Human Prostate Cancer Tissues Research Articles

Activation of overexpressed glucagon-like peptide-1 receptor attenuates prostate cancer growth by inhibiting cell cycle progression.

Aims/IntroductionIncretin therapy is a common treatment for type 2 diabetes mellitus. We have previously reported an anti‐prostate cancer effect of glucagon‐like peptide‐1 receptor (GLP‐1R) agonist exendin‐4. The attenuation of cell proliferation in the prostate cancer cell line was dependent on GLP‐1R expression. Here, we examined the relationship between human prostate cancer severity and GLP‐1R expression, as well as the effect of forced expression of GLP‐1R using a lentiviral vector.Materials and MethodsProstate cancer tissues were extracted by prostatectomy and biopsy. GLP‐1R was overexpressed in ALVA‐41 cells using a lentiviral vector (ALVA‐41‐GLP‐1R cells). GLP‐1R expression was detected by immunohistochemistry and quantitative polymerase chain reaction. Cell proliferation was examined by growth curves and bromodeoxyuridine incorporation assays. Cell cycle distribution and regulators were examined by flow cytometry and western blotting. In vivo experiments were carried out using a xenografted model.ResultsGLP‐1R expression levels were significantly inversely associated with the Gleason score of human prostate cancer tissues. Abundant GLP‐1R expression and functions were confirmed in ALVA‐41‐GLP‐1R cells. Exendin‐4 significantly decreased ALVA‐41‐GLP‐1R cell proliferation in a dose‐dependent manner. DNA synthesis and G1‐to‐S phase transition were inhibited in ALVA‐41‐GLP‐1R cells. SKP2 expression was decreased and p27Kip1 protein was subsequently increased in ALVA‐41‐GLP‐1R cells treated with exendin‐4. In vivo experiments carried out by implanting ALVA‐41‐GLP‐1R cells showed that exendin‐4 decreased prostate cancer growth by activation of GLP‐1R overexpressed in ALVA41‐GLP‐1R cells.ConclusionsForced expression of GLP‐1R attenuates prostate cancer cell proliferation by inhibiting cell cycle progression in vitro and in vivo. Therefore, GLP‐1R activation might be a potential therapy for prostate cancer.

Transient Sox9 Expression Facilitates Resistance to Androgen-Targeted Therapy in Prostate Cancer.

Patients with metastatic prostate cancer are increasingly presenting with treatment-resistant, androgen receptor-negative/low (AR-/Low) tumors, with or without neuroendocrine characteristics, in processes attributed to tumor cell plasticity. This plasticity has been modeled by Rb1/p53 knockdown/knockout and is accompanied by overexpression of the pluripotency factor, Sox2. Here, we explore the role of the developmental transcription factor Sox9 in the process of prostate cancer therapy response and tumor progression. Unique prostate cancer cell models that capture AR-/Low stem cell-like intermediates were analyzed for features of plasticity and the functional role of Sox9. Human prostate cancer xenografts and tissue microarrays were evaluated for temporal alterations in Sox9 expression. The role of NF-κB pathway activity in Sox9 overexpression was explored. Prostate cancer stem cell-like intermediates have reduced Rb1 and p53 protein expression and overexpress Sox2 as well as Sox9. Sox9 was required for spheroid growth, and overexpression increased invasiveness and neural features of prostate cancer cells. Sox9 was transiently upregulated in castration-induced progression of prostate cancer xenografts and was specifically overexpressed in neoadjuvant hormone therapy (NHT)-treated patient tumors. High Sox9 expression in NHT-treated patients predicts biochemical recurrence. Finally, we link Sox9 induction to NF-κB dimer activation in prostate cancer cells. Developmentally reprogrammed prostate cancer cell models recapitulate features of clinically advanced prostate tumors, including downregulated Rb1/p53 and overexpression of Sox2 with Sox9. Sox9 is a marker of a transitional state that identifies prostate cancer cells under the stress of therapeutic assault and facilitates progression to therapy resistance. Its expression may index the relative activity of the NF-κB pathway.

Prostate field cancerization and exosomes: Association between CD9, early growth response 1 and fatty acid synthase.

