Prostate Cancer Specimens Research Articles

Nucleophosmin 1 cooperates with BRD4 to facilitate c-Myc transcription to promote prostate cancer progression

Nucleophosmin 1 (NPM1) is a multifunctional protein that promotes tumor progression in various cancers and is associated with a poor prognosis of prostate cancer (PCa). However, the mechanism by which NPM1 exerts its malignant potential in PCa remains elusive. Here, we showed that NPM1 is overexpressed in PCa cell lines and tissues and that the dysregulation of NPM1 promotes PCa proliferation. We also demonstrated that NPM1 transcriptionally upregulates c-Myc expression in PCa cells that is diminished by blockade of bromodomain-containing protein 4 (BRD4). Furthermore, we detected a correlation between NPM1 and c-Myc in patient PCa specimens. Mechanistically, NPM1 influences and cooperates with BRD4 to facilitate c-Myc transcription to promote PCa progression. In addition, JQ1, a bromodomain and extra-terminal domain (BET) inhibitor, in combination with NPM1 inhibition suppresses PCa progression in vitro and in vivo. These results indicate that NPM1 promotes PCa progression through a c-Myc -mediated pathway via BRD4, and blockade of the NPM1–c-Myc oncogenic pathway may be a therapeutic strategy for PCa.

Anillin actin-binding protein expression correlates with poor prognosis for prostate cancer patients

ObjectiveOctamer transcription factor 1 (OCT1), a transcription factor that interacts with androgen receptor, is involved in prostate cancer (PCa) progression. The OCT1 target gene, anillin actin-binding protein (ANLN), is highly expressed in castration-resistant PCa tissue; however, it remains unclear whether ANLN expression in hormone-sensitive PCa tissue could be used as a predictive biomarker for poor prognosis of patients. We aimed to investigate ANLN expression in PCa tissue obtained via radical prostatectomy. MethodsImmunohistochemical staining for ANLN was performed on 86 PCa specimens, followed by evaluation using immunoreactivity (IR) scores. Prognosis was analyzed by the log-rank test using the Kaplan–Meier method to generate a cancer-specific survival curve. The correlations between ANLN IR and clinical parameters as well as OCT1 IR were analyzed using the Chi-squared test. ResultsThe median IR score was 0 for ANLN. Accordingly, given the low median IR score, an IR score of ≥3 was defined as positive. There were 17 (19.8%) ANLN-positive cases, and these cases had a significantly poorer prognosis. Multivariate analysis revealed that the Gleason score, pathologic T and N stages, and positive ANLN expression were significant predictors of poor prognosis. Notably, patients with both positive ANLN and high OCT1 expression had a significantly decreased overall survival (p=0.001). ConclusionANLN, which is a OCT1 target gene especially in castration-resistant PCa, is expressed in a small number of hormone-sensitive PCa cases. Both positive ANLN expression and high OCT1 expression are significantly correlated with poor prognosis for PCa patients.

Melittin inhibits tumor cell migration and enhances cisplatin sensitivity by suppressing IL-17 signaling pathway gene LCN2 in castration-resistant prostate cancer.

Melittin is a small molecule polypeptide extracted from the abdominal cavity of bees, which is used to treat inflammatory diseases and relieve pain. However, the antitumor effect of melittin and its mechanisms remain unclear, especially in castration-resistant prostate cancer (CRPC). Through CCK-8 assay, colony formation assay, wound healing assay and Transwell migration assay, we explored the effect of melittin on CRPC cell lines. In addition, with microarray analysis, gene ontology analysis and kyoto encyclopedia of genes and genomes analysis, this study identified key genes and signaling pathways that influence the growth of PC-3 cells. Meanwhile, the effect of melittin on CRPC was also verified through subcutaneous tumor formation experiments. Finally, we also tested the relevant indicators of human prostate cancer (PCa) specimens through immunohistochemistry and H＆E stating. Here, melittin was verified to inhibit the cell proliferation and migration of CPRC. Moreover, RNA-sequence analysis demonstrated that Interleukin-17 (IL-17) signaling pathway gene Lipocalin-2 (LCN2) was downregulated by melittin treatment in CRPC. Further investigation revealed that overexpression of LCN2 was able to rescue tumor suppression and cisplatin sensitivity which melittin mediated. Interestingly, the expression of LCN2 is highly related to metastasis in PCa. In brief, our study indicates that LCN2 plays an oncogenic role in CRPC and melittin may be selected as an attractive candidate for CRPC therapy.

Unravelling the Role of P300 and TMPRSS2 in Prostate Cancer: A Literature Review.

