Prostatic Hyperplasia Tissues Research Articles

GREM1 knockdown regulates the proliferation, apoptosis and EMT of benign prostatic hyperplasia by suppressing the STAT3/c-Myc signaling

BackgroundGremlin 1 (GREM1) has been reported to be highly expressed in prostate hyperplasia tissues. However, the role and molecular mechanism of GREM1 in benign prostatic hyperplasia (BPH) is still unclear. MethodsIn this study, expression of GREM1 in BPH-1 cells was detected by western blot assay. Cell counting kit-8 assay was performed to assess cell proliferation. Flow cytometry and western blot were used to assess cell apoptosis and cell cycle. The EMT process was detected by western blot assay and immunofluorescence staining. In addition, colivelin was used as a STAT3 activator and the expressions of STAT3/c-Myc signaling were assessed by western blot assay. ResultsThe data showed that GREM1 silencing inhibited BPH-1 cell proliferation and promoted cell apoptosis. Moreover, GREM1 silencing repressed the cell cycle progression and the development of EMT. In addition, knockdown of GREM1 suppressed the expression of the STAT3/c-Myc signaling in BPH-1 cells and colivelin treatment rehabilitated this signaling. Moreover, c-Myc overexpression or colivelin reversed the effects of GREM1 silencing on BPH-1 cell proliferation, cell apoptosis, cell cycle, as well as EMT. ConclusionTo sum up, GREM1 silencing may alleviate the BPH progress by inhibiting the STAT3/c-Myc signaling.

Preliminary study on miRNA in prostate cancer

ObjectiveTo screen for miRNAs differentially expressed in prostate cancer and prostate hyperplasia tissues and to validate their association with prostate cancer.MethodsPatients diagnosed by pathology in the Department of Urology of the First Affiliated Hospital of Xinjiang Medical University from October 2021 to June 2022 were selected, and their general clinical information, blood samples, and prostate tissue samples were collected. miRNA microarray technology was performed to obtain differentially expressed miRNAs in prostate cancer and hyperplasia tissues, and miRNAs to be studied were screened by microarray results and review of relevant literature. The detection of miRNA expression in the patients’ blood and prostate tissue samples was measured. The miRNA-222-mimics were transfected into PC3 cells, and cell biology experiments such as CCK8, scratch, Transwell, and flow cytometry were performed to detect the effects of overexpressed miRNA-222 on the growth and proliferation, invasive ability, apoptotic ability, and metastatic ability of prostate cancer cells.ResultsThe results of the miRNA microarray showed that there were many differentially expressed miRNAs in prostate cancer and hyperplasia tissues, and four miRNAs, miRNA-144, miRNA-222, miRNA-1248, and miRNA-3651 were finally selected as the subjects by reviewing relevant literature. The results showed that the expression of miRNA-222 in prostate cancer tissues was lower than that in prostate hyperplasia tissues (P < 0.05). The expression of miRNA-222, miRNA-1248, and miRNA-3651 in blood samples of prostate cancer patients was lower than that in prostate hyperplasia patients (P < 0.05). The analysis results indicated that the f/t ratio and the relative expression of miRNA-222 and miRNA-1248 were independent influences of prostate cancer (P < 0.05), in which overexpression of miRNA-222 decreased the proliferative, invasive, and metastatic abilities of PC3 cells and enhanced the level of apoptosis of cancer cells.ConclusionsAlthough there was no significant change in the overall incidence of prostate cancer in this study, significant changes occurred in the incidence of prostate cancer with different characteristics. In addition, the nomogram prediction model of prostate cancer-specific survival rate constructed based on four factors has a high reference value, which helps physicians to correctly assess the patient-specific survival rate and provides a reference basis for patient diagnosis and prognosis evaluation.

Characterization of the Tumor Microenvironment and the Biological Processes with a Role in Prostatic Tumorigenesis.

Prostate intratumoral heterogeneity, driven by epithelial–mesenchymal plasticity, contributes to the limited treatment response, and it is therefore necessary to use the biomarkers to improve patient prognostic survival. We aimed to characterize the tumor microenvironment (T lymphocyte infiltration, intratumoral CD34, and KI-67 expressions) by immunohistochemistry methods and to study the biological mechanisms (cell cycle, cell proliferation by adhesion glycoproteins, cell apoptosis) involved in the evolution of the prostate tumor process by flow-cytometry techniques. Our results showed that proliferative activity (S-phase) revealed statistically significant lower values of prostate adenocarcinoma (PCa) and benign prostatic hyperplasia (BPH) reported at non-malignant adjacent cell samples (PCa 4.32 ± 4.91; BPH 2.35 ± 1.37 vs. C 10.23 ± 0.43, p < 0.01). Furthermore, 68% of BPH cases and 88% of patients with PCa had aneuploidy. Statistically increased values of cell proliferation (CD34+ CD61+) were observed in prostate adenocarcinoma and hyperplasia cases reported to non-malignant adjacent cell samples (PCa 28.79 ± 10.14; BPH 40.65 ± 11.88 vs. C 16.15 ± 2.58, p < 0.05). The CD42b+ cell population with a role in cell adhesion, and metastasis had a significantly increased value in PCa cases (38.39 ± 11.23) reported to controls (C 26.24 ± 0.62, p < 0.01). The intratumoral expression of CD34 showed a significantly increased pattern of PCa tissue samples reported to controls (PCa 26.12 ± 6.84 vs. C 1.50 ± 0.70, p < 0.01). Flow cytometric analysis of the cell cycle, apoptosis, and adhesion glycoproteins with a critical role in tumoral cell proliferation, T cell infiltrations, Ki-67, and CD 34 expressions by IHC methods are recommended as techniques for the efficient means of measurement for adenocarcinoma and hyperplasia prostate tissue samples and should be explored in the future.