Intracapsular and well‑defined adenocarcinomas of the prostate are often surrounded by tissue areas that harbor molecular aberrations, including those of genetic, epigenetic and biochemical nature. This is known as field cancerization, or a field effect and denotes a state of pre‑malignancy. Such alterations in histologically normal tumor‑adjacent prostatic tissues have been recognized as clinically important and are potentially exploitable as biomarkers of disease and/or targets for preventative/therapeutic intervention. The authors have previously identified and validated two protein markers of field cancerization: The expressional upregulation of the transcription factor early growth response1 (EGR‑1) and the lipogenic enzyme fatty acid synthase (FASN). However, the molecular etiology of prostate field cancerization, including EGR‑1 and FASN upregulation, remains largely unknown. It was thus hypothesized that extracellular vesicles, notably exosomes, released by tumor lesions may induce molecular alterations in the surrounding tissues, resulting in field cancerization, priming the tissue, and ultimately promoting multifocal tumorigenesis, which is often observed in prostate cancer. Towards testing this hypothesis, the current study, to the best of our knowledge, for the first time, presents correlative protein expression data, generated in disease‑free, tumor‑adjacent and cancerous human prostate tissues by quantitative immunofluorescence, between the exosomal marker CD9, and EGR‑1 and FASN. Despite the pilot character of the present study, and the static nature and heterogeneity of human tissues, the data suggest that CD9 expression itself is part of a field effect. In support of this hypothesis, the results suggest a possible contribution of exosomes to the induction of field cancerization in the prostate, particularly for EGR‑1. These findings were corroborated in established cell models of cancerous (LNCaP) and non‑cancerous (RWPE‑1) human prostate epithelial cells. The findings of this study warrant further investigation into the functional interface between exosomes and field cancerization, as a detailed understanding of this characterization may lead to the development of clinical applications related to diagnosis and/or prognosis and targeted intervention to prevent progression from pre‑malignancy to cancer.

KLF16 Affects the MYC Signature and Tumor Growth in Prostate Cancer.

BackgroundKLF16, a member of the Kruppel-like factor (KLF) family, functions in the regulation of dopaminergic transmission, metabolism, and endocrinology. However, the role of KLF16 in prostate cancer (PCa) remains unknown.MethodsWe screened the expression of KLFs in PCa based on bioinformatics analysis. The protein levels of KLF16 in PCa specimens were confirmed by immunohistochemistry. Inhibiting KLF16 by RNA interference with shRNA was used to determine the effects of KLF16 on PCa cell growth in vitro and in vivo. RNA sequencing was used to investigate the signaling regulated by KLF16 in PCa. Bioinformatics analysis was also used to determine the possible correlations of KLF16 and signaling in PCa cohorts.ResultsBioinformatics analysis showed that KLF16 may be required for PCa development. Notably, the expression of KLF16 was elevated in human PCa tissues. In vitro and in vivo experiments both demonstrated that depleting KLF16 significantly inhibited the growth of PCa cells. Downregulation of KLF16 significantly decreased the expression of MYC signaling in PCa cells. Furthermore, KLF16 expression was correlated with MYC signaling activity.ConclusionKLF16 was overexpressed in PCa tissues compared to normal tissues. KLF16 knockdown suppressed PCa cell growth in vitro and in vivo, and a deficiency of KLF16 inhibited activation of MYC signaling.

Simultaneous Detection of Zinc and Its Pathway Metabolites Using MALDI MS Imaging of Prostate Tissue.

Levels of zinc, along with its mechanistically related metabolites citrate and aspartate, are widely reported as reduced in prostate cancer compared to healthy tissue and are therefore pointed out as potential cancer biomarkers. Previously, it has only been possible to analyze zinc and metabolites by separate detection methods. Through matrix-assisted laser desorption/ionization mass spectrometry imaging (MSI), we were for the first time able to demonstrate, in two different sample sets (n = 45 and n = 4), the simultaneous spatial detection of zinc, in the form of ZnCl3–, together with citrate, aspartate, and N-acetylaspartate on human prostate cancer tissues. The reliability of the ZnCl3– detection was validated by total zinc determination using laser ablation inductively coupled plasma MSI on adjacent serial tissue sections. Zinc, citrate, and aspartate were correlated with each other (range r = 0.46 to 0.74) and showed a significant reduction in cancer compared to non-cancer epithelium (p < 0.05, log2 fold change range: −0.423 to −0.987), while no significant difference between cancer and stroma tissue was found. Simultaneous spatial detection of zinc and its metabolites is not only a valuable tool for analyzing the role of zinc in prostate metabolism but might also provide a fast and simple method to detect zinc, citrate, and aspartate levels as a biomarker signature for prostate cancer diagnostics and prognostics.