Prostate cancer is one of the most common malignant diseases in men, and it contributes significantly to the increased mortality rate in men worldwide. This study aimed to review the roles of p300 and TMPRSS2 (transmembrane protease, serine 2) in the AR (androgen receptor) pathway as they are closely related to the development and progression of prostate cancer. This paper represents a library-based study conducted by selecting the most suitable, up-to-date scientific published articles from online journals. We focused on articles that use similar techniques, particularly those that use prostate cancer cell lines and immunohistochemical staining to study the molecular impact of p300 and TMPRSS2 in prostate cancer specimens. The TMPRSS2:ERG fusion is considered relevant to prostate cancer, but its association with the development and progression as well as its clinical significance have not been fully elucidated. On the other hand, high p300 levels in prostate cancer biopsies predict larger tumor volumes, extraprostatic extension of disease, and seminal vesicle involvement at prostatectomy, and may be associated with prostate cancer progression after surgery. The inhibition of p300 has been shown to reduce the proliferation of prostate cancer cells with TMPRSS2:ETS (E26 transformation-specific) fusions, and combining p300 inhibitors with other targeted therapies may increase their efficacy. Overall, the interplay between the p300 and TMPRSS2 pathways is an active area of research.

Collagen VI deposition mediates stromal T cell trapping through inhibition of T cell motility in the prostate tumor microenvironment

The tumor extracellular matrix (ECM) is a barrier to anti-tumor immunity in solid tumors by disrupting T cell-tumor cell interaction underlying the need for elucidating mechanisms by which specific ECM proteins impact T cell motility and activity within the desmoplastic stroma of solid tumors. Here, we show that Collagen VI (Col VI) deposition correlates with stromal T cell density in human prostate cancer specimens. Furthermore, motility of CD4+ T cells is completely ablated on purified Col VI surfaces when compared with Fibronectin and Collagen I. Importantly, T cells adhered to Col VI surfaces displayed reduced cell spreading and fibrillar actin, indicating a reduction in traction force generation accompanied by a decrease in integrin β1 clustering. We found that CD4+ T cells largely lack expression of integrin α1 in the prostate tumor microenvironment and that blockade of α1β1 integrin heterodimers inhibited CD8+ T cell motility on prostate fibroblast-derived matrix, while re-expression of ITGA1 improved motility. Taken together, we show that the Col VI-rich microenvironment in prostate cancer reduces the motility of CD4+ T cells lacking integrin α1, leading to their accumulation in the stroma, thus putatively inhibiting anti-tumor T cell responses.

Abstract B030: Spatially-resolved cell-cell interactions between tumor microenvironment and immunologically “hot” and “cold” locally advanced prostate tumors

Abstract Background: Immune-checkpoint inhibitors (ICI) such as anti-PD-L1 have shown promise in solid tumors with high mutational burden and immune infiltration such as melanoma. However, ICI have shown have only shown mild success in prostate cancer. One likely explanation is that majority of prostate cancer have low mutational burden leading to poor immune infiltration. However, exceptions to the immune infiltration have been observed in a subset of locally advanced prostate cancer specimens. We hypothesized that specific immune subsets, particular myeloid progenitor cells, drive microenvironmental differences between hot and cold tumors. Methods: A total of four formalin-fixed paraffin-embedded tissue punch samples were obtained from biobank at UCLA and were sectioned at 5µm thickness. The sections were deparaffinized and de-crosslinked per manufacture’s instructions, followed by spatial transcriptome profiling using the Visium (10x Genomics) platform. Libraries were constructed from RNA and sequenced on Illumina platform. The reads were processed using Spaceranger. Spot level read counts were first filtered by gene detection rate, total gene count and mitochondrial content. Spots passing QC were normalized and clustered based on the top 2000 most highly variable genes. Cell-cell communication was assessed using the LIANA ligand-receptor analysis framework. To identify regions of inflammation, cell type deconvolution was performed using SpatialDWLS (dampened weighted least squares) and gene-set enrichment was performed using ssgsea from GSVA package. All analysis and statistics were perform using R version 4.1. Results: The number of spots passed QC ranged from approximately 3400 – 4000. Clustering by gene expression resulted in 14 – 23 clusters that showed high concordance with expert pathologist annotation of tumor and tumor subtype mapping to intraductal carcinoma and invasive carcinoma. Cell-type deconvolution by SpatialDWLS found that different tissue regions harbored approximately 1.5-4.5% of immune cells. The myeloid fraction was 0.8%- 3% and T-cells of total immune cells, representing 0.4% - 0.9% of the total cell population. These regions with myeloid cell enrichment were associated with elevated interferon alpha and gamma response, as well as IL2-STAT5 signaling in geographically proximal regions. Conclusion: The presence of myeloid cells in the microenvironments of immunologically “hot” tumors may represent a major factor contributing to immunosuppression in tumors predicted to respond to ICI. Ongoing efforts profiling “hot” tumors will assess the feasibility of modulating myeloid cells in order to recruit cytotoxic T cells. Citation Format: Anson Ku, Rong Rong Huang, Huihui Ye, Adam Sowalsky. Spatially-resolved cell-cell interactions between tumor microenvironment and immunologically “hot” and “cold” locally advanced prostate tumors [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B030.