Expression of Endocan in Tissue Samples from Prostate Adenocarcinoma and Prostate Hyperplasia: A comparative retrospective study.

In this study, we aimed to determine whether there is a significant difference in endocan expression levels between prostate adenocarcinoma and prostate hyperplasia tissues by using an immunohistochemical method. Materials and Methods:All of 51 patients, who were getting treatment for the last 5 years, participated in the study. 31 of 51 patients underwent transrectal sonography (TRUSG) -assisted prostate biopsy because of prostate adenocarcinoma as diagnosed with elevated PSA levels and histopathological examination. The remaining 20 patients comprised the control group. The control group included patients with benign prostate hyperplasia based on pathological examination. Results:It was found that there was strong positive epithelial staining in 74.2% of patients with prostate cancer while in 5% of controls, indicating a statistically significant difference (P < .001). It was also found that the rate of strong positive endothelial staining was 77.4% in the patient group whereas 5% in the control group (P < .001). Also, the rate of strong positive stromal staining was 64.5% in the patient group while 5% in the control group (P < .001). Conclusion:We found that tissue endocan expression level was statistically significantly higher in patients with prostate cancer compared to those with benign prostate hyperplasia by using an immunohistochemical method.

The expression of YWHAZ and NDRG1 predicts aggressive outcome in human prostate cancer

Some prostate cancers (PCas) are histo-pathologically grouped within the same Gleason Grade (GG), but can differ significantly in outcome. Herein, we aimed at identifying molecular biomarkers that could improve risk prediction in PCa. LC ESI–MS/MS was performed on human PCa and benign prostatic hyperplasia (BPH) tissues and peptide data was integrated with omic analyses. We identified high YWHAZ and NDRG1 expression to be associated with poor PCa prognosis considering all Gleason scores (GS). YWHAZ and NDRG1 defined two subpopulations of PCa patients with high and intermediate risk of death. Multivariable analyses confirmed their independence from GS. ROC analysis unveiled that YWHAZ outperformed GS beyond 60 months post-diagnosis. The genomic analysis of PCa patients with YWHAZ amplification, or increased mRNA or protein levels, revealed significant alterations in key DNA repair genes. We hereby state the relevance of YWHAZ in PCa, showcasing its role as an independent strong predictor of aggressiveness.

Expression of breast cancer metastasis suppressor 1 and osteopontin protein in prostate cancer and its clinical significance

Objective To investigate the expression level and clinical significance of breast cancer metastasis suppressor 1 (BRMS1) and osteopontin (OPN) in prostate carcinoma. Methods The expression level of BRMS1 and OPN protein was detected in 46 specimens of prostate carcinoma (PCa) tissues and 29 benign prostatic hyperplasia (BPH) tissues by immunohistochemical methods. The relationship between protein level and clinicopathological data was analyzed in prostate cancer patients. Results The expression rate of BRMS1 in the PCa tissues was 21.7% (10/46), which was significantly lower than that in the BPH tissues [86.2% (25/29), P=0.000]. The expression rate of OPN in PCa tissues was 70.0% (32/46), which was significantly higher than that in the BPH tissues [27.6% (8/29), P=0.000]. The expression level of BRMS1 was correlated with distant metastasis (P=0.046), but not with gender, Gleason score, PSA level, tumor size. The expression level of OPN was correlated with the Gleason score, tumor size and distant metastasis (P=0.046, 0.029, 0.035), but not with gender and PSA level. The expression level of BRMS1 was negatively correlated with OPN (P=0.021). There is a high tendency of distant metastasis in patients with BRMS1 negative and OPN positive expression. Conclusion BRMS1 and OPN may play an important but opposite role in the metastasis of prostate cancer, the combined test of the two protein can be used as an effective auxiliary index to evaluate the malignancy of prostate cancer. Key words: Prostatic cancer; Breast cancer metastasis suppressor 1; Osteopontin

Expression of forkhead transcription factor O4 in prostate cancer and its effect on prostate cancer cell invasion