MiR-142-3p functions as an oncogene in prostate cancer by targeting FOXO1.

Prostate cancer (PCa) is a heterogeneous malignancy, and is a primary cause of cancer-related death in males. Forkhead box transcription factor O1 (FOXO1) exerts antitumor effects in various cancers, including PCa. However, the regulatory mechanism of miR-142-3p on FOXO1 expression in human PCa has not been characterized. In this study, we showed that FOXO1 protein levels were downregulated in PCa tissues and cells. Moreover, FOXO1 expression was a predictor of disease-free survival in patients with PCa and was a predictor of prognosis. Increased expression of FOXO1 suppressed cellular proliferation and induced cell cycle arrest at G0/G1 in vitro. However, FOXO1 mRNA and protein levels were inconsistent in human PCa tissues and cell lines. We showed that miR-142-3p levels were negatively correlated with FOXO1 protein levels in PCa. We also showed that miR-142-3p suppressed FOXO1 expression by directly targeting its 3′-untranslated region. Furthermore, suppression of miR-142-3p inhibited cell proliferation and induced cell cycle arrest, and these effects were blocked by FOXO1 knockdown. In vivo experiments showed that miR-142-3p knockout impaired tumor growth. Our results validate that FOXO1 acted as a tumor suppressor in PCa and demonstrated that FOXO1 was regulated by miR-142-3p, and miR-142-3p may be a potential target for treatment of PCa.

Copy number-based quantification assay for non-invasive detection of PVT1-derived transcripts.

BackgroundOne of the most important susceptibility loci for cancer is the 8q24 human chromosomal region. The non-protein coding gene locus plasmacytoma variant translocation 1 (PVT1) is located at 8q24 and is dysregulated in prostate cancer. PVT1 gives rise to multiple transcripts which may have different functions. Here, we describe a real-time quantitative polymerase chain reaction (qPCR)-based assay for copy number-based quantitation of PVT1 exons 4A, 4B, and 9 to enable accurate, reproducible, and quantifiable detection.MethodsPVT1 exons 4A, 4B, and 9 were cloned into a plasmid vector to create standards for subsequent creation of linear standard curves representing a broad range of concentrations. PCR was carried out using SYBR-Green signal detection to quantify PVT1 exons 4A, 4B, and 9. The efficacy of this assay was evaluated by using it to detect these transcripts in prostate epithelial and prostate cancer cell lines, normal and cancerous human prostate tissues, human serum, mouse plasma, and urine samples.ResultsThe results indicate that the assay can be used to quantify both low and high copy numbers of PVT1-derived transcripts. This is the first report of a copy number-based quantification assay for non-invasive detection of PVT1 derived transcripts.ConclusionsThis novel assay holds promise for routine non-invasive testing in diseases where PVT1 is dysregulated.

DEPTOR is an in vivo tumor suppressor that inhibits prostate tumorigenesis via the inactivation of mTORC1/2 signals

The DEPTOR-mTORC1/2 axis has been shown to play an important, but a context dependent role in the regulation of proliferation and the survival of various cancer cells in cell culture settings. The in vivo role of DEPTOR in tumorigenesis remains elusive. Here we showed that the levels of both DEPTOR protein and mRNA were substantially decreased in human prostate cancer tissues, which positively correlated with disease progression. DEPTOR depletion accelerated proliferation and survival, migration, and invasion in human prostate cancer cells. Mechanistically, DEPTOR depletion not only activated both mTORC1 and mTORC2 signals to promote cell proliferation and survival, but also induced an AKT-dependent epithelial–mesenchymal transition (EMT) and β-catenin nuclear translocation to promote cell migration and invasion. Abrogation of mTOR or AKT activation rescued the biological consequences of DEPTOR depletion. Importantly, in a Deptor-KO mouse model, Deptor knockout accelerated prostate tumorigenesis triggered by Pten loss via the activation of mTOR signaling. Collectively, our study demonstrates that DEPTOR is a tumor suppressor in the prostate, and its depletion promotes tumorigenesis via the activation of mTORC1 and mTORC2 signals. Thus, DEPTOR reactivation via a variety of means would have therapeutic potential for the treatment of prostate cancer.

MicroRNA‑301a‑3p overexpression promotes cell invasion and proliferation by targeting runt‑related transcription factor 3 in prostate cancer.