Abstract A049: Immunohistochemical detection of ERG, ETV1, ETV4, and PTEN in prostate tumor specimens reveals the heterogeneity of multi-focal prostate cancer

Abstract Introduction: ETS gene rearrangements involving ERG, ETV1 and ETV4, as well as PTEN deletions are among the most common genomic alterations in prostate cancer. Although these events are found to be mutually exclusive, the multifocality of prostate cancer suggests that they may co-occur in some tumors. The immunohistochemistry (IHC) detection of both ERG overexpression and PTEN protein loss are reliable substitutes for fluorescence in situ hybridization (FISH) assay to probe genomic alterations. The lack of specific antibodies that can discriminate between multiple ETS factors, however, has limited the evaluation for the co-occurrence of these genomic alterations in prostate cancer. Improving our ability to identify the multifocality or inter-focal heterogeneity of tumors can enhance our understanding of biological mechanisms of tumor initiation and progression, and help stratify patients for treatment. Methods: Using monoclonal antibodies (mAbs) that we developed against ETV1 and ETV4, we examined their expression in relation to ERG and PTEN expression by IHC using two TMAs constructed from primary prostate cancer specimens of independent patient cohorts: one was constructed from multiple 1 mm cores representing distinct tumor focus from multifocal tumors from 50 African American (AA) and 50 Caucasian American (CA) men; the other, from a single 2 mm core of individual tumors from a cohort of 152 AA and 304 CA men. Positive expression for each ETS factor and PTEN were evaluated for association with race and clinico-pathologic features that include PSA at diagnosis, Gleason score, pathologic T stage, biochemical recurrence, and metastasis. Univariate analysis of BCR free survival as a function of ERG, ETV1 and ETV4 and PTEN expression was performed for both patient cohorts. Results: ERG(+) expression alone or in combination with PTEN loss were significantly higher in CA men in both patient cohorts. When expression of ETS factors were evaluated together with PTEN status, positive expression of ETS factors and PTEN loss were significantly more prevalent among CA men in both patient cohorts ETV1 and ETV4 expression were detected at 5% or less in both patient cohorts. Cases harboring tumor foci that were positive for more than one marker evaluated frequently showed either intra-tumor or inter-tumor heterogeneity. Univariate analysis of PTEN-ETS protein expression status and BCR free survival showed that patients with loss of PTEN expression trends towards poorer BCR free survival regardless of ETS expression status. Conclusion: We showed that the IHC detection of multiple aberrantly expressed proteins such as ERG, ETV1, ETV4 and PTEN in prostate tumor specimens using highly specific mAbs can reveal the heterogeneity and multi-focality in prostate cancer, which could be used to enhance the stratification of patients for novel treatment strategies. Citation Format: Shyh-Han Tan, Cara Schafer, Denise Young, Jiji Jiang, Yingjie Song, Albert Dobi, Gyorgy Petrovics, Bettina F. Drake, Gregory T. Chesnut, Isabell A. Sesterhenn. Immunohistochemical detection of ERG, ETV1, ETV4, and PTEN in prostate tumor specimens reveals the heterogeneity of multi-focal prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A049.

Abstract IA003: Evolution of AR gene structure during prostate cancer progression

Abstract Castration-resistant prostate cancer (CRPC) emerges when endocrine therapies are no longer effective at suppressing activity of the androgen receptor (AR) transcription factor. Our laboratory has implemented DNA sequencing approaches to identify genomic mechanisms contributing to CRPC in prostate cancer cell lines, patient-derived xenografts (PDXs), and clinical specimens. These approaches have revealed AR as one of the most frequently-rearranged genes in CRPC. However the patterns of AR gene rearrangements observed between patients and even between anatomically distinct metastases within a single patient are remarkably complex and heterogeneous. To address this complexity, we analyzed a cohort of prostate cancer specimens using linked-read DNA-sequencing. This approach found that a subset of clinical CRPC metastases had accumulated complex AR rearrangements in conjunction with very high AR copy number, a phenomenon that also occurred in PDX models grown under selective pressure of castration. Using orthogonal approaches of optical genome mapping and fluorescence in situ hybridization, we found that AR residing on extrachromosomal circular DNA (eccDNA) was an underlying mechanism. This study identifies eccDNA as an engine for accumulation of AR gene copy number and structural complexity during CRPC progression. Citation Format: Scott M Dehm. Evolution of AR gene structure during prostate cancer progression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr IA003.