To examine the expression of forkhead transcription factor O4 (FOXO4) in prostate cancer and to explore its effect on prostate cancer cell invasion. Methods: Immunohistochemistry was used to detect the expression of FOXO4 in prostate hyperplasia tissues and prostate cancer tissues. Western blot was used to detect the expression of FOXO4 in prostate hyperplasia cell line BPH-1 and prostate cancer cell lines: PC-3 and DU145. PC-3 cells with high relative expression of FOXO4 were transfected with FOXO4 siRNA and scramble siRNA; DU145 cells with low expression of FOXO4 were transfected with FOXO4 plasmid and blank vector. Matrigel Transwell assay was used to detect the invasive ability of transfected cells. The expression of endothelial-mesenchymal transition (EMT)-related proteins E-cadherin, N-cadherin, and vimentin in the transfected cells was detected by Western blot. Results: The expression of FOXO4 in prostate cancer cells and tissues was significantly lower than that in the prostate hyperplasia cells and tissues (both P<0.05). In the prostate cancer tissues, the expression of FOXO4 in cancer tissues with prostate cancer specific antigen (PSA) value <4 was significantly higher than that in the tissues with 4≤PSA≤10 and PSA>10 (all P<0.05). The expression of FOXO4 in cancer tissues with Gleason score <8 was significantly higher than that in the cancer tissues with Gleason ≥8 (P<0.05). The expression of FOXO4 in clinical stage T1-T2 prostate cancer tissues was higher than that in the clinical stage T3-T4 prostate cancer tissues (P<0.05). The expression of FOXO4 in prostate cancer tissues without lymph node metastasis was significantly higher than that in the prostate cancer tissues with lymph node metastasis (P<0.05). Down-regulation of FOXO4 in PC-3 cells could significantly promote the EMT and invasion, with the decreased expression of E-cadherin and the increased expression of N-cadherin and vimentin (all P<0.05); Up-regulation of FOXO4 in DU145 cells could inhibit the EMT and invasion of cells, with the increased expression of E-cadherin and the decreased expression of N-cadherin and vimentin (all P<0.05). Conclusion: FOXO4 is involved in prostate cancer progression, and it can inhibit prostate cancer cell invasion by regulating EMT of prostate cancer cells.

Abstract A058: Integrative prostate cancer tissue proteomics dissects clear and distinct proteomes for human prostate cancer and benign prostatic hyperplasia

Abstract Current approaches in proteomics techniques and MS systems have revived the quest for novel biomarkers in prostate cancer (PCa) boosting the molecular characterization of the disease. To reveal fundamental differences between benign and malignant growth of prostate cells, we combined a new protein extraction procedure that disrupts the crosslinked proteins from the previously formaldehyde-fixed, paraffin-embedded tissue samples with in-depth proteomic analysis (ESI-MS/MS). Human PCa and benign prostatic hyperplasia (BPH) tissue samples were obtained using phase-transfer surfactant-aided extraction/tryptic digestion of formalin-fixed and paraffin-embedded sections mounted on microscope slides. Data analysis was based on label-free spectral counting, identifying with a minimum of two peptides, 1331 and 1239 proteins in PCa and BPH tissue proteomes, respectively. 71 proteins were exclusively present in PCa samples, while 122 proteins where exclusively present in BPH samples. In order to prioritize candidate markers for PCa, we compared protein expression based on normalized spectral counts between tissue samples. We set as cut-offs proteins that were found with a minimum of three peptides within the PCa and BPH proteomes. This filter resulted in the selection of two clusters of 11 and 16 proteins, respectively. The data sets highlighted distinct proteins that were previously studied in the context of prostate cancer progression, including SSBP1, GDF15, NDRG1, C4A, and APOE for PCa and DUSP3, MME, SRI, and DSG1 for BPH, thus providing further confirmation for the robustness of our quantification method. We next subjected our candidate list to bioinformatics analysis (Oncomine). Accordingly, the 5 proteins mentioned for PCa were significantly upregulated (fold change &amp;gt;1.5, P≤0.05) in prostate adenocarcinoma vs. normal prostate gland. Whole-exome analysis (cBioportal) revealed amplification as the most frequent genetic alteration and RNASeq data also confirmed a significant upregulation for these proteins (P≤0.05). Strikingly, proteins associated with BPH were significantly downregulated (fold change &amp;gt;1.5, P≤0.05) across the same comparison and RNA-seq data also confirmed a significant downregulation for these proteins (P≤0.05). This report showcases significant and extensive differences in protein expression patterns between BPH and prostate carcinoma. Proteome analysis of prostate tissues should help to disclose the molecular mechanisms underlying prostate malignant growth, resulting in new sets of biomarkers for diagnostic, prognostic, and therapeutic use. Citation Format: Sofia Lage Vickers, Juan Antonio Bizzotto, Alejandra Paez, Javier Cotignola, Carlos Scorticati, Osvaldo Mazza, Pia Valacco, Geraldine Gueron, Elba Vazquez. Integrative prostate cancer tissue proteomics dissects clear and distinct proteomes for human prostate cancer and benign prostatic hyperplasia [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A058.