MicroRNAs (miRNAs) are known to serve a role in tumorigenic programs. The dysregulated expression of miR‑301a‑3p may affect the progression of various types of human cancer; however, the expression and the role of miR‑301a‑3p in prostate cancer are still unclear. The present study aimed to clarify the role and molecular mechanism of miR‑301a‑3p in prostate cancer. The results demonstrated that the expression of miR‑301a‑3p was significantly upregulated in human prostate cancer tissues and in several prostate cancer cell lines. Invitro overexpression of miR‑301a‑3p notably increased prostate cancer cell proliferation and invasion. Bioinformatics analysis revealed that runt‑related transcription factor 3 (RUNX3) may be a target of miR‑301a‑3p, which was confirmed by Dual‑luciferase reporter assay. Western blot analysis also demonstrated that miR‑301a‑3p regulated the protein expression levels of RUNX3. In addition, the results indicated that miR‑301a‑3p may regulate the Wnt signaling pathway, and rescue experiments indicated that RUNX3 contributed to the effects of miR‑301a‑3p on cell proliferation and invasion through Wnt signaling. In conclusion, these findings suggested that miR‑301a‑3p may promote prostate cancer cell invasion and proliferation by targeting RUNX3, and provided insight into understanding prostate cancer pathogenesis. miR‑301a‑3p may be a potential therapeutic candidate to treat prostate cancer.

The role of WNT10B in normal prostate gland development and prostate cancer

WNT signaling is implicated in embryonic development, and in adult tissue homeostasis, while its deregulation is evident in disease. This study investigates the unique roles of canonical WNT10B in both normal prostate development and prostate cancer (PCa) progression. Organ culture and rat ventral prostates (VPs) were used to study Wnt10b ontogeny and growth effect of WNT10B protein. PB-SV40 LTag rat VPs were utilized for Wnt expression polymerase chain reaction (PCR) array and immunohistochemistry. Human localized PCa tissue microarrays (TMAs) were investigated for differential WNT10B expression. Human RNA-seq data sets were queried for differential expression of WNT10B in metastatic and localized PCa. Knockdown of WNT10B in PC3 cells was utilized to study its effects on proliferation, stemness, epithelial to mesenchymal transition (EMT), and xenograft propagation. Wnt10b expression was highest at birth and rapidly declined in the postnatal rat VP. Exogenous WNT10B addition to culture developing VPs decreased growth suggesting an antiproliferative role. VPs from PB-SV40 LTag rats with localized PCa showed a 25-fold reduction in Wnt10b messenger RNA (mRNA) expession, confirmed at the protein level. Human PCa TMAs revealed elevated WNT10B protein in prostate intraepithelial neoplasia compared with normal prostates but reduced levels in localized PCa specimens. In contrast, RNA-seq data set of annotated human PCa metastasis found a significant increase in WNT10B mRNA expression compared with localized tumors suggesting stage-specific functions of WNT10B. Similarly, WNT10B mRNA levels were increased in metastatic cell lines PC3, PC3M, as well as in HuSLC, a PCa stem-like cell line, as compared with disease-free primary prostate epithelial cells. WNT10B knockdown in PC3 cells reduced expression of EMT genes, MMP9 and stemness genes NANOG and SOX2 and markedly reduced the stem cell-like side population. Furthermore, loss of WNT10B abrogated the ability of PC3 cells to propagate tumors via serial transplantation. Taken together, these results suggest a dual role for WNT10B in normal development and in PCa progression with opposing functions depending on disease stage. We propose that decreased WNT10B levels in localized cancer allow for a hyperproliferative state, whereas increased levels in advanced disease confer a stemness and malignant propensity which is mitigated by knocking down WNT10B levels. This raises the potential for WNT10B as a novel target for therapeutic intervention in metastatic PCa.

Genome atlas analysis based profiling of Akt pathway genes in the early and advanced human prostate cancer.

Recent studies conducted in the mouse and cellular models suggest a stage-specific, differential effect of Akt activity modulation on tumor growth and metastasis in various cancers. In prostate cancer (PCa), although the deletion of Akt1 gene in a neuroendocrine model of TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) blunted oncogenic transformation and tumor growth, Akt1 suppression in the advanced PCa resulted in the activation of transforming growth factor-β pathway and enhanced metastasis to the lungs. Such a dual role for the Akt isoforms and its signaling partners has not been investigated in human PCa. In the current study, we performed genomic database analysis of Akt isoforms and associated pathway molecules in human prostate adenocarcinoma, castration-resistant PCa, neuroendocrine PCa and metastatic PCa for mutations, genetic alterations, mRNA and protein expressions and activating phosphorylations from cBioportal. Results from the protein data analysis from the cBioportal were compared to the results of our data on human PCa tissue analysis and the cellular effects of Akt1 suppression using MK-2206 on PCa cell aggressiveness. Our study indicates the existence of a dual role for Akt1 in PCa and warrants a large-scale analysis of the early and advanced stage PCa clinical samples for further clarity.