Abstract A001: Differential alternative RNA splicing and transcription events between Black and White prostate cancer patients involve genes promoting cancer aggressiveness and associate with patient survival

Abstract Black patients suffer disproportionately from prostate cancer (PCa) progression to lethal disease compared with Asian and White patients. Comparing the molecular landscape of PCa in Black and White men has the potential to identify targets for development of new precision medicine interventions. Alternative RNA splicing and transcription events (ARS/Ts) can alter gene function. Cancer may exploit ARS/Ts to enhance proliferation, metastasis, and drug resistance. Here, we isolated RNA from fresh frozen PCa specimens and tumor-associated normal (TAN) specimens from 35 self-reported Black and 37 self-reported White patients diagnosed with Gleason-high or -low PCa and undergoing radical prostatectomy or biopsy. Analysis of follow-up clinical data for patients participating in our study show an increased hazard ratio of PCa progression among Black patients compared to White patients (HR = 2.13 and p < 0.05). We performed comparative transcriptomic analysis between Gleason-high or -low tumor and TAN samples among Blacks, among Whites, or between Blacks and Whites with Gleason-high or –low PCa. Among differential ARS/Ts identified between tumors and TANs among Blacks, 532 are unique to Gleason-high and 673 are unique to Gleason-low. Among differential ARS/Ts between Blacks and Whites, 360 are unique to Gleason-high and 242 are unique to Gleason-low. Interestingly, we found that even in TANs, there are 802 differential ARS/Ts between Blacks and Whites, with 459 and 343 in Gleason-high and Gleason-low, respectively. Among the genes undergoing differential ARS/Ts between Blacks and Whites are FGFR1, AKT1, H6PD, MDM2, and RAD51D in Gleason-high and CDK7 and VEGFA in Gleason-low. Notably, the number of genes undergoing ARS/Ts between Blacks and Whites with Gleason-high or -low tumors was higher than the number of genes undergoing differential aggregate gene expression between Blacks and Whites. Using gene set enrichment analysis, we found that genes undergoing ARS/Ts function in cancer relevant pathways. For example, the genes undergoing ARS/Ts between Blacks and Whites with Gleason-high or -low tumors were enriched in spermatogenesis and P53 hallmark pathways, respectively. Importantly, survival analysis shows a number of differential ARS/Ts between Blacks and Whites with Gleason-high or -low tumors are individually associated with progression-free survival. In summary, we have investigated the biology of PCa and TAN tissue between Black and White patients at the exon level. A number of genes undergoing newly identified differential ARS/Ts between Blacks and Whites are in genes reported to promote cancer aggressiveness and a number are in genes whose function in cancer was unreported prior to our findings, and a number are associated with patient progression-free survival. Our findings have identified novel targets with potential for precision oncology for PCa. Estimation of genetic ancestry of study participants and in vitro and in vivo validation of prioritized targets for PCa biology are underway. Citation Format: Muthana Al Abo, Wen-Chi Foo, Lauren Howard, Monika Anand, Daniel J. George, Steven R. Patierno, Jennifer A Freedman. Differential alternative RNA splicing and transcription events between Black and White prostate cancer patients involve genes promoting cancer aggressiveness and associate with patient survival [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A001.

Targeting PHB1 to inhibit castration-resistant prostate cancer progression in vitro and in vivo

BackgroundCastration-resistant prostate cancer (CRPC) is currently the main challenge for prostate cancer (PCa) treatment, and there is an urgent need to find novel therapeutic targets and drugs. Prohibitin (PHB1) is a multifunctional chaperone/scaffold protein that is upregulated in various cancers and plays a pro-cancer role. FL3 is a synthetic flavagline drug that inhibits cancer cell proliferation by targeting PHB1. However, the biological functions of PHB1 in CRPC and the effect of FL3 on CRPC cells remain to be explored.MethodsSeveral public datasets were used to analyze the association between the expression level of PHB1 and PCa progression as well as outcome in PCa patients. The expression of PHB1 in human PCa specimens and PCa cell lines was examined by immunohistochemistry (IHC), qRT-PCR, and Western blot. The biological roles of PHB1 in castration resistance and underlying mechanisms were investigated by gain/loss-of-function analyses. Next, in vitro and in vivo experiments were conducted to investigate the anti-cancer effects of FL3 on CRPC cells as well as the underlying mechanisms.ResultsPHB1 expression was significantly upregulated in CRPC and was associated with poor prognosis. PHB1 promoted castration resistance of PCa cells under androgen deprivation condition. PHB1 is an androgen receptor (AR) suppressive gene, and androgen deprivation promoted the PHB1 expression and its nucleus-cytoplasmic translocation. FL3, alone or combined with the second-generation anti-androgen Enzalutamide (ENZ), suppressed CRPC cells especially ENZ-sensitive CRPC cells both in vitro and in vivo. Mechanically, we demonstrated that FL3 promoted trafficking of PHB1 from plasma membrane and mitochondria to nucleus, which in turn inhibited AR signaling as well as MAPK signaling, yet promoted apoptosis in CRPC cells.ConclusionOur data indicated that PHB1 is aberrantly upregulated in CRPC and is involved in castration resistance, as well as providing a novel rational approach for treating ENZ-sensitive CRPC.