Abstract 1797: Characterization of a novel androgen receptor coregulator RIPK1

Abstract Using bicalutamide-androgen receptor (AR) DNA binding domain-ligand binding domain as bait, we observed enrichment of FxxFY motif-containing peptides. Protein database searches revealed the presence of receptor-interacting protein kinase 1 (RIPK1) harboring one FxxFY motif. RIPK1 interacted directly with AR and suppressed AR transactivation in a dose-dependent manner. Domain mapping experiments showed that the FxxFY motif in RIPK1 is critical for interactions with AR and the death domain of RIPK1 plays a crucial role in its inhibitory effect on transactivation. In terms of tissue expression, RIPK1 levels were markedly higher in benign prostate hyperplasia and non-cancerous tissue regions relative to the tumor area. With the aid of computer modeling for screening of chemicals targeting activation function 2 (AF-2) of AR, we identified oxadiazole derivatives as good candidates and subsequently generated a small library of these compounds. A number of candidates could effectively suppress AR transactivation and AR-related functions in vitro and in vivo with tolerable toxicity via inhibiting AR-peptide, AR-coregulator and AR N-C interactions. Combination of these chemicals with antiandrogen had an additive suppressive effect on AR transcriptional activity. Our collective findings may pave the way in creating new strategies for the development and design of anti-AR drugs. Citation Format: Cheng-Lung Hsu, Jai-Shin Liu, Ting-Wei Lin, Chun-Cheng Lin, Wen-Guey Wu. Characterization of a novel androgen receptor coregulator RIPK1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1797.

TRANSCRIPTION FACTOR BRN-3α AND PROSTATE CANCER, RELATION WITH HORMONE RECEPTORS AND AKT/M-TOR SIGNALING PATHWAY ACTIVATION

Introduction. The transcription factor Brn-3α (product of the POU4F1) plays a significant role in carcinogenesis. The purpose of the study was to investigate Brn-3α, AR, ERα expressions in tissues of benign prostatic hyperplasia and prostate cancer as well as to determine their association with AKT/mTOR signaling pathway activation. Material and methods . The study included 30 patients with locally advanced prostate cancer and 15 patients with benign prostatic hyperplasia. The expression levels of Brn-3α, AR, ERα and the components of AKT/m-TOR signaling pathway were determined using RT-PCR assay. Results. Overexpression of Brn-3α in prostate cancer tissues was found to be associated with high level of ERα mRNA. High expression of AR was revealed in tissues of benign prostatic hyperplasia and prostate cancer. High expression levels of AKT, m-TOR and PTEN were observed in prostate cancer tissues. However, no significant changes in PTEN mRNA level were found in tissues of prostatic hyperplasia. Conclusion. Brn-3α transcription factor was associated with prostate cancer and accompanied with high expression levels of ERα, AKT and m-TOR.

Expressions and significance of Mcl-1 and Caspase-3 and theirs correlation in tissues of prostate cancer

Objective To study the relationship of the expressions of Mcl-1 and Caspase-3 in tissues of prostate cancer(PCa) and benign prostatic hyperplasia (BPH), investigate the correlation of Mcl-l and Caspase-3. Methods The expressions of Mcl-1 and Caspase-3 were observed separately in 30 of BPH, and 35 of PCa by means of immunohistochemical methods. Results Mcl-1 positive rate in BPH was 13.33%(4/30), and in PCa was 82.86%(29/35). Caspase-3 positive rate in BPH was 73.33%(22/30), and in PCa was 25.71%(9/35). Expression of Mcl-1 and Caspase-3 in PCa was unrelated with preperative PSA, distant metastases, Gleason score and clincal stage(P>0.05). There was a negative relationship between the expression of Mcl-1 and Caspase-3 in tissues of PCa(rs=-0.748, P<0.05). Conclusions The expression of Mcl-1 is raised while the expression of Caspase-3 is low in PCa tissue, showing that they may have a common mechanism in the process of apoptosis. Key words: Prostatic Neoplasms; Prostatic Hyperplasia; Caspase 3

Expression and significance of Zinc finger MYND-type containing 8 in prostate cancer via the regulation of glucocorticoid receptor

Objective To identify Zinc finger MYND-type containing 8 (ZMYND8) as a direct target for glucocorticoid receptor (GR) and illustrate the correlations between ZMYND8 and clinical characteristics of prostate cancer. Methods Bioinformatics software and Western blotting were used to analyze the relationship between ZMYND8 and GR in GR-knock-out prostate cancer cells. In order to evaluated the correlation of ZMYND8 with clinical characteristics, immunohistochemistry was used to detect ZMYND8 protein in 80 cases of prostate cancer and benign prostate hyperplasia (BPH) tissues. Results ZMYND8 functioned as a cancer promoter via the regulation of GR, and the expression of ZMYND8 protein was significantly increased in prostate cancer and metastatic tumor tissue (immunoreactivity score: no cancer: 5.700±2.054, cancer: 7.340±2.163, P=0.001; metastatic cancer: 6.710±2.369, non-metastatic cancer: 8.110±1.595, P=0.028). Conclusion Our study showed that ZMYND8 is regulated by GR, and ZMYND8 is a cancer promoter in prostate cancer. Key words: Zinc finger MYND-type containing 8; Glucocorticoid receptor; Prostate cancer; Metastasis; Castration-resistance

