Validation of Novel Biomarkers for Prostate Cancer Progression by the Combination of Bioinformatics, Clinical and Functional Studies

Abstract

The identification and validation of biomarkers for clinical applications remains an important issue for improving diagnostics and therapy in many diseases, including prostate cancer. Gene expression profiles are routinely applied to identify diagnostic and predictive biomarkers or novel targets for cancer. However, only few predictive markers identified in silico have also been validated for clinical, functional or mechanistic relevance in disease progression. In this study, we have used a broad, bioinformatics-based approach to identify such biomarkers across a spectrum of progression stages, including normal and tumor-adjacent, premalignant, primary and late stage lesions. Bioinformatics data mining combined with clinical validation of biomarkers by sensitive, quantitative reverse-transcription PCR (qRT-PCR), followed by functional evaluation of candidate genes in disease-relevant processes, such as cancer cell proliferation, motility and invasion. From 300 initial candidates, eight genes were selected for validation by several layers of data mining and filtering. For clinical validation, differential mRNA expression of selected genes was measured by qRT-PCR in 197 clinical prostate tissue samples including normal prostate, compared against histologically benign and cancerous tissues. Based on the qRT-PCR results, significantly different mRNA expression was confirmed in normal prostate versus malignant PCa samples (for all eight genes), but also in cancer-adjacent tissues, even in the absence of detectable cancer cells, thus pointing to the possibility of pronounced field effects in prostate lesions. For the validation of the functional properties of these genes, and to demonstrate their putative relevance for disease-relevant processes, siRNA knock-down studies were performed in both 2D and 3D organotypic cell culture models. Silencing of three genes (DLX1, PLA2G7 and RHOU) in the prostate cancer cell lines PC3 and VCaP by siRNA resulted in marked growth arrest and cytotoxicity, particularly in 3D organotypic cell culture conditions. In addition, silencing of PLA2G7, RHOU, ACSM1, LAMB1 and CACNA1D also resulted in reduced tumor cell invasion in PC3 organoid cultures. For PLA2G7 and RHOU, the effects of siRNA silencing on proliferation and cell-motility could also be confirmed in 2D monolayer cultures. In conclusion, DLX1 and RHOU showed the strongest potential as useful clinical biomarkers for PCa diagnosis, further validated by their functional roles in PCa progression. These candidates may be useful for more reliable identification of relapses or therapy failures prior to the recurrence local or distant metastases.

Genetic variants in the genes encoding rho GTPases and related regulators predict cutaneous melanoma‐specific survival

“…Our analysis identified four significant SNPs mapped to four genes encoding a member of Rho GTPases ( RHOU ), two members of GAPs ( ARHGAP22 and ARHGAP44 ), and a member of GEFs ( ARHGEF10 ). RHOU is upregulated by the Wnt1 signaling in Wnt1‐transformed mouse mammary cells to promote filopodium formation and stress fiber dissolution, and has been reported to regulate tumor cell invasion in prostate cancer by functioning similarly as the Cdc42 small GTPase . While ARHGAP22 and ARHGAP44 trigger local Rac‐GTP hydrolysis, thus reducing actin polymerization required for filopodia formation .…”

“…RHOU is upregulated by the Wnt1 signaling in Wnt1-transformed mouse mammary cells to promote filopodium formation and stress fiber dissolution, 28 and has been reported to regulate tumor cell invasion in prostate cancer by functioning similarly as the Cdc42 small GTPase. 29 While ARHGAP22 and ARHGAP44 trigger local Rac-GTP hydrolysis, thus reducing actin polymerization required for filopodia formation. 14,30 For example, in melanoma cell movement, ARHGAP22 can be activated to suppress mesenchymal movement by inactivating Rac.…”

Lipoprotein‐associated phospholipase A2: The story continues

“…Further functional studies revealed that silencing PLA2G7 expression produced antiproliferative, proapoptotic, and antimigratory effects on prostate cancer cells and that statin treatment combined with PLA2G7 silencing synergistically exerted an antiproliferative effect . Recently, Alinezhad et al also identified PLA2G7 as a valid biomarker for prostate cancer and subsequently evaluated its functions in disease‐relevant processes. Similarly, diminished tumor cell invasion and proliferation were detected after silencing PLA2G7 .…”

Radiogenomics and radiotherapy response modeling

“…This list was later updated with examples of good reporting (Altman et al , 2012). A further validation would involve testing using functional biological assays of the presented hypothesis of the identified biomarker (Alinezhad et al , 2016). For genetic risks, a checklist of 25 items recommended for strengthening the reporting of Genetic RIsk Prediction Studies (GRIPS) was further developed particularly to improve the knowledge synthesis and application of information from multiple genetic studies that might differ in design, conduct or analysis (Janssens et al , 2011).…”