Co-expression and clinical utility of AR-FL and AR splice variants AR-V3, AR-V7 and AR-V9 in prostate cancer

BackgroundAndrogen receptor (AR) splice variants (AR-Vs) have been discussed as a biomarker in prostate cancer (PC). However, some reports question the predictive property of AR-Vs. From a mechanistic perspective, the connection between AR full length (AR-FL) and AR-Vs is not fully understood. Here, we aimed to investigate the dependence of AR-FL and AR-V expression levels on AR gene activity. Additionally, we intended to comprehensively analyze presence of AR-FL and three clinically relevant AR-Vs (AR-V3, AR-V7 and AR-V9) in different stages of disease, especially with respect to clinical utility in PC patients undergoing AR targeted agent (ARTA) treatment.MethodsAR-FL and AR-V levels were analyzed in PC and non-PC cell lines upon artificial increase of AR pre-mRNA using either drug treatment or AR gene activation. Furthermore, expression of AR-FL and AR-Vs was determined in PC specimen at distinct stages of disease (primary (n = 10) and metastatic tissues (n = 20), liquid biopsy samples (n = 422), mCRPC liquid biopsy samples of n = 96 patients starting novel treatment). Finally, baseline AR-FL and AR-V status was correlated with clinical outcome in a defined cohort of n = 65 mCRPC patients undergoing ARTA treatment.ResultsWe revealed rising levels of AR-FL accompanied with appearance and increase of AR-Vs in dependence of elevated AR pre-mRNA levels. We also noticed increase in AR-FL and AR-V levels throughout disease progression. AR-V expression was always associated with high AR-FL levels without any sample being solely AR-V positive. In patients undergoing ARTA treatment, AR-FL did show prognostic, yet not predictive validity. Additionally, we observed a substantial clinical response to ARTA treatment even in AR-V positive patients. Accordingly, multivariate analysis did not demonstrate independent significance of AR-Vs in neither predictive nor prognostic clinical utility.ConclusionWe demonstrate a correlation between AR-FL and AR-V expression during PC progression; with AR-V expression being a side-effect of elevated AR pre-mRNA levels. Clinically, AR-V positivity relies on high levels of AR-FL, making cells still vulnerable to ARTA treatment, as demonstrated by AR-FL and AR-V positive patients responding to ARTA treatment. Thus, AR-FL and AR-V might be considered as a prognostic, yet not predictive biomarker in mCRPC patients.

MP20-03 microRNA-15b-5p/ MUSCARINIC RECEPTORS/ YAP SIGNALING CONTRIBUTES TO CASTRATION-RESISTANT GROWTH OF PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023MP20-03 microRNA-15b-5p/ MUSCARINIC RECEPTORS/ YAP SIGNALING CONTRIBUTES TO CASTRATION-RESISTANT GROWTH OF PROSTATE CANCER Yusuke Goto, Shunichi Asai, Shinichi Sakamoto, Tomokazu Sazuka, Yusuke Imamura, Naohiko Seki, and Tomohiko Ichikawa Yusuke GotoYusuke Goto More articles by this author , Shunichi AsaiShunichi Asai More articles by this author , Shinichi SakamotoShinichi Sakamoto More articles by this author , Tomokazu SazukaTomokazu Sazuka More articles by this author , Yusuke ImamuraYusuke Imamura More articles by this author , Naohiko SekiNaohiko Seki More articles by this author , and Tomohiko IchikawaTomohiko Ichikawa More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate cancer (PCa) innervation contributes to the progression of PCa. However, the precise impact of innervation on PCa cells is still poorly understood. We have recently shown that muscarinic receptors promote castration-resistant growth of PCa through FAK-YAP signaling axis. However, mechanisms of upregulation of muscarinic receptors leading to castration-resistant growth of PCa cells were unclear. METHODS: Small RNA-sequencing and bioinformatics screening were used to identify candidate microRNAs to regulate muscarinic receptors. Luciferase reporter assay was performed to see direct regulation of muscarinic receptors by microRNAs. Western blot and qRT-PCR verified the effect of the microRNA on downstream YAP signaling. Cell proliferation and sphere assay demonstrated the effect of microRNA on the muscarinic-receptor-driven castration-resistant growth of PCa. RESULTS: Small RNA-seq showed that 255 microRNAs were significantly downregulated under castrated condition in LNCaP cells. Using bioinformatic approach, microRNA-15b-5p was found to be one of the candidate microRNAs that directly regulate m3. microRNA-15b-5p was confirmed to downregulate with castrate condition, in contrast to the upregulation of muscarinic receptor 3 (m3) with castration in vitro. Indeed, microRNA-15b-5p was downregulated in castration-resistant PCa (CRPC) compared with hormone-sensitive PCa (HSPC) specimens. Moreover, microRNA-15b-5p directly binds to m3, and microRNA-15b-5p inhibited YAP activation which was induced by m3 stimulation. Furthermore, microRNA-15b-5p abolished castration-resistant growth of PCa cells which was induced by m3 stimulation, in 2D cell proliferation assay and 3D cell sphere assay. CONCLUSIONS: microRNA-15b-5p/m3/YAP signaling axis is a novel pathway which promotes castration-resistant growth of PCa. Our findings provide novel mechanisms of muscarinic-signal-driven CRPC progression. Source of Funding: JSPS KAKENHI Grant Number: 21K16753 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e276 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yusuke Goto More articles by this author Shunichi Asai More articles by this author Shinichi Sakamoto More articles by this author Tomokazu Sazuka More articles by this author Yusuke Imamura More articles by this author Naohiko Seki More articles by this author Tomohiko Ichikawa More articles by this author Expand All Advertisement PDF downloadLoading ...