Abstract 5707: STAT3 and STAT5a are potential therapeutic targets in castration-resistant prostate cancer

Abstract Background: Currently, there is no effective pharmacotherapy for metastatic castration-resistant prostate cancer (mCRPC). The mechanisms underlying mCRPC are not clearly understood, thus hindering rational-based drug design. Evidence suggests that signal transducer and activator of transcription 3 (STAT3) and 5a/b (STAT5a/b), key components of the JAK/STAT pathway, play a significant role in aggressive PC. However, expression of STAT3/5 in prostate tumor samples have not been studied. Here, we evaluated the possible role of STAT3/5a in aggressive PC. Materials and Methods: Expression of STAT3 and STAT5a in high grade PC (HGPC, n=15) and in benign prostatic hyperplasia (BPH, n=15) tissue sections were evaluated by immunohistochemistry (IHC) according to the protocol approved by Institutional Review Board. Age ranged from 59 to 95 years (median = 81) in the former and 57 years to 86 years (median = 68 years) in the latter category. In addition, the effects of STAT inhibitor, Pimozide, on androgen-sensitive LNCaP and its castration-resistant subline C4-2 PC cell growth were assessed in vitro. Results: A strong nuclear immunoreactivity for STAT3 and STAT5a in 93% (n=14) and 80% (n=12) of HGPC cases, respectively, were observed. Only one BPH case showed strong STAT3 expression. Focal and weak positivity for STAT3 and STAT5a were noted in 47% (n=7) and 67% (n=10) of BPH cases. None of the BPH cases showed strong STAT5a expression. HGPC cases showed significantly higher STAT3 positivity compared with BPH (p=0.01). No significant differences were observed for STAT5a expression between HGPC and BPH group (p=0.4). However, multifocal or diffuse and strong STAT3 (p&amp;lt;0.0001) and STAT5a (p&amp;lt;0.0001) expression was significantly higher in HGRPC than BPH. Pimozide exposure significantly inhibited LNCaP cell proliferation at 10 μM and C4-2 cell proliferation at 20 μM concentration. Conclusion: Our results demonstrate that increased expression of STAT3 and STAT5a may serve as a predictive biomarker for efficacy of the JAK/STAT targeted therapy. Since JAK/STAT and androgen receptor signaling are functionally synergistic in contributing PC progression, the inhibition of JAK/STAT-alone or in combination with AR may lead to a novel treatment modality for mCRPC. Citation Format: Sambit K. Mohanty, Kader Yagiz, Luthringer Luthringer, Mahul B. Amin, Serhan Alkan, Bekir Cinar. STAT3 and STAT5a are potential therapeutic targets in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5707. doi:10.1158/1538-7445.AM2017-5707

Abstract 226: Verification of prostate cancer genomics biomarker candidates at protein level using PRISM-SRM

Abstract Mass spectrometry (MS) based targeted proteomics such as selected reaction monitoring (SRM) provides an antibody-independent strategy for sensitive, specific and multiplexed verification of genomics biomarker candidates at the protein level. In order to identify a panel of proteins with the potential to discriminate between aggressive and indolent forms of prostate cancer and predict prostate cancer progression, we have selected 52 protein candidates from existing prostate cancer genomics data sets and validated cancer drivers, and performed quantitative proteomics analysis in tumor and control tissue samples using the highly sensitive PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing)-SRM approach. PRISM-SRM assays have been developed for the 52 prostate cancer biomarker candidates including: prostate cancer prognosis associated genes, prostate cancer associated genes that were up-regulated in transcriptomics studies, and other cancer-related genes (including the ERG or ETV1 isoforms). Two sets of tissue samples were analyzed using PRISM-SRM with heavy isotope-labeled synthetic peptides as internal standards: 1) 10 high Gleason-score (7-9) primary prostate tumors and 10 benign prostatic hyperplasia (BPH) tissues (OCT-embedded specimens); and 2) 10 primary tumors from patients showing metastatic progression, 10 primary tumors from patients who showed biochemical recurrence (BCR), and 10 primary tumors from patients with no BCR or metastatic progression after more than ten years of follow-up after radical prostatectomy (FFPE whole mount prostate specimens). Overall, PRISM-SRM analyses of all the patient tissue samples enabled the detection of 48 out of 52 biomarker candidates, suggesting extremely low level of expression of the remaining 6 genes (HXC6, OSTP, TWST1, and ERG8); in comparison regular LC-SRM can only detect 21 of these candidates at the protein level. In the 10 high Gleason-score tumors and 10 BPH controls, 13 proteins were found differentially abundant with P&amp;lt;0.05. In the 10X10X10 FFPE sample analysis, there were three proteins discriminating between “metastatic progression” and “no progression” tumors, one protein discriminated between BCR and “no progression” tumors, and four proteins discriminated between metastatic progression and BCR tumors (P&amp;lt;0.05). These promising biomarker candidates will be further evaluated, individually and in panels, in independent, larger cohort for their potential prognostic applications. In summary, PRISM-SRM provides a highly sensitive method for quantification and rapid screening of multiple potential biomarker candidates at the protein level. This approach holds great potential for rapidly translating genomics-based discovery candidates into protein-based biomarkers. Citation Format: Hui Wang, Yuqian Gao, Athena Schepmoes, Gyorgy Petrovics, Jennifer Cullen, Thomas Fillmore, Tujin Shi, Wei-Jun Qian, Richard Smith, Brandi Weaver, Robin Leach, Ian Thompson, Sudhir Srivastava, Jacob Kagan, Albert Dobi, Karin Rodland, Shiv Srivastava, Tao Liu. Verification of prostate cancer genomics biomarker candidates at protein level using PRISM-SRM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 226. doi:10.1158/1538-7445.AM2017-226

