 in prostate cancer progression, providing a novel biomarker for early detection of prostate cancer.

High throughput assessment of biomarkers in tissue microarrays using artificial intelligence: PTEN loss as a proof-of-principle in multi-center prostate cancer cohorts.

Phosphatase and tensin homolog (PTEN) loss is associated with adverse outcomes in prostate cancer and has clinical potential as a prognostic biomarker. The objective of this work was to develop an artificial intelligence (AI) system for automated detection and localization of PTEN loss on immunohistochemically (IHC) stained sections. PTEN loss was assessed using IHC in two prostate tissue microarrays (TMA) (internal cohort, n = 272 and external cohort, n = 129 patients). TMA cores were visually scored for PTEN loss by pathologists and, if present, spatially annotated. Cores from each patient within the internal TMA cohort were split into 90% cross-validation (N = 2048) and 10% hold-out testing (N = 224) sets. ResNet-101 architecture was used to train core-based classification using a multi-resolution ensemble approach (×5, ×10, and ×20). For spatial annotations, single resolution pixel-based classification was trained from patches extracted at ×20 resolution, interpolated to ×40 resolution, and applied in a sliding-window fashion. A final AI-based prediction model was created from combining multi-resolution and pixel-based models. Performance was evaluated in 428 cores of external cohort. From both cohorts, a total of 2700 cores were studied, with a frequency of PTEN loss of 14.5% in internal (180/1239) and external 13.5% (43/319) cancer cores. The final AI-based prediction of PTEN status demonstrated 98.1% accuracy (95.0% sensitivity, 98.4% specificity; median dice score = 0.811) in internal cohort cross-validation set and 99.1% accuracy (100% sensitivity, 99.0% specificity; median dice score = 0.804) in internal cohort test set. Overall core-based classification in the external cohort was significantly improved in the external cohort (area under the curve = 0.964, 90.6% sensitivity, 95.7% specificity) when further trained (fine-tuned) using 15% of cohort data (19/124 patients). These results demonstrate a robust and fully automated method for detection and localization of PTEN loss in prostate cancer tissue samples. AI-based algorithms have potential to streamline sample assessment in research and clinical laboratories.

LncRNA MCM3AP-AS1 Promotes Cell Proliferation and Invasion Through Regulating miR-543-3p/SLC39A10/PTEN Axis in Prostate Cancer.

ObjectiveLong-chain noncoding RNAs (lncRNAs) are key players in a wide range of biological processes, especially the pathogenesis and development of tumors. LncRNA MCM3AP-AS1 has been demonstrated to be involved in the invasion of various tumors including prostate cancer (PCa). However, its functions in PCa have not been fully elucidated.MethodsqRT-PCR was conducted to measure the expression levels of lncRNA MCM3AP-AS1 and miR-543-3p in PCa tissue samples and cell lines. The expression levels of E-cadherin and SLC39A10 proteins were detected by Western blots. CCK-8 test, cell scratch test and trans-well test were used to evaluate the proliferation, invasion and migration abilities of PCa cells, respectively. Annexin V-FITC/PI experiments were carried out to determine the status of apoptosis. Bioinformatics analysis and Luciferase assay were used to explore the relationship between lncRNA MCM3AP-AS1, miR-543-3p and SLC39A10.ResultsIn PCa tissue samples and cell lines, lncRNA MCM3AP-AS1 was up-regulated while miR-543-3p was down-regulated. Over-expression of MCM3AP-AS1 could promote the proliferation and invasion of PCa cells. Correlation analysis showed that the expression of MCM3AP-AS1 and miR-543-3p was significantly and inversely correlated. We further verified that miR-543-3p inhibitor was able to reverse si-MCM3AP-AS1-mediated inhibitory effects on the PCa cell proliferation, migration and invasion through regulating the downstream protein axis SLC39A10/PTEN/Akt. Finally, in vivo experiments indicated that knocking down of MCM3AP-AS1 could largely reduce tumor volumes, and decreased the ratio of Ki67-positive cells and the expression of SLC39A10 in tumor samples.ConclusionLncRNA MCM3AP-AS1 can promote the proliferation, migration and invasion abilities of PCa cells through regulating the miR-543-3p/SLC39A10/PTEN axis, which suggests that lncRNA MCM3AP-AS1 might be a potential target for prostate cancer therapy.

Circular RNA cir-ITCH Is a Potential Therapeutic Target for the Treatment of Castration-Resistant Prostate Cancer.

cir-ITCH, a well-known tumor-suppressive circular RNA, plays a critical role in different cancers. However, its expression and functional role in prostate cancer (PCa) are unclear. Herein, we explored the potential mechanism and tumor-inhibiting role of cir-ITCH in PCa. Using reverse transcriptase polymerase chain reaction assay, we analyzed the expression of cir-ITCH in PCa and paired adjacent nontumor tissue samples resected during surgical operation, as well as in two cell lines of human PCa (LNCaP and PC-3) and the immortalized normal prostate epithelial cell line (RWPE-1). Cell viability and migration of PCa cell lines were evaluated using CCK-8 and wound-healing assays. Expression of key proteins of the Wnt/β-catenin and PI3K/AKT/mTOR pathways was detected using western blotting. We found that cir-ITCH expression was typically downregulated in the tissues and cell lines of PCa compared to that in the peritumoral tissue and in RWPE-1 cells, respectively. The results showed that cir-ITCH overexpression significantly inhibits the proliferation, migration, and invasion of human PCa cells and that reciprocal inhibition of expression occurred between cir-ITCH and miR-17. Proteins in the Wnt/β-catenin and PI3K/AKT/mTOR pathways were downregulated by overexpression of cir-ITCH both in androgen receptor-positive LNCaP cells and androgen receptor-negative PC-3 cells. Taken together, these data demonstrated that cir-ITCH plays a tumor-suppressive role in human PCa cells, partly through the Wnt/β-catenin and PI3K/AKT/mTOR pathways. Thus, cir-ITCH may serve as a novel therapeutic target for the treatment of PCa, especially castration-resistant prostate cancer.

Abstract 3560: PLK4 is a HIF-1α target gene: A novel mechanism inducing centrosome amplification in tumor cells

Abstract Background: The presence of supernumerary or abnormally large centrosomes, also known as centrosome amplification (CA), drives tumor progression by enhancing chromosomal instability, and altering cellular microtubule cytoskeletons and tissue cytoarchitectures to promote the migration and invasion capabilities of cancer cells. Although the prevalence of CA is documented in a variety of cancer types, it remains unclear how CA is induced in tumor cells.This study aims to examine the role of intratumoral hypoxia in inducing CA via HIF-1α. Methods: We (a) exposed cultured breast, pancreatic, colorectal and prostate cancer cells to hypoxia, and (b) genetically manipulated hypoxia [via overexpression (OE) or knock-out (KO) of HIF-1α], and quantitated CA employing immunocytofluorescence methods. We also quantitated the levels of CA-associated genes and proteins by qRT-PCR and western blot, respectively. Next, to discern the biological pathway through which HIF-1α induces CA, we performed ChIP and Luciferase reporter assays. We immunohistochemically labeled breast (n=24), colon (38), prostate (n=52), and pancreatic (n=34) cancer tissue samples for HIF-1α and PLK4. Serial sections from the same tumors were also immunofluorescently labeled for γ-tubulin and CA was quantitated. Using public microarray datasets (TCGA), we investigated whether centrosomal gene expression is enriched in tumors characterized by a high expression of a hypoxia gene expression signature. Results: Cells cultured in hypoxic conditions exhibited higher CA (p<0.05) than the cells cultured in normoxic conditions. CA levels increased when cells with HIF-1α OE were cultured in normoxic conditions and no significant induction of CA was observed when the HIF-1α KO cells were cultured under hypoxic conditions. ChIP and Luciferase assay confirmed that HIF-1α induced CA by directly upregulating the expression of PLK4. Moreover, when tumor cells were exposed to hypoxia after knock-down of PLK4, no significant changes in CA and CA-associated proteins were observed. In clinical patient samples (breast, colon, pancreatic and prostate cancer), we observed that HIF-1α expression was significantly (p<0.05) correlated with PLK4 expression at the protein level (breast ρ=0.81, colon ρ=0.76, prostate ρ=0.68, and pancreatic ρ=0.67). Findings from our in silico analyses further validated these observations by showing a strong correlation between the expressions of HIF-1α and PLK4 at the transcriptional level. We also found that centrosomal gene expression is enriched in tumors with high hypoxia score (TCGA dataset of breast, colon, pancreatic and prostate cancer). Conclusion: Collectively, our findings suggest that hypoxia drives CA in tumor cells through HIF-1α-mediated transcriptional upregulation of PLK4. Given the development of several promising HIF-1α inhibitors, this research could potentially aid in patient risk stratification for clinical decision-making, and enable design of new therapies. Citation Format: Karuna Mittal, Jaspreet Kaur, Shaligram Sharma, Ishita Choudhary, Guanhao Wei, Precious Imhansi Jacob, Da Hoon Choi, Nagini Maganti, Shrikant Pawar, Michael S. Toss, Emiel A. Janssen, Meenakshi V. Gupta, Michelle D. Reid, Emad A. Rakha, Padmashree Rida, Ritu Aneja. PLK4 is a HIF-1α target gene: A novel mechanism inducing centrosome amplification in tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3560.