Senescent Tumor Cells Are Frequently Present at the Invasion Front: Implications for Improving Disease Control in Patients with Locally Advanced Prostate Cancer

Introduction: Local tumor invasion is a critical factor for the outcome of men with prostate cancer. In particular, seminal vesicle invasion (SVI) has been reported to be associated with a more unfavorable prognosis. A better understanding of the functional state of invading prostate cancer cells is crucial to develop novel therapeutic strategies for patients with locally advanced disease. Methods: The prognostic impact of local tumor progression was ascertained in over 1,000 men with prostate cancer. Prostate cancer specimens were stained by double-immunohistochemistry for the proliferation marker Ki-67 and the senescence marker p16^INK4A. The migratory properties of senescent prostate cancer cells were analyzed in vitro using a wound healing assay and immunofluorescence microscopy for p16^INK4A. Results: We confirm the notion that patients with SVI have a more unfavorable prognosis than patients with extraprostatic extension alone. Surprisingly, we found that the tumor invasion front frequently harbors p16^INK4A-positive and Ki-67-negative, i.e., senescent, tumor cells. While the intraprostatic tumor periphery was a hotspot for both proliferation and expression of p16^INK4A, the area of SVI showed less proliferative activity but was at the same time a hotspot of cells with increased nuclear p16^INK4A expression. Senescence was associated with an accelerated migration of prostate cancer cells in vitro. Conclusion: This proof-of-concept study shows that invading prostate cancer cells frequently show signs of cellular senescence. This finding may open new avenues for neoadjuvant and adjuvant treatment concepts in men with locally advanced prostate cancer.

MiR-124-3p inhibits tumor progression in prostate cancer by targeting EZH2

Prostate cancer (PCa) is widespread cancer with significant morbidity and mortality rates. MicroRNAs (miRNAs) have been identified as important post-transcriptional modulators in various malignancies. This study investigated the miR-124-3p effect on PCa cell proliferation, infiltration, and apoptosis. EZH2 and miR-124-3p expression levels were measured in PCa tissues. PCa cell lines DU145 and PC3 were transfected with miR-124-3p inhibitors or analogs. EZH2 and miR-124-3p linkage was validated by conducting the luciferase enzyme reporter test. The cell viability and apoptosis were assessed by flow cytometry and MTT test. Cell movement was noted during infiltration using transwell assays. EZH2, AKT, and mTOR contents were assessed using qRT-PCR and western blotting. In clinical PCa specimens, miR-124-3p and EZH2 contents were inversely correlated. Further research has demonstrated that EZH2 is the miR-124-3p direct target. Furthermore, miR-124-3p overexpression reduced EZH2 levels and lowered cell viability, infiltration, and promoted cell death, whereas miR-124-3p silencing had the opposite effect. Overexpression of miR-124-3p decreased the phosphorylation level of AKT and mTOR, whereas miR-124-3p downregulation produced the opposite result. Our findings depict that miR-124-3p prevents PCa proliferative and invasive processes while promoting apoptosis by targeting EZH2.

ARPC5 is transcriptionally activated by KLF4, and promotes cell migration and invasion in prostate cancer via up-regulating ADAM17 : ARPC5 serves as an oncogene in prostate cancer.

Prostate cancer (PCa) is one of the most common cancers in men worldwide. Actin-related protein 2/3 complex subunit 5 (ARPC5) has been validated as a critical regulator in several kinds of human tumors. However, whether ARPC5 is implicated in PCa progression remains largely unknown. PCa specimens and PCa cell lines were obtained for detecting gene expressions using western blot and quantitative reverse transcriptase PCR (qRT-PCR). PCa cells transfected with ARPC5 shRNA or a disintegrin and metalloprotease 17 (ADAM17) overexpressed plasmids were harvested for assessing cell proliferation, migration and invasion by using cell counting kit-8 (CCK-8), colony formation and transwell assays, respectively. The interaction relationship between molecules was testified with chromatin immunoprecipitation and luciferase reporter assay. Xenograft mice model was conducted for confirming the role of ARPC5/ADAM17 axis in vivo. Upregulated ARPC5 was observed in PCa tissues and cells, as well as forecasted poor prognosis of PCa patients. Depletion of ARPC5 inhibited PCa cell proliferation, migration and invasion. Krüppel-like factor 4 (KLF4) was identified to be a transcriptional activator of ARPC5 via binding with its promoter region. Furthermore, ADAM17 served as a downstream effector of ARPC5. ADAM17 overexpression overturned ARPC5 knockdown-induced repressive impacts on PCa progression in vitro and in vivo. Collectively, ARPC5 was activated by KLF4 and upregulated ADAM17 to promote PCa progression, which might act as a promising therapeutic target and prognostic biomarker for PCa.

Synchronous bladder and prostate cancer specimens obtained from radical cystoprostatectomy: A single‑center retrospective analysis.

The aim of the present study was to analyze incidence, histopathological features and clinical outcomes of patients undergoing radical cystoprostatectomy (RCP) for bladder cancer, in which incidental prostate cancer (PCa) was found. How these types of cancer impacted the patients' management and whether prostate-sparing cystectomy could be an option for these patients was determined. The current study retrospectively analyzed the data of a cohort of patients from 'Umberto I' Hospital of Nocera Inferiore who underwent RCP for bladder transitional cell carcinoma. Patients with a preoperative diagnosis or clinical suspicion of PCa were excluded. Patients affected by incidental PCa in the RCP specimens were identified, and then their demographic, histopathological and clinical outcome data were collected. Overall, it was revealed that of the 303 patients undergoing RCP for bladder cancer, 69 (22.7%) had incidental PCa, with a median age of 71.6 (age range, 54-89 years). In total, 23 (33.33%) of the 69 patients with incidental PCa were considered to have clinically significant prostate disease. In conclusion, it was relatively common to identify incidental PCa in RCP specimens but no preoperative predictive factors were identified that were able to determine 'non-aggressive' PCa status. Therefore, the present results demonstrate the need for a careful and complete prostate removal during RCP. Nevertheless, since organ-sparing surgeries are widely performed in young population, due to the impossibility of predicting aggressive prostate cancer, these patients require close monitoring through lifelong PSA surveillance, particularly focusing on the possible relapse of PCa after RCP.

Membrane dual-targeting probes: A promising strategy for fluorescence-guided prostate cancer surgery and lymph node metastases detection.

Fluorescence-guided surgery (FGS) with tumor-targeted imaging agents, particularly those using the near-infrared wavelength, has emerged as a real-time technique to highlight the tumor location and margins during a surgical procedure. For accurate visualization of prostate cancer (PCa) boundary and lymphatic metastasis, we developed a new approach involving an efficient self-quenched near-infrared fluorescence probe, Cy-KUE-OA, with dual PCa-membrane affinity. Cy-KUE-OA specifically targeted the prostate-specific membrane antigen (PSMA), anchored into the phospholipids of the cell membrane of PCa cells and consequently showed a strong Cy7-de-quenching effect. This dual-membrane-targeting probe allowed us to detect PSMA-expressing PCa cells both invitro and invivo and enabled clear visualization of the tumor boundary during fluorescence-guided laparoscopic surgery in PCa mouse models. Furthermore, the high PCa preference of Cy-KUE-OA was confirmed on surgically resected patient specimens of healthy tissues, PCa, and lymph node metastases. Taken together, our results serve as a bridge between preclinical and clinical research in FGS of PCa and lay a solid foundation for further clinical research.

TEAD3 inhibits the proliferation and metastasis of prostate cancer via suppressing ADRBK2

TEAD3 acts as a transcription factor in many tumors to promote tumor occurrence and development. But in prostate cancer (PCa), it appears as a tumor suppressor gene. Recent studies have shown that this may be related to subcellular localization and posttranslational modification.We found that TEAD3 was down-expressed in PCa. Immunohistochemistry of clinical PCa specimens confirmed that TEAD3 expression was the highest in benign prostatic hyperplasia (BPH) tissues, followed by primary PCa tissues, and the lowest in metastatic PCa tissues, and its expression level was positively correlated with overall survival. MTT assay, clone formation assay, and scratch assay confirmed that overexpression of TEAD3 could significantly inhibit the proliferation and migration of PCa cells. Next-generation sequencing results indicated that Hedgehog (Hh) signaling pathway was significantly inhibited after overexpression of TEAD3. Rescue assays suggested that ADRBK2 could reverse the proliferation and migration ability caused by overexpression of TEAD3.TEAD3 is downregulated in PCa and associated with poor patient prognosis. Overexpression of TEAD3 inhibits the proliferation and migration ability of PCa cells via restraining the mRNA level of ADRBK2.These results indicate that TEAD3 was down-expressed in PCa patients and was positively correlated with a high Gleason score and poor prognosis. Mechanistically, we found that the upregulation of TEAD3 inhibits the proliferation and metastasis of prostate cancer by inhibiting the expression of ADRBK2.