Decreased expression of ferroportin in prostate cancer.

The present study examined the expression levels of ferroportin, a transmembrane protein that transports iron from the inside of a cell to the outside, in the prostate cancer PC3, DU145 and LNCAP cell lines, in the normal prostate RWPE2 cell line, and in tissue samples from different differentiation stages of prostatic carcinoma and prostatic hyperplasia. The study also investigated the role of ferroportin protein expression in the diagnosis and prognosis of prostate cancer. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were employed to measure the mRNA and protein expression levels of ferroportin in the PC3, DU145, LNCAP and RWPE2 cells. Immunohistochemistry was used to determine ferroportin protein expression in the prostate cancer and prostatic hyperplasia tissues. Compared with the normal prostate RWPE2 cells, ferroportin protein expression was significantly lower in the prostate cancer PC3, DU145 and LNCAP cells (P<0.05). Compared with the prostatic hyperplasia tissues, ferroportin protein expression was significantly reduced in the prostate cancer tissues (P<0.05). Overall, the expression levels of ferroportin in the prostate cancer tissues were lower than those in the normal prostate tissues, which may provide valuable clinical information for the diagnosis and prediction of disease progression in prostate cancer, and may indicate a potential therapeutic target for treating prostate cancer by regulating iron metabolism.

Abstract 1848: Evaluation of the frequency of RNASE L in 462 and 541 variants in Iranian prostate cancer patients

Abstract Introduction: Several genetic loci are suspected to be involved in prostate cancer, including the hereditary prostate cancer. RNASEL regulates cell proliferation and apoptosis through the IFN-regulated 2-5A pathway that mediates anti proliferative activities and has been suggested to be a candidate tumor suppressor gene. There are numerous nucleotide variants identified in the RNASEL gene. The two most commonly found variants in the U.S. non-Hispanic Caucasian population are the non-synonymous variants: 1385 G&amp;gt;A; Arg462Gln; and 1623T&amp;gt;G; Asp 541 Glu has been reported. The Arg 462 Gln variant reduces the ability of the cell to cause apoptosis in response to activation by 2-5 (A) and also has three times less enzymatic activity than normal, whereas the Asp 541 Glu variant has no known effect on RNASEL protein function. There continues to be much debate over whether these common variants increase the risk of prostate cancer. It has been also reported that the Arg 462 Gln AA genotype associated with both increased prostate cancer in U.S. Caucasian sample groups and decreased prostate cancer risk in Caucasian and Japanese sample groups Moreover, the Asp 541 Glu variant within RNASEL has been reported that the GG and TT genotypes were associated with an increased risk for prostate cancer in Japanese and European-American samples, respectively. On the other hand, a significant negative association of the TT genotype with prostate cancer in Swedish Caucasian samples has been reported .However; the role of these common mutations is debatable. The aim of this study was to investigate the role of common variants of RNASE L gene; Arg 462 Gln and Asp 541 Glu in Iranian men with prostate cancer. Patients and Methods:Blocks of paraffin-embedded tissue from 46 patients with adenoma carcinoma prostate cancer and 50 patients with hyperplasia prostate tissue and also fifty healthy blood controls with PSA&amp;lt;4 was enrolled in this current research study. ARMS-PCR amplification was performed with specific primers. Results and Conclusions: This is the first study that investigates the association of prostate cancer risk with RNASEL variants in Iranian prostate cancer patients. Our result indicated that the frequency of the AA, GG and AG genotypes in the RNASEL 462 SNP in Tumors were 30%, 13.3% and 56.66% respectively, in hyperplasia was 30.3% ,15.15% and 54.54% respectively and in controls was 26%, 14% and 60% respectively .The frequency of the TT, GG and TG genotypes in the RNASEL 541 SNP in Tumors were 3%, 0 % and 93.47% respectively, in hyperplasia were 10 %,0% and 90% and in blood controls were 8%, 0% and 92% respectively. Our finding indicated that these variations were not a significant association with prostate cancer risk in Iranian patients and the other variations of this gene maybe involve in Iranian prostate cancer patients. Note: This abstract was not presented at the meeting. Citation Format: Sirweh Molla, Massoud Ghaffarpour, Somayeh Saeeda, Seyed Werya Hossieni, Sina Mirzaahmadi, Mohammad Reza Rahmany. Evaluation of the frequency of RNASE L in 462 and 541 variants in Iranian prostate cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1848. doi:10.1158/1538-7445.AM2014-1848