Reduced cytoplasmic expression of MAGE-A2 predicts tumor aggressiveness and survival: an immunohistochemical analysis.

Melanoma antigen gene A2 (MAGE-A2) is one of the most cancer-testis antigens overexpressed in various types of cancers. Silencing the MAGE-A2 expression inhibited the proliferation of prostate cancer (PCa) cells and increased the chemosensitivity. However, the expression pattern of MAGE-A2 in PCa tissue samples and its prognostic and therapeutic values for PCa patients is still unclear. In this study, for the first time, the staining pattern and clinical significance of MAGE-A2 were evaluated in 166 paraffin-embedded prostate tissues, including 148 cases of PCa and 18 cases of high-grade prostatic intraepithelial neoplasia (HPIN), by immunohistochemical analysis. The simultaneous expression of both nuclear and cytoplasmic patterns of MAGE-A2 with different staining intensities was observed among studied cases. Increased expression of MAGE-A2 was significantly found in PCa tissues compared to HPIN cases (P < 0.0001). Among PCa samples, the strong staining intensity of nuclear expression was predominantly observed in comparison with cytoplasmic expression in PCa tissues (P < 0.0001). A significant and inverse correlation was found between the cytoplasmic expression of MAGE-A2 and increased Gleason score (P = 0.002). Increased cytoplasmic expression of MAGE-A2 was associated with longer biochemical recurrence-free survival (BCR-FS) and disease-free survival (DFS) of patients (P = 0.002, P = 0.001, respectively). In multivariate analysis, Gleason score and cytoplasmic expression of MAGE-A2 were independent predictors of the BCR-FS (P = 0.014; P = 0.028, respectively). Taken together, cytoplasmic expression of MAGE-A2 was inversely proportional to the malignant grade and duration of recurrence of the disease in patients with PCa.

Carbon Nanospheres Exert Antitumor Effects Associated with Downregulation of 4E-BP1 Expression on Prostate Cancer.

IntroductionAlthough carbon nanospheres (CNPs) are promising nanomaterials in cancer treatment, how they affect prostate cancer (PCa) remains unclear.MethodsIn this study, scanning electron microscopy (SEM), X-ray diffraction (XRD), and Raman spectroscopy were used to confirm the successful synthesis of CNPs. CCK-8, flow cytometry, Transwell, wound healing, Western blot and immunohistochemistry (IHC) assays were performed to evaluate the antitumor effect of CNPs toward the two kinds of prostate cancer cell lines PC3 and DU145.ResultsOur results showed that CNPs inhibited cell growth, invasion, and migration and induced apoptosis and autophagy in PCa cells. Multifactor detection of a single Akt phosphorylation pathway and Western blot results suggested the suppression of 4E-BP1 in PCa cells after incubation with CNPs. The results from animal experiments also suggested the antitumor effect of CNPs and reduced 4E-BP1 expression in PCa tissue samples from BALB/c nude mice administered a local subcutaneous injection of CNPs.

Increased nuclear factor I/B expression in prostate cancer correlates with AR expression.

Most prostate cancers express androgen receptor (AR), and our previous studies have focused on identifying transcription factors that modify AR function. We have shown that nuclear factor I/B (NFIB) regulates AR activity in androgen-dependent prostate cancer cells in vitro. However, the status of NFIB in prostate cancer was unknown. We immunostained a tissue microarray including normal, hyperplastic, prostatic intraepithelial neoplasia, primary prostatic adenocarcinoma, and castration-resistant prostate cancer tissue samples for NFIB, AR, and synaptophysin, a marker of neuroendocrine differentiation. We interrogated publically available data sets in cBioPortal to correlate NFIB expression and AR and neuroendocrine prostate cancer (NEPCa) activity scores. We analyzed prostate cancer cell lines for NFIB expression via Western blot analysis and used nuclear and cytoplasmic fractionation to assess where NFIB is localized. We performed co-immunoprecipitation studies to determine if NFIB and AR interact. NFIB increased in the nucleus and cytoplasm of prostate cancer samples versus matched normal controls, independent of Gleason score. Similarly, cytoplasmic AR and synaptophysin increased in primary prostate cancer. We observed strong NFIB staining in primary small cell prostate cancer. The ratio of cytoplasmic-to-nuclear NFIB staining was predictive of earlier biochemical recurrence in prostate cancer, once adjusted for tumor margin status. Cytoplasmic AR was an independent predictor of biochemical recurrence. There was no statistically significant difference between NFIB and synaptophysin expression in primary and castration-resistant prostate cancer, but cytoplasmic AR expression was increased in castration-resistant samples. In primary prostate cancer, nuclear NFIB expression correlated with cytoplasmic NFIB and nuclear AR, while cytoplasmic NFIB correlated with synaptophysin, and nuclear and cytoplasmic AR. In castration-resistant prostate cancer samples, NFIB expression correlated positively with an AR activity score, and negatively with the NEPCa score. In prostate cancer cell lines, NFIB exists in several isoforms. We observed NFIB predominantly in the nuclear fraction of prostate cancer cells with increased cytoplasmic expression seen in castration-resistant cell lines. We observed an interaction between AR and NFIB through co-immunoprecipitation experiments. We have described the expression pattern of NFIB in primary and castration-resistant prostate cancer and its positive correlation with AR. We have also demonstrated AR interacts with NFIB.

Darolutamide antagonizes androgen signaling by blocking enhancer and super-enhancer activation.

Prostate cancer (PCa) is one of the most frequent tumor types in the male Western population. Early‐stage PCa and late‐stage PCa are dependent on androgen signaling, and inhibitors of the androgen receptor (AR) axis represent the standard therapy. Here, we studied in detail the global impact of darolutamide, a newly approved AR antagonist, on the transcriptome and AR‐bound cistrome in two PCa cell models. Darolutamide strongly depleted the AR from gene regulatory regions and abolished AR‐driven transcriptional signaling. Enhancer activation was blocked at the chromatin level as evaluated by H3K27 acetylation (H3K27ac), H3K4 monomethylation (H3K4me1), and FOXA1, MED1, and BRD4 binding. We identified genomic regions with high affinities for the AR in androgen‐stimulated, but also in androgen‐depleted conditions. A similar AR affinity pattern was observed in healthy and PCa tissue samples. High FOXA1, BRD4, H3K27ac, and H3K4me1 levels were found to mark regions showing AR binding in the hormone‐depleted setting. Conversely, low FOXA1, BRD4, and H3K27ac levels were observed at regulatory sites that responded strongly to androgen stimulation, and AR interactions at these sites were blocked by darolutamide. Beside marked loss of AR occupancy, FOXA1 recruitment to chromatin was also clearly reduced after darolutamide treatment. We furthermore identified numerous androgen‐regulated super‐enhancers (SEs) that were associated with hallmark androgen and cell proliferation‐associated gene sets. Importantly, these SEs are also active in PCa tissues and sensitive to darolutamide treatment in our models. Our findings demonstrate that darolutamide is a potent AR antagonist blocking genome‐wide AR enhancer and SE activation, and downstream transcription. We also show the existence of a dynamic AR cistrome that depends on the androgen levels and on high AR affinity regions present in PCa cell lines and also in tissue samples.

E2F5 promotes prostate cancer cell migration and invasion through regulation of TFPI2, MMP-2 and MMP-9.

Previously, our laboratory demonstrated that a deregulated E2F5/p38/SMAD3 axis was associated with uncontrolled cellular proliferation in prostate cancer (PCa). Here, we investigate the role of E2F5 in PCa in further details. RNAi-mediated E2F5 knockdown and pathway-focused gene expression profiling in PC3 cells identified TFPI2 as a downstream target of E2F5. Manipulation of E2F5 expression was also found to alter MMP-2 and MMP-9 levels as detected by Proteome Profiler array, western blot and reverse transcription coupled quantitative polymerase chain reaction Site-directed mutagenesis, dual-luciferase assays and chromatin immunoprecipitation with anti-E2F5-IgG coupled with qPCR confirmed recruitment of E2F5 on TFPI2, MMP-2 and MMP-9 promoters. RNAi-mediated knockdown of E2F5 expression in PC3 caused a significant alteration of cell migration while that of TFFI2 resulted in a modest change. Abrogation of E2F5 and TFPI2 expression was associated with significant changes in the gelatinolytic activity of active forms of MMP-2 and MMP-9. Moreover, E2F5, MMP-2 and MMP-9 levels were elevated in biopsies of PCa patients relative to that of benign hyperplasia, while TFPI2 expression was reduced. MMP-9 was coimmunoprecipitated with anti-TFPI2-IgG in PCa tissue samples suggesting a direct interaction between the proteins. Finally, artemisinin treatment in PC3 cells repressed E2F5 along with MMP-2/MMP-9 while triggering TFPI2 expression which alleviated PC3 aggressiveness possibly through inhibition of MMP activities. Together, our study reinstates an oncogenic role of E2F5 which operates as a dual-function transcription factor for its targets TFPI2, MMP-2 and MMP-9 and promotes cellular invasiveness. This study also indicates a therapeutic potential of artemisinin, a natural compound which acts by correcting dysfunctional E2F5/TFPI2/MMP axis in PCa.

Abstract A38: RNA-seq analysis identified candidate pathogens for prostate cancer

Abstract There is an abundance of evidence that chronic inflammation is linked to prostate cancer (PC), and pathogen infection is considered to be one of the possible causes for PC. Prostate infections by bacteria or viruses have been widely investigated. However, most of these studies have showed conflicting and controversial results. Most studies have focused on the PCR-based targeted detection of bacteria or viruses, and pathogen for PC remains to be found. To identify pathogen for PC, we investigated the transcriptomes of human prostate samples using whole-genome next-generation sequencing. The prostate specimens obtained by radical prostatectomy from 20 PC patients were sliced and divided into four sections. High-quality RNAs were extracted from the sections including PC. RNA-seq libraries were constructed according to the Illumina protocol and next-generation sequenced on a HiSeq 2000 instrument. To explore the potential of other pathogens being associated with PC, we performed de novo assembly of unmapped reads from the RNA-seq data from 20 PC samples. RNA-seq analysis identified four bacteria as candidate pathogens, which included Cutibacterium acnes, uncultured Chroococcidiopsis, Micrococcus luteus, and Moraxella osloensis. Among these candidate pathogens, Cutibacterium acnes was detected in 19 of 20 PC tissue samples by immunohistochemistry. We also performed PCR to detect other candidate pathogens (uncultured Chroococcidiopsis, Micrococcus luteus, and Moraxella osloensis) in PC samples for validation. Our findings provide further evidence of Cutibacterium acnes infections in human PCs as a promising pathogen for PC, and suggest that uncultured Chroococcidiopsis, Micrococcus luteus, and Moraxella osloensis could be novel candidate pathogens for PC. Further research is required and ongoing to clarify the molecular mechanisms underlying bacterial pathogenesis of PC. Citation Format: Shingo Ashida, Chiaki Kawada, Masanori Daibata, Keiji Inoue, Hidewaki Nakagawa. RNA-seq analysis identified candidate pathogens for prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A38.