Abstract 5251: STAT1 and miR155 expression in human prostate cancer tissue

Abstract Background: Transcription factors (TFs) and microRNAs (miRNAs) form a gene regulatory network at transcriptional and posttranscriptional level. Increasing evidence shows that miRNAs regulates molecular pathways in cancer by targeting various oncogenes and tumor suppressor genes. However, the mutual regulations between TFs and miRNAs in cancer cells have not been well characterized. Signal transducer and activator of transcription 1 (STAT1) has been reported as a tumor suppressor, but recent evidence suggests that may also play a tumor-promoting role. An STAT1-miR155 feedback loop has been identified in normal tissues. We aimed to explore the STAT1 and miR155 expression in prostate cancer by a bioinformatics analysis combined with immunohistochemical evaluation of STAT1 in normal and prostate cancer tissues. Design: The expression of miR155 was explored in the dbDEMC2 database of Differentially Expressed miRNAs in Human Cancers. Potential targets of this miRNA were searched in the microRNA-target interactions database (miRTarBase), and STAT1 was selected. The protein expression level of STAT1 was analyzed in normal and tumor tissues, from the TCGA databases, by GEPIA bioinformatics tools; and by immunohistochemistry in a prostate tissue microarray (TMA), constructed from prostatectomy samples of patients with localized prostate cancer (PCa), including representative areas from PCa and benign prostate tissue (BPT). Immunohistochemical expression of STAT1 was evaluated by two pathologists, an H-score was obtained for each spot by microscopic assessment of the percentage of epithelial cells with positive staining. Differences in STAT1 expression were assessed by comparing H-Scores between BPT and PCa tissues using Mann-Whitney test, a p value less than 0.05 was considered statistically significant. The Ethical Review Board of the academic institution approved this study. Results: The analysis of miR155 and STAT1 expression in the Genome Cancer Atlas Database showed that both are overexpressed in a variety of tumors including prostate cancer. STAT1 expression at protein level was evaluated in 143 tissue cores representing PCa, and 78 tissue cores representing BPT. Mean STAT1 expression in cores with PCa, was significantly higher than mean of STAT1 expression in cores with BPT (p less than 0.0001). There was a higher mean staining score in PCa (210, 95% CI: 196,1-227,8), compared to BPT (105,5, 95% CI: 87,1-123,9). STAT1 staining was present predominantly in the cytoplasm, also in the membrane and nucleus, of prostate cancer tumor cells. Conclusions: Although miR155 and STAT1 are supposed to be involved in a negative auto-regulatory feedback mechanism, they were both found overexpressed in most of the prostate cancer tissues, which could be explained by the loss of the regulatory feedback loop equilibrium in the tumor environment. Additional studies to detect the in situ expression of miR155 are in progress to validate these results. Citation Format: Ines Benedetti, Lia Barrios Garcia, Juan Rebollo. STAT1 and miR155 expression in human prostate cancer tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5251.

Abstract 349: Oncolytic reovirus variants induce direct oncolysis in human prostate cancer