Inhibition of androgen induces autophagy in benign prostate epithelial cells

5-α Reductase inhibitor can reduce the volume of benign prostatic hyperplasia by lowering benign prostatic hyperplasia level and consequently inducing epithelial cells apoptosis. The present study investigated whether autophagy and apoptosis of benign prostatic hyperplasia epithelial cells are influenced by low benign prostatic hyperplasia levels. PWR-1E prostate epithelial cells transfected with GFP-LC3 plasmid were subjected to androgen deprivation conditions. Then the autophagic puncta were evaluated by fluorescence microscopy, and the cellular apoptosis rate was detected by 4, 6-diamidino-2-phenylindole staining after blocking of autophagic process by 3-methyladenine. Furthermore, autophagy status was also determined in hyperplasia prostate tissues from 5-α reductase inhibitor-treated patients by immunohistochemistry. In the androgen deprivation medium, autophagic punta increased markedly in PWR-1E cells, and blockage of autophagy by 3-methyladenine significantly promoted PWR-1E cells' apoptosis rate. In vivo, the expression of LC3 protein (an important autophagic marker) in hyperplasia prostate tissue significantly increased after 5-α reductase inhibitor treatment. Meanwhile, the prostate-specific antigen, as an inner control, decreased. 5-α Reductase inhibitor treatment increases autophagy and possibly decreases the apoptosis of prostate epithelial cells.

Abstract 3117: Overexpression of the stress transcription co-activator LEDGF/p75 contributes to the upregulation of the stress protective genes HSP27, ERp57 and CYGB in prostate cancer.

Abstract Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer related death in men in the US. PCa tumor aggressiveness, response to treatment, and mortality are worst in African-American men, compared to other ethnic groups, thus presenting a major health disparity. Our group reported previously that lens epithelium derived growth factor p75 (LEDGF/p75), a stress survival transcription coactivator, has elevated expression in PCa cells and tissues and promotes resistance to cell death induced by oxidative stress and chemotherapy. This is likely due to its ability to transactivate promoter regions of stress and antioxidant genes such as peroxiredoxin 6 (Prdx6) and heat shock protein 27 (HSP27). We hypothesized that high expression of LEDGF/p75 in prostate tumor cells and tissues contributes to the elevated expression of its putative target genes, some of which were identified in previous studies by our group and others. These include genes associated with stress protection and chemotherapy resistance such as HSP27, endoplasmic reticulum protein 57 (ERp57), and cytoglobin (CYGB). To test this hypothesis we performed an expression analysis of LEDGF/p75, HSP27, ERp57 and CYGB proteins in human PCa cell lines using immunoblotting, and in prostate tumor tissues using immunohistochemistry on tumor tissue microarrays (TMAs). PCa cell lines included stable PC3 clones overexpressing LEDGF/p75, as well as docetaxel (DTX) resistant PC3-DR and DU145-DR cells overexpressing this protein, with their corresponding parental DTX sensitive cell lines. The TMAs contained tissue specimens corresponding to prostate tumor, hyperplasia, normal-adjacent, and disease-free normal prostate tissues. Correlations between the expression of LEDGF/p75 and HSP27 or ERp57 or CYGB in cell lines as well as in tumors and normal tissues was determined. Our results indicated that overexpression of LEDGF/p75 in PC3 stable clones and in DTX resistant PCa cells was associated with increased protein expression of HSP27, ERp57 and CYGB. Statistically significant correlation was also observed between the protein levels of LEDGF/p75 and HSPp27 or ERp57 or CYGB in the TMAs. Luciferase-based transcription reporter assays confirmed that the promoter regions of HSP27 and ERp57 are activated by LEDGF/p75 in PCa cells. Chromatin immunoprecipitation assays also demonstrated binding of LEDGF/p75 to HSP27 promoter in PCa cells. Additional promoter activation studies are in progress to demonstrate that LEDGF/p75 transcriptionally regulates ERp57 and CYGB genes. These results enhance our understanding of the contribution of LEDGF/p75 overexpression to the activation of stress protective genes and its implications for chemotherapy resistance in metastatic PCa. Citation Format: Anamika Basu, Christina Cajigas, Benanette Medina, Heather Rojas, Hiya Banerjee, Melanie Mediavilla-Varela, Laisum Leoh, Carlos Casiano. Overexpression of the stress transcription co-activator LEDGF/p75 contributes to the upregulation of the stress protective genes HSP27, ERp57 and CYGB in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3117. doi:10.1158/1538-7445.AM2013-3117

Abstract 3307: Transcriptional silencing of c-Myc by promoter-directed siRNA efficiently target prostate cancer stem cells.