Prognostic value of microRNA-20b expression level in patients with prostate cancer.

miR-20b is a member of the miR-106a-363 gene cluster located in the mammalian X chromosome, the larger miR-17 family, and the miR-17-92 and miR-106b-25 gene clusters. Previous studies have indicated that miR-20b may function as oncogene or tumor suppressor in different types of cancers. The present study analyzed the association between miR-20b and clinicopathological characteristics of patients with prostate cancer. A total of 127 pairs of prostate cancer tissue samples and adjacent prostate tissue samples were collected from April 2013 to March 2018. The associations between miR-20b expression levels and clinicopathological factors were assessed using the χ2‑test. Survival was estimated using the Kaplan-Meier method, and the differences in survival according to miR-20b expression were compared using the log-rank test. Prognostic values of miR-20b expression and clinical outcomes were evaluated by Cox regression analysis. The relative expression of miR-20b in prostate cancer tissues was significantly higher than that in adjacent noncancerous prostate tissues (P<0.001). miR-20b expression was observed to be significantly associated with Gleason score (P<0.001), lymph node metastasis (P<0.001), and TNM stage (P=0.002). The log-rank test indicated that patients with increased miR-20b expression experienced poor overall survival (P=0.037). Multivariate Cox regression analysis showed that miR-20b expression level (HR=2.181, 95% CI: 1.772-9.021, P=0.016) was an independent factor in predicting the overall survival of prostate cancer patients. The present study demonstrated that tissue miR-20b expression level could be a promising biomarker of prognosis in prostate cancer.

Upregulated Wnt-11 and miR-21 Expression Trigger Epithelial Mesenchymal Transition in Aggressive Prostate Cancer Cells.

Prostate cancer (PCa) is the second-leading cause of cancer-related death among men. microRNAs have been identified as having potential roles in tumorigenesis. An oncomir, miR-21, is commonly highly upregulated in many cancers, including PCa, and showed correlation with the Wnt-signaling axis to increase invasion. Wnt-11 is a developmentally regulated gene and has been found to be upregulated in PCa, but its mechanism is unknown. The present study aimed to investigate the roles of miR-21 and Wnt-11 in PCa in vivo and in vitro. First, different Gleason score PCa tissue samples were used; both miR-21 and Wnt-11 expressions correlate with high Gleason scores in PCa patient tissues. This data then was confirmed with formalin-fixed paraffin cell blocks using PCa cell lines LNCaP and PC3. Cell survival and colony formation studies proved that miR-21 involves in cells’ behaviors, as well as the epithelial-mesenchymal transition. Consistent with the previous data, silencing miR-21 led to significant inhibition of cellular invasiveness. Overall, these results suggest that miR-21 plays a significant role related to Wnt-11 in the pathophysiology of PCa.

LncRNA UNC5B-AS1 promotes malignant progression of prostate cancer by competitive binding to caspase-9.

This study aims to investigate the expression of LncRNA UNC5B-AS1 in prostate cancer (PCa) and to further investigate whether it can prompt malignant progression of PCa via regulating caspase-9. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was conducted to examine UNC5B-AS1 expression in 50 pairs of tumor tissue specimens and paracancerous ones collected from PCa patients, and the interplay between UNC5B-AS1 expression and clinical indicators of PCa was also analyzed. Meanwhile, UNC5B-AS1 levels in PCa cell lines were also further verified by qRT-PCR. In addition, UNC5B-AS1 knockdown model was constructed using lentivirus in PCa cell lines, and cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), transwell and flow cytometry assays were performed to figure out the impact of UNC5B-AS1 on the biological function of PCa cells. Finally, cell recovery experiment was conducted to explore the underlying mechanism and the association between UNC5B-AS1 and caspase-9. QRT-PCR results suggested that UNC5B-AS1 expression in PCa tissue samples was remarkably higher than in adjacent ones, with a statistically significant difference. Compared with patients with low expression of UNC5B-AS1, patients with highly-expressed UNC5B-AS1 had a higher incidence of distant metastasis and more advanced pathological stage. At the same time, proliferation and invasion, as well as migration ability of cells in sh-UNC5B-AS1 group, was conspicuously attenuated while cell apoptosis ability was conversely enhanced. Furthermore, qRT-PCR results revealed that caspase-9 and UNC5B-AS1 showed a negative correlation in gene expression level in PCa tissues. The results of the luciferase reporter gene experiment demonstrated that UNC5B-AS1 can be targeted by caspase-9 through their binding site. Additionally, cell recovery experiment indicated that UNC5B-AS1 and caspase-9 can be mutually regulated, which then together affect the malignant progression of PCa. UNC5B-AS1 expression was found remarkably increased in both PCa tissues and cell lines, which was remarkably associated with pathological stage and incidence of distant metastasis of PCa patients. In addition, UNC5B-AS1 was able to accelerate the malignant progression of PCa by modulating caspase-9 expression.

LncRNA PROX1-AS1 promotes proliferation, invasion, and migration in prostate cancer via targeting miR-647.

Long noncoding RNAs (lncRNAs) act as an important role in many diseases. In this research, lncRNA PROX1-AS1 was explored to identify how it functioned in the development of prostate cancer (PC). Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was utilized to detect PROX1-AS1 expression in PC patients. Then, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, colony formation assay, and transwell assay were performed to identify its function in PC cells. Furthermore, the potential mechanism was also explored using mechanism assays. PROX1-AS1 expression level was significantly higher in PC tissue samples and cell lines. Results of MTT assay, colony formation assay, and transwell assay showed that cell proliferation and invasion were inhibited through the silence of PROX1-AS1 in PC cells, while cell proliferation and invasion were promoted through the overexpression of PROX1-AS1 in PC cells. Furthermore, the expression of miR-647 was upregulated via the silence of PROX1-AS1 in PC cells, while the expression of miR-647 was downregulated via the overexpression of PROX1-AS1 in PC cells. Further mechanism assays showed that miR-647 was a direct target of PROX1-AS1 in PC. Correlation analysis showed that miR-647 expression was negatively correlated with PROX1-AS1 expression in PC tissues. Results above suggested that PROX1-AS1 could enhance cell proliferation and invasion of PC cells by sponging miR-647 and might be applied as a novel target for the treatment of PC.

Leucine Zipper-EF-Hand Containing Transmembrane Protein 1 Is a Potential Prognostic Biomarker and Promotes Cell Progression in Prostate Cancer.

PurposeThe leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) is a mitochondrial protein that has been associated with the occurrence and development of malignant tumors. Previous studies have shown that LETM1 expression is increased in several types of human cancer and is associated with a poor clinical outcome. However, the role of LETM1 in prostate cancer (PCa) has not yet been determined. In this study, we investigated the clinicopathological significance of LETM1 expression and its role in PCa progression.MethodsWe assessed the expression of LETM1 and genes related to cancer stemness, epithelial-mesenchymal transition (EMT), cell cycle, and PI3K/Akt signaling in 133 paraffin-embedded PCa tissue samples and cancer cells by using immunohistochemistry, immunofluorescence, and Western blotting.ResultsLETM1 expression was significantly increased in PCa, and it was positively correlated with Gleason score, pathologic tumor (pT) stage, clinical stage, and high microvessel density. Survival analysis showed that patients with PCa with a high level of LETM1 expression exhibited a low overall survival. Cox regression analysis indicated that LETM1 is an independent poor prognostic PCa factor. Additionally, the expression of LETM1 was correlated with cancer cell stemness-associated genes, EMT-related genes, cell cycle regulatory genes, and PI3K/Akt signaling gene expression in PCa. Furthermore, knocking down LETM1 expression down-regulated the expression of stemness-related proteins, while inhibiting tumor spheroid formation, EMT-like changes, cell proliferation, migration, and invasion in PCa cells. Importantly, the PI3K inhibitor LY294002 strongly inhibited the expression of LETM1, pPI3K-p85, and pAkt (Thr308, Ser473) in PCa cells.ConclusionThese results indicate that LETM1 expression is associated with cancer cell stemness, promotes EMT-like changes and cell proliferation and is a potential prognostic biomarker for PCa.

PLCε knockdown enhances the radiosensitivity of castration‑resistant prostate cancer via the AR/PARP1/DNA‑PKcs axis.