Abstract The acquisition of therapy resistance and the formation of distant metastases represent major problems in prostate cancer. To date, immunotherapeutic approaches have been relatively disappointing in the treatment of prostate cancer. Therapies based on oncolytic viruses are being exploited for several tumor entities and have been studied in multiple clinical trials. Mammalian orthoreovirus is a non-enveloped double stranded RNA virus that has not been linked to serious disease in humans. Reovirus is relatively safe to use and several clinical trials are currently ongoing to study the oncolytic effect of reovirus in various tumor types. Type 3 Dearing (T3D) is a promising candidate for the use as an oncolytic agent. Besides wildtype T3D reovirus (R124), spontaneous mutants Jin1, 2 and 3 were generated which harbor mutations in spike protein Sigma-1. In this way, Jin mutants are able to infect cells independently of the Junction Adhesion Molecule-A (JAM-A) entry receptor on the tumor cell surface, which is often downregulated in different types of cancer. In this study, the direct oncolytic effect of wildtype (R124) and Jin mutant reovirus was examined in human prostate cancer cells in vitro and in our ‘near-patient’ model of ex vivo cultured human prostate cancer tissue slices. Both R124 and Jin3 dose-dependently induced prostate cancer cell death in PC-3M-Pro4luc2, DU145 and 22Rv1 cells in vitro. Interestingly, JAM-A negative cancer cells were insensitive for R124, but were effectively killed by Jin1 mutant, indicating the beneficial effects of the Jin mutant in JAM-A low/negative tumors. Moreover, tumor tissue slices were generated from explanted patient prostate cancer tissues and patient-derived xenografts (PDX) and cultured with reovirus. Oncolytic effects were observed in ex vivo cultured tumor slices upon viral replication and subsequent stimulation of apoptosis. Therefore, ex vivo culture of patient-derived tumor tissue allows the assessment of individual anti-tumor response of the oncolytic virotherapy (i.e. personalized therapeutics). Taken together, our results indicate that both wildtype R124 virus and mutant Jin reovirus can infect and replicate in human prostate cancer cells and patient-derived prostate cancer tissues. The Jin mutants are of particular interest in JAM-A negative tumors. The indirect immunomodulatory effects of reovirus in human prostate cancer are currently ongoing. In conclusion, our selected oncolytic reoviruses may represent a promising novel treatment strategy in human prostate cancer alone or combined with immunostimulatory agents. Citation Format: Arjanneke F. van de Merbel, Geertje van der Horst, Maaike H. van der Mark, Diana J. van den Wollenberg, Jack A. Schalken, Anne Collins, Norman J. Maitland, Rob C. Hoeben, Gabri van der Pluijm. Oncolytic reovirus variants induce direct oncolysis in human prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 349.

Downregulated LINC00460 inhibits cell proliferation and promotes cell apoptosis in prostate cancer.

LINC00460 has been confirmed to contribute to cancer development. However, the role and function of LINC00460 in prostate cancer is not identified. The purpose of this study was to evaluate the expression and effect of LINC00460 on prostate cancer cell malignant behaviors. The expression of LINC00460 in cancer tissues and cell lines were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assay. The LINC00460 expression was downregulated by siRNA. The cell counting kit-8 (CCK-8) assay was used to detect cell proliferation. The cell migration and invasion were detected by migration and Matrigel invasion assays. The Western blot assay was used to detect the altered expression levels of Ki67, Cyclin D1, PI3K, p-AKT, T-AKT, Bcl2, and Bax. LINC00460 was increased in human prostate cancer tissues and cell lines. LINC00460 high expression was related to Tumor Size (T1-T2/T3-T4; p=0.004), and high Gleason Score (≤8/>8, p=0.000). Downregulation of LINC00460 by siRNA could inhibit cancer cell proliferation and decreased Ki67 and Cyclin D1 expression. Meanwhile, downregulation of LINC00460 promoted apoptosis of cell lines and was related to PI3K/AKT pathway. LINC00460 could regulate cell proliferation and cell apoptosis, which might be a novel marker in prostate cancer.

Targeting POH1 inhibits prostate cancer cell growth and enhances the suppressive efficacy of androgen deprivation and docetaxel.

POH1, a member of the JAMM domain containing deubiquitinases, functions in malignant progression of certain types of cancer. However, the role of POH1 in prostate cancer (PCa) remains unclear. We performed RNA interference against the JAMM members in PC3 cells and analyzed cell proliferation. POH1 knockdown was established to evaluate the effects of POH1 on cell growth in vitro and in vivo. RNA-sequencing was utilized to explore the molecular details underlying the biological function of POH1 in PCa. The expression of POH1 in PCa tissues was detected by immunohistochemistry. The POH1 inhibitor capzimin was evaluated to explore whether pharmacologically inhibiting POH1 significantly affected PCa cell proliferation alone or enhanced the inhibitory efficacy of docetaxel and androgen deprivation. Functional analyses identified POH1 as a JAMM deubiquitinase that is required for PCa proliferation. Importantly, expression of POH1 was higher in human PCa tissues (PCas) than that in normal prostate tissues, and a positive correlation was detected between elevated POH1 expression and higher pathological grades in PCas. In vivo experiments further demonstrated that depleting POH1 significantly suppressed the growth of PCa cell xenografts. POH1 deficiency profoundly inhibited the expression of a set of genes involving the cell cycle and caused G0/G1 phase arrest. Furthermore, the POH1 inhibitor capzimin phenotypically recapitulated the effects of POH1 knockdown and improved the efficacy of docetaxel and androgen deprivation in PCa cells. POH1 was overexpressed in PCas and was correlated with pathological grades in human PCas. Inhibiting POH1 by gene silencing or pharmacological inhibition with capzimin suppressed PCa cell growth. Exploring the inhibition of POH1 in combination with other drugs may provide a strategy to benefit patients with PCa.