Abstract Prostate cancer is the most frequent epithelial cancer in elderly men in Western countries. Depending on the stage, prostate cancer treatment requires surgery, hormone therapy, radio- and chemo-therapy. c-MYC is a transcription factor activated by mitogenic signaling pathways and playing a central function in stem cell biology. Over-activity of c-Myc plays a central role in tumorigenesis by affecting cell proliferation, metabolic adaptation and survival. Amplification of c-MYC is one of the most common genetic alterations occurring in cancer genomes. c-Myc expression is significantly elevated in invasive prostate adenocarcinomas compared to benign prostatic hyperplasia and normal prostate tissue. Particularly, c-Myc is highly expressed in prostate cancer cells having the CD44+/CD24- phenotype, which is described as a hallmark of cancer progenitor/stem cells (CSCs). Importantly, several studies link therapy resistance and disease progression to the CSC subpopulation within the highly heterogeneous cellular composition of the tumor. CSCs are likely the tumor initiating population that produces metastasis and disease recurrence. These lines of evidence suggest a central role of c-Myc in the maintenance of CSC compartment in human tumors and justify c-Myc as a therapeutic target. Previously, we showed that the c-Myc gene could be silenced with small interfering RNAs (siRNA) targeting its promoter. Interestingly, single transfection with myc targeting siRNA (myc-siRNA) induced long lasting effects on cell proliferation and clonogenicity, indicative of a persistent loss of proliferative and clonogenic potential. In this study, we investigated the effects of transcriptional silencing of c-Myc on the CSC subpopulation in human prostate cancer cell lines in vitro and in vivo. We found that treatment with myc-siRNA significantly reduced the fraction of CSCs defined by expression of stem cell surface markers CD44 and CD24, in vitro sphere forming ability and self-renewal. Furthermore, combined analysis of senescence and cell surface markers showed that senescence occurred prevalently in the CD44+/CD24- cell subpopulation leading to its depletion, and reduced self renewal capability, in vivo tumorigenicity and metastatic potential. Moreover, repeated intraperitoneal administration of myc-siRNA over a 4-week period reduced tumor growth in a xenogeneic prostate cancer model. These results are consistent with the role of c-Myc in the maintenance of the CSC subpopulation in human prostate tumors and suggest that c-Myc downregulation induces an irreversible loss of clonogenic and tumor-initiating capability linked to the induction of cell senescence in CSCs. Our findings show also that an RNAi-based transcriptional therapy directed to genes directly involved in the maintenance of the cancer stem cells could be an efficient approach to block progression and relapse of prostate cancer. Citation Format: Gianluca Civenni, Anastasia Malek, Domenico Albino, Manuela Sarti, Sandra Pinton, Stefano Di Marco, Giuseppina M. Carbone, Carlo V. Catapano. Transcriptional silencing of c-Myc by promoter-directed siRNA efficiently target prostate cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3307. doi:10.1158/1538-7445.AM2013-3307

Non metastatic cell protein 1 expression and clinical significance in prostate cancer

Objective To explore the role of non metastatic cell protein 1 (NME1) in diagnosing prostate cancer (PCa) and judging early metastasis of PCa.Methods The gene chip was used to screen NME1 in PCa and benign prostatic hyperplasia (BPH) tissues,the differential expression of NME1 prostein in PCa and adjacent tissues was analyzed by using fluorescence 2-D difference gel electrophoresis (2DDIGE) technology,and validated by using immunohistochemistry (IHC).The NME1 IHC score and clinicopathological parameters of PCa patients were analyzed.Results Relative to BPH tissues the expression of NME1 was down-regulated in PCa tissues by gene chip screening (P ＜ 0.01),and relative to adjacent tissues the same phenomenon was observed in PCa tissues by using 3D-DIGE technology (P ＜0.01).The expression of NME1 was negatively correlated with serum PSA levels (P ＜ 0.05),Gleason score (P ＜0.01) and TNM stage (P ＜ 0.05),positively correlated with metastasis (P ＜ 0.01),but not correlated with age (P ＞ 0.05).NME1-positive PCa patients had a lower overall survival rate (Log Rank =11.117,P ＜0.01),but there was no significant difference in biochemical recurrence rate between NMEl-negative and NMEl-positive PCa patients (Log Rank =0.247,P ＞ 0.05).Conclusion The low expression of NME1 is closely related with the malignancy of PCa,and the loss of NME1 may promote tumor cell growth.Serum PSA levels in combination with NME1 levels can be used to diagnose PCa early and judge the degree of the malignancy of PCa.The high expression of NME1 may promote PCa metastasis. Key words: Non metastatic cell protein 1 gene; Prostate carcinoma; Diagnosis