Radiotherapy (RT) has been used as a therapeutic option for treatment of prostate cancer (PCa) for a number of years; however, patients frequently develop RT resistance, particularly in castration-resistant PCa (CRPC), although the underlying mechanisms remain unknown. Understanding the underlying mechanism of RT resistance in CRPC may potentially highlight novel targets to improve therapeutic options for patients with PCa. In the present study, the expression levels of phospholipase Cε (PLCε), androgen receptor (AR) and DNA-dependent protein kinase catalytic subunit (PKcs) were examined in PCa tissue samples and PCa cells, and the effects of PLCε knockdown on AR and DNA damage repair (DDR)-related molecules were determined. The association between PLCε, AR and Poly (ADP-ribose) polymerase 1 (PARP1), as well as their respective roles in radiation resistance, were assessed using gene knockdown and pharmaceutical inhibitors or activators. A chromatin immunoprecipitation assay was used to determine the epigenetic regulatory effects of PLCε on PARP1. Animal experiments were performed to assess whether the mechanisms observed in vitro could be replicated in vivo. The expression levels of PLCε, AR and DNA-PKcs were significantly upregulated in PCa, particularly in CRPC. PLCε knockdown reduced the viability and increased apoptosis of cells subjected to radiation. Additionally, PLCε deficiency suppressed DDR progression by downregulating an AR and PARP1 positive feedback loop and the associated downstream molecules following radiation. PLCε depletion also increased the presence of histone H3 lysine 27 trimethylation in the PARP1 promoter region, suggesting increased methylation of the PARP1 gene and thus resulting in reduced expression of PARP1. In vivo, PLCε knockdown significantly potentiated the effects of radiation on tumor growth. Taken together, the results of the present study demonstrated that PLCε knockdown enhanced the radiosensitivity of CRPC by downregulating the AR/PARP1/DNA-PKcs axis.

Deep learning-based approach for automated assessment of PTEN status.

294 Background: PTEN loss is associated with adverse outcomes in prostate cancer and has the potential to be clinically implemented as a prognostic biomarker. Deep learning algorithms applied to digital pathology can provide automated and objective assessment of biomarkers. The objective of this work was to develop an artificial intelligence (AI) system for automated detection and localization of PTEN loss in prostate cancer samples. Methods: Immunohistochemistry (IHC) was used to measure PTEN protein levels on prostate tissue microarrays (TMA) from two institutions (in-house n=272 and external n=125 patients). TMA cores were visually scored for PTEN loss by pathologists and, if present, spatially annotated. In-house cohort (N=1239 cores) were divided into 70/20/10 training/validation/testing sets. Two algorithms were developed: a) Class I=core-based, to label each core for biomarker status and b) Class II=pixel-based, to spatially distinguish areas of PTEN loss within each core. ResNet101 architecture was used to train a multi-resolution ensemble of classifiers at 5x, 10x, and 20x for Class I task and a single classifier at simulated 40x for Class II segmentation. Results: For Class I algorithm, accuracy of PTEN status was 88.3% and 93.4% in validation and testing cohorts, respectively (Table). AI-based probability of PTEN loss was higher in cores with complete loss vs partial loss. Accuracy was improved to 90.7% in validation and 93.5% in test cohorts using the Class II region-based algorithm, with median dice scores 0.833 and 0.831, respectively. Direct application to external set demonstrated a high false positive rate. Loading trained model and conservatively re-training (“fine-tuning”) on 48/320 external cohort cores improved accuracy to 93.4%. Conclusions: Results demonstrate feasibility and robustness for fully automated detection and localization of PTEN loss in prostate cancer tissue samples and possibility for time/cost-effectiveness of sample processing/scoring in research and clinical laboratories.[Table: see text]

Expression of miR-148b-3p is correlated with overexpression of biomarkers in prostate cancer.

Prostate cancer (PCa) is one of the leading causes of death among men. Genes such as PCA3, PSA, and Fra-1 are suggested to serve as potential tools for the detection of PCa, as they are deregulated during this pathology. A similar event occurs with small non-coding RNAs, called miRNAs, specifically miR-195-5p, miR-133a-3p, and miR-148b-3p, which were analyzed in a Chinese population and suggested to be possible candidates for PCa diagnosis. We evaluated the expression levels of three miRNAs and three genes in tissue samples of PCa and benign prostate disease, such as benign prostatic hyperplasia, or prostatitis, in order to determine their potential as candidates for PCa detection. Our results showed a statistically significant overexpression of 279-fold increase in PSA levels and a 1,012-fold increase in PCA3 levels in PCa patients compared to benign prostate disease patients (p = 0.001 and p = 0.002, respectively). We observed a positive correlation between the expression of miR-148b-3p and the expression of PSA and PCA3 genes, two established biomarkers in PCa. The expression of miR-148b-3p was not related to clinical characteristics, such as age and weight, as observed for the other miRNAs analyzed, suggesting its potential as a biomarker for detection of this pathology.