Identification of the Dysregulated Pathways and Key Gene in Prostate Cancer by Transcriptome Analysis and Cell Biology Experiments

Background: Prostate cancer (PCa) is a common cancer in elderly men with the first increasing new cases and the second leading cause of cancer death, but the molecular mechanisms underlying the pathogenesis of prostate cancer remain unclear. Methods: Here we mainly used Weight gene co-expression network analysis (WGCNA), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein to protein interaction (PPI), gene set enrichment analysis (GSEA) and cell biology experiments to analyze prostate cancer data in GEO and The Cancer Genome Atlas (TCGA) databases and revealed the main dysregulated pathways and key genes in prostate carcinogenesis. Results: We found that the focal adhesion pathway was the main pathway in PCa. FERMT2 was shown to be the key gene for prostate tumorigenesis both in GSE6919 and TCGA datasets. By using WGCNA and GSEA analysis, we found that FERMT2 was related to the focal adhesion pathway and the ECM interaction pathway. Cell biology experiments demonstrated that FERMT2 inhibited tumor cell proliferation and migration. Conclusion: Our findings revel that downregulation of FERMT2 and the focal adhesion pathway are the main characteristics of PCa and FERMT2 might be the potential biomarker or treatment target for PCa.Trial registration: The study is not a clinical trial.

The Oncogenic Potential of TCTEX1D4 is Modulated by the Phosphoprotein Phosphatase PP1

Protein phosphatase 1 (PP1) regulates several cellular events via interaction with multiple regulatory subunits. The human prostate proteome includes various PP1-interacting proteins; however, a very limited number of interactions is yet characterized and their role in prostate tumorigenesis remains poorly understood. Tctex1 domain-containing protein 4 (TCTEX1D4) was previously identified as a PP1-interacting protein, but its function, as well as the relevance of its interaction with PP1, are virtually unknown. In this study we addressed the role of the PP1/TCTEX1D4 complex in prostate tumorigenesis. We found distinct expression levels and subcellular distributions for TCTEX1D4 and PP1γ in human prostate epithelial normal-like and malignant cells. Moreover, we showed that TCTEX1D4 participates in the regulation of cell proliferation and modulation of microRNAs expression and that its interaction with PP1 controls its function. Taken together, our study provides first evidence for the involvement of the PP1/TCTEX1D4 complex in prostate tumorigenesis.

Transcriptional regulation and ubiquitination-dependent regulation of HnRNPK oncogenic function in prostate tumorigenesis

Background Heterogeneous nuclear ribonucleoprotein K (HnRNPK) is a nucleic acid-binding protein that regulates diverse biological events. Pathologically, HnRNPK proteins are frequently overexpressed and clinically correlated with poor prognosis in various types of human cancers and are therefore pursued as attractive therapeutic targets for select patients. However, both the transcriptional regulation and degradation of HnRNPK in prostate cancer remain poorly understood. Methods qRT-PCR was used to detect the expression of HnRNPK mRNA and miRNA; Immunoblots and immunohistochemical assays were used to determine the levels of HnRNPK and other proteins. Flow cytometry was used to investigate cell cycle stage. MTS and clonogenic assays were used to investigate cell proliferation. Immunoprecipitation was used to analyse the interaction between SPOP and HnRNPK. A prostate carcinoma xenograft mouse model was used to detect the in vivo effects of HnRNPK and miRNA. Results In the present study, we noted that HnRNPK emerged as an important player in the carcinogenesis process of prostate cancer. miR-206 and miR-613 suppressed HnRNPK expression by targeting its 3’-UTR in PrCa cell lines in which HnRNPK is overexpressed. To explore the potential biological function, proliferation and colony formation of PrCa cells in vitro and tumor growth in vivo were also dramatically suppressed upon reintroduction of miR-206/miR-613. We have further provided evidence that Cullin 3 SPOP is a novel upstream E3 ubiquitin ligase complex that governs HnRNPK protein stability and oncogenic functions by promoting the degradation of HnRNPK in polyubiquitination-dependent proteolysis in the prostate cancer setting. Moreover, prostate cancer-associated SPOP mutants fail to interact with and promote the destruction of HnRNPK proteins. Conclusion Our findings reveal new posttranscriptional and posttranslational modification mechanisms of HnRNPK regulation via miR-206/miR-613 and SPOP, respectively. More importantly, given the critical oncogenic role of HnRNPK and the high frequency of SPOP mutations in prostate cancer, our results provide a molecular rationale for the clinical investigation of novel strategies to combat prostate cancer based on SPOP genetic status.

Suppression of ANT2 by miR-137 Inhibits Prostate Tumorigenesis

Prostate cancer (PCa) is a serious disease that affects men’s health. To date, no effective and long-lasting treatment option for this condition is available in clinical practice. ANT2 is highly expressed in a variety of hormone-related cancers, but its relationship and regulatory mechanism with PCa are unclear. In this study, we found that ANT2 expression was significantly upregulated in PCa tissues relative to control samples. Genetic knockdown of ANT2 effectively inhibited, while overexpression promoted, proliferation, migration, and invasion of PCa cells. In addition, miR-137 expression was reduced in prostate cancer tissues relative to control tissues. We identified a regulatory site for miR-137 in the 3′-UTR of ANT2 mRNA; luciferase reporter assays indicated that ANT2 is a direct target gene for miR-137. Transfecting cells with miR-137 mimics and/or an ANT2-encoding plasmid revealed that ANT2 promotes proliferation, migration, and invasion of PCa, whereas co-expression of miR-137 mimics inhibited these behaviors. These observations suggest that miR-137 mimics inhibit development of PCa by antagonizing expression of ANT2. Furthermore, tumorigenic assays in nude mice showed that miR-137 inhibitors abolished the inhibitory effect of ANT2 knockdown on PCa tumor growth. Collectively, our findings suggest that ANT2, a target gene of miR-137, is intimately involved in development of PCa, providing new evidence for the mechanism underlying pathogenesis of PCa as well as new options for targeted therapy.