Prostate cancer cell-platelet bidirectional signaling promotes calcium mobilization, invasion and apoptotic resistance via distinct receptor-ligand pairs

Platelets play a crucial role in cancer and thrombosis. However, the receptor-ligand repertoire mediating prostate cancer (PCa) cell-platelet interactions and ensuing consequences have not been fully elucidated. Microvilli emanating from the plasma membrane of PCa cell lines (RC77 T/E, MDA PCa 2b) directly contacted individual platelets and platelet aggregates. PCa cell-platelet interactions were associated with calcium mobilization in platelets, and translocation of P-selectin and integrin αIIbβ3 onto the platelet surface. PCa cell-platelet interactions reciprocally promoted PCa cell invasion and apoptotic resistance, and these events were insensitive to androgen receptor blockade by bicalutamide. PCa cells were exceedingly sensitive to activation by platelets in vitro, occurring at a PCa cell:platelet coculture ratio as low as 1:10 (whereas PCa patient blood contains 1:2,000,000 per ml). Conditioned medium from cocultures stimulated PCa cell invasion but not apoptotic resistance nor platelet aggregation. Candidate transmembrane signaling proteins responsible for PCa cell-platelet oncogenic events were identified by RNA-Seq and broadly divided into 4 major categories: (1) integrin-ligand, (2) EPH receptor-ephrin, (3) immune checkpoint receptor-ligand, and (4) miscellaneous receptor-ligand interactions. Based on antibody neutralization and small molecule inhibitor assays, PCa cell-stimulated calcium mobilization in platelets was found to be mediated by a fibronectin1 (FN1)-αIIbβ3 signaling axis. Platelet-stimulated PCa cell invasion was facilitated by a CD55-adhesion G protein coupled receptor E5 (ADGRE5) axis, with contribution from platelet cytokines CCL3L1 and IL32. Platelet-stimulated PCa cell apoptotic resistance relied on ephrin-EPH receptor and lysophosphatidic acid (LPA)-LPA receptor (LPAR) signaling. Of participating signaling partners, FN1 and LPAR3 overexpression was observed in PCa specimens compared to normal prostate, while high expression of CCR1 (CCL3L1 receptor), EPHA1 and LPAR5 in PCa was associated with poor patient survival. These findings emphasize that non-overlapping receptor-ligand pairs participate in oncogenesis and thrombosis, highlighting the complexity of any contemplated clinical intervention strategy.

Clinical and molecular significance of the RNA m6A methyltransferase complex in prostate cancer.

N6-methyladenosine (m6A) is the most abundant internal mRNA modification and is dynamically regulated through distinct protein complexes that methylate, demethylate, and/or interpret the m6A modification. These proteins, and the m6A modification, are involved in the regulation of gene expression, RNA stability, splicing and translation. Given its role in these crucial processes, m6A has been implicated in many diseases, including in cancer development and progression. Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in men and recent studies support a role for m6A in PCa. Despite this, the literature currently lacks an integrated analysis of the expression of key components of the m6A RNA methyltransferase complex, both in PCa patients and in well-established cell line models. For this reason, this study used immunohistochemistry and functional studies to investigate the mechanistic and clinical significance of the METTL3, METTL14, WTAP and CBLL1 components of the m6A methyltransferase complex in PCa specimens and cell lines. Expression of METTL3 and CBLL1, but not METTL14 and WTAP, was associated with poorer PCa patient outcomes. Expression of METTL3, METTL14, WTAP and CBLL1 was higher in PCa cells compared with non-malignant prostate cells, with the highest expression seen in castrate-sensitive, androgen-responsive PCa cells. Moreover, in PCa cell lines, expression of METTL3 and WTAP was found to be androgen-regulated. To investigate the mechanistic role(s) of the m6A methyltransferase complex in PCa cells, short hairpin RNA (shRNA)-mediated knockdown coupled with next generation sequencing was used to determine the transcriptome-wide roles of METTL3, the catalytic subunit of the m6A methyltransferase complex. Functional depletion of METTL3 resulted in upregulation of the androgen receptor (AR), together with 134 AR-regulated genes. METTL3 knockdown also resulted in altered splicing, and enrichment of cell cycle, DNA repair and metabolic pathways. Collectively, this study identified the functional and clinical significance of four essential m6A complex components in PCa patient specimens and cell lines for the first time. Further studies are now warranted to determine the potential therapeutic relevance of METTL3 inhibitors in development to treat leukaemia to benefit patients with PCa.