Highly expressed long non-coding RNA FEZF1-AS1 promotes cells proliferation and metastasis through Notch signaling in prostate cancer.

Growing studies indicated that long non-coding RNAs (lncRNAs) acted as imperative players in neoplasms initiation and progression. This research was designed to study the potential involvements of lncRNA FEZF1-AS1 (FEZF1-AS1) in the pathogenesis of prostate cancer (PCa). Real-time PCR was performed to detect the expressions of FEZF1-AS1 in PCa specimens and cell lines. Correlations between G- FEZF1-AS1 expressions and clinical characteristics and overall survivals were determined using statistical methods. The CCK-8 assays, colony formation assay, flow cytometry, transwell, and wound scratch assays were carried out to study cells viability, cells migration, and invasion. Western blot and RT-PCR were used for the determination of the influence of FEZF1-AS1 on Notch signaling pathway. We found that FEZF1-AS1 expressions were distinctly reduced in human PCa tissues and cell lines compared with their non-tumor counterparts, and its higher levels were strongly associated with lymph node metastasis (p=0.012) and Angiolymphatic invasion (p=0.022). Then, Kaplan-Meier assays showed that patients with higher expressions of FEZF1-AS1 were shown to predict unfavorable overall survival. Cox proportional hazards risks assays revealed that FEZF1-AS1 acted as an independent prognostic factor for PCa. Functional investigations suggested that knockdown of FEZF1-AS1 could suppress cells proliferation, trigger late apoptosis, and inhibit cells invasion and migration. Mechanistic assays demonstrated that FEZF1-AS1 exhibited its tumor-promotive roles by activating the Notch signaling pathway. We suggested that FEZF1-AS1 served as a tumor promoter in PCa and may develop a novel therapeutic target for PCa patients.

1955-P: GLP-1R Activation Attenuates Prostate Cancer Growth via Inhibiting Cell Cycle Progression

Incretin therapy is one of the most popular treatment for type 2 diabetes. We have previously reported anti-prostate cancer effect of GLP-1R agonist Exendin-4(Ex-4) (Diabetes 2014, PLOS ONE 2015), and LEADER trial suggested that GLP-1R agonist could reduce prostate cancer. In our previous report, the attenuation of prostate cancer cell proliferation was depending on GLP-1R expression, and Ex-4 did not attenuate cell proliferation in ALVA-41, a prostate cancer cell without intrinsic GLP-1R expression. Then, we next examined anti-prostate cancer effect in ALVA-41 forced expressed GLP-1R using lentivirus vector (ALVA-41-GLP-1R). We confirmed abundant GLP-1R expression in ALVA-41-GLP-1R using RT-PCR and immunohistochemistry, and intracellular cAMP level was increased by Ex-4 in ALVA-41-GLP-1R. Ex-4 significantly decreased the proliferation of ALVA-41-GLP-1R in a dose dependent manner, but not in ALVA-41-control. BrdU assay revealed that DNA synthesis was attenuated in ALVA-41-GLP-1R, and G1 to S phase entry was inhibited in cell cycle distribution in flow cytometry analysis. Further, p27kip protein was increased and Skp2, ubiquitin ligase for p27kip, expression was decreased in ALVA-41-GLP-1R treated by Ex-4. In vivo experiments using xenograft model mice transplanting ALVA-41-GLP-1R revealed that Ex-4 decreased prostate cancer growth via activation of GLP-1R forced expressed in ALVA41. Further, GLP-1R expression level in human prostate cancer tissues was inversely associated with Gleason grading system of human prostate cancer. These data suggested that forced expressed GLP-1R attenuated prostate cancer cell proliferation via inhibiting cell cycle progression in vivo and in vitro, and GLP-1R activation could be one of optional therapy for prostate cancer. Disclosure T. Shigeoka: None. T. Nomiyama: None. T. Kawanami: None. Y. Hamaguchi: None. T. Tanaka: None. S. Irie: None. K. Nabeshima: None. M. Tanaka: None. T. Yanase: None. Funding Japan Society for the Promotion of Science

Identification of candidate diagnostic and prognostic biomarkers for human prostate cancer: RPL22L1 and RPS21.