Integrative genomic and epigenomic analyses identified IRAK1 as a novel target for chronic inflammation-driven prostate tumorigenesis

The impacts of many inflammatory genes in prostate cancer (PCa) remain understudied despite the increasing evidence that associates chronic inflammation with PCa initiation, progression, and therapy resistance. The overarching goal of this study was to identify dysregulated inflammatory genes that correlate with PCa progression and decipher their molecular mechanisms as well as clinical significance in PCa using integrative genomics, transcriptomics, and epigenomics approach. Our Weighted Gene Co-expression Analysis (WGCNA) and multivariate analysis identified 10 inflammatory genes: IRAK1, PPIL5/LRR1, HMGB3, HMGB2, TRAIP, IL1F5/IL36RN, ILF2, TRIM59, NFKBIL2/TONSL, and TRAF7 that were significantly associated with PCa progression. We explored the potentials of IRAK1 and other inflammatory genes as diagnostic and/or prognostic biomarkers by performing both KM survival and AUROC curve analyses. Our results indicate the clinical significance of these inflammatory genes in predicting the development and progression of PCa. IRAK1 was found to be overexpressed and hypomethylated in most PCa samples. A significantly high percentage of castration-resistant PCa (CRPC) and neuroendocrine PCa (NEPC) samples display copy number variations, especially amplification of the IRAK1 gene compared to the indolent prostate adenocarcinoma (PRAD) samples. Furthermore, we identified missense and frameshift mutations of IRAK1 in a few PRAD samples with potential functional implications. In conclusion, the results from this study suggest that IRAK1 dysregulation may be an important contributor to chronic prostatitis (inflammation) and PCa progression.

HGK promotes metastatic dissemination in prostate cancer

Metastasis is the process of cancer cell dissemination from primary tumors to different organs being the bone the preferred site for metastatic homing of prostate cancer (PCa) cells. Prostate tumorigenesis is a multi-stage process that ultimately tends to advance to become metastatic PCa. Once PCa patients develop skeletal metastases, they eventually succumb to the disease. Therefore, it is imperative to identify essential molecular drivers of this process to develop new therapeutic alternatives for the treatment of this devastating disease. Here, we have identified MAP4K4 as a relevant gene for metastasis in PCa. Our work shows that genetic deletion of MAP4K4 or pharmacological inhibition of its encoded kinase, HGK, inhibits metastatic PCa cells migration and clonogenic properties. Hence, MAP4K4 might promote metastasis and tumor growth. Mechanistically, our results indicate that HGK depleted cells exhibit profound differences in F-actin organization, increasing cell spreading and focal adhesion stability. Additionally, HGK depleted cells fails to respond to TNF-α stimulation and chemoattractant action. Moreover, here we show that HGK upregulation in PCa samples from TCGA and other databases correlates with a poor prognosis of the disease. Hence, we suggest that it could be used as prognostic biomarker to predict the appearance of an aggressive phenotype of PCa tumors and ultimately, the appearance of metastasis. In summary, our results highlight an essential role for HGK in the dissemination of PCa cells and its potential use as prognostic biomarker.

The Transcriptional Regulation and Ubiquitination Dependent Regulation of Hnrnpk Oncogenic Function in Prostate Tumorigenesis

Background Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is a nucleic acid-binding protein that regulates diverse biological events. Pathologically, hnRNPK proteins are frequently overexpressed and clinically correlated with poor prognosis to various types of human cancers, therefore pursued as attractive therapeutic targets for selective patients. However, both the transcriptional regulation and degradation of hnRNPK in prostate cancer are remain poorly understood. Methods qRT-PCR was used to detect the expression of hnRNPK and miRNA; Immunoblots and immunohistochemical assays were used to determine the levels of hnRNPK and other proteins. Flow cytometry was used to investigate cell cycle stage. MTS and clonogenic assays were used to investigate cell proliferation. Immunoprecipitation was used to analyze the interaction between SPOP and hnRNPK. A prostate carcinoma xenograft mouse model was used to detect the in vivo effects of hnRNPK and miRNA. Results In the present study, we observed that hnRNPK emerged as an important player in carcinogenesis process of PrCa. miR-206 and miR-613 suppressed hnRNPK expression by targeting the 3’-UTR of hnRNPK in PrCa cell lines, where hnRNP K is overexpressed. In biological effects studies, proliferation and colony formation of PrCa cells in vitro, and tumor growth in vivo, were also dramatically suppressed upon reintroduction of miR-206/ miR-613. We have further provided clear evidence that Cullin 3 SPOP as a novel upstream E3 ubiquitin ligase complex that governs hnRNPK proteins stability and oncogenic functions through promoting the degradation of HnRNP K in a poly-ubiquitinaion dependent proteolysis in the prostate cancer setting. Moreover, prostate cancer-associated SPOP mutants fail to interact with and promote the destruction of hnRNPK proteins. Conclusion Our finding reveal new post-transcriptional and post-translational modifications mechanism of hnRNPK regulation via miR-206/ miR-613 and SPOP, respectively. More important, given the critical oncogenic role of hnRNPK and high frequency of SPOP mutation in prostate cancer, our results provide a molecular rationale for the clinical investigation of novel strategies to combat prostate cancer based on SPOP genetic status.