Prostate cancer (PCa) is one of the most common malignancies in men worldwide. This study was designed to investigate the potential of Ribosomal Protein L22-like1 (RPL22L1) and Ribosomal Protein S21 (RPS21) as diagnostic and prognostic biomarkers for PCa. First, RPL22L1 and RPS21 were screened as the key molecular of PCa by bioinformatics analysis. Subsequently, the prostate tissue samples were stained for antibodies against RPL22L1 and RPS21. The unbiased signal quantification was performed by ImageJ software, and the results showed that the expression of RPL22L1 and RPS21 exhibited significant differences between the PCa tissues and the normal prostate tissues. Receiver-operating characteristics (ROC) curves were prepared, and then the area under the curve (AUC) values of RPL22L1 and RPS21 were calculated as 0.798 and 0.768, and the likelihood ratio (LR) values of RPL22L1 and RPS21 were calculated as 2.86 and 2.53. These data implied that the over-expression of RPL22L1 and RPS21 is associated with the presence of PCa. The further analysis suggested that the expression of RPL22L1 and RPS21 were significantly higher in high Gleason grade than they were in low Gleason grade. In addition, in vitro studies were undertaken to evaluate the roles of RPL22L1 and RPS21 in PCa. The results revealed that these genes promote PCa cell proliferation, migration and invasion, and inhibit PCa cell apoptosis. Taken together, these data showed that RPL22L1 and RPS21 exhibited higher expression in human prostate cancer tissue, and involved in PCa cell proliferation and invasion. This research provided a novel insight into diagnostic and prognostic biomarkers for PCa.

MiR-129-5p inhibits prostate cancer proliferation via targeting ETV1.

BackgroundProstate cancer is one of the most commonly diagnosed diseases in males.MethodsRT-qPCR was used to detect miR-129-5p expression in tumor tissues and adjacent normal tissues from patients with prostate cancer. The cell proliferation assay and colony forming assay were used to study the role of miR-129-5p in mediating prostate cancer cell growth. Bioinformatic analysis and dual luciferase assay were performed to predict and confirm ETV1 as a target gene of miR-129-5p.ResultsWe found that miR-129-5p levels were decreased significantly in human prostate cancer tissues compared with matched normal tissues from patients with prostate cancer. Overexpression of miR-129-5p suppressed prostate cancer cell growth while antagonist of miR-129-5p promoted cell proliferation in immortal prostate cell line RWPE-1. In addition, elevation of miR-129-5p decreased ETV1 expression in prostate cancer cells while downregulation of miR-129-5p increased ETV1 expression in RWPE-1. Mechanistically, ETV1 is confirmed a direct target of miR-129-5p in prostate cancer cells. Through repression of ETV1 expression, miR-129-5p could inactivate YAP signaling in prostate cancer cells. In addition, overexpression of ETV1 attenuated miR-129-5p induced cell proliferation in prostate cancer cells. Correlation analysis further revealed that there was a negative correlation between miR-129-5p levels and ETV1 mRNA levels in tumor tissues from patients with prostate cancer.ConclusionOur results identified miR-129-5p as a tumor suppressor in prostate cancer via repression of ETV1.

Targeting Tumor Markers with Antisense Peptides: An Example of Human Prostate Specific Antigen

The purpose of this paper was to outline the development of short peptide targeting of the human prostate specific antigen (hPSA), and to evaluate its effectiveness in staining PSA in human prostate cancer tissue. The targeting of the hPSA antigen by means of antisense peptide AVRDKVG was designed according to a three-step method involving: 1. The selection of the molecular target (hPSA epitope), 2. the modeling of an antisense peptide (paratope) based on the epitope sequence, and 3. the spectroscopic evaluation of sense–antisense peptide binding. We then modified standard hPSA immunohistochemical staining practice by using a biotinylated antisense peptide instead of the standard monoclonal antibody and compared the results of both procedures. Immunochemical testing on human tissue showed the applicability of the antisense peptide technology to human molecular targets. This methodology represents a new approach to deriving peptide ligands and potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines.