Deregulated E2F5/p38/SMAD3 Circuitry Reinforces the Pro-Tumorigenic Switch of TGFβ Signaling in Prostate Cancer

Abstract

Transforming growth factor-β signaling exerts divergent effects on normal and cancer cells, although mechanism underlying this differential behavior remains unclear. In this study, expression of 94 genes pertaining to the TGF-β signaling pathway was compared between tumor and benign tissue samples from the human prostate gland to identify major discriminators driving prostate carcinogenesis. E2F5 was identified as one of the most deregulated genes in prostate cancer tissues, predominantly in samples with Gleason-score 6. Expression of other deregulated components of TGF-β signaling was examined by qRT-PCR, Western blot, and immune-staining. Function of E2F5 and p38 in prostate cancer was investigated using siRNA-treatment of PC3 cell-line followed by analyses of associated components and cell cycle. Observations revealed that E2F5 overexpression was accompanied by significantly higher phosphorylation of SMAD3 at Ser-208 in the linker region (pSMAD3L) and p38 in tumor tissue. A striking difference in SMAD3 phosphorylation, marked by preponderance of pSMAD3L and pSMAD3C (Ser-423 and 425) in tumor and benign tissues, respectively was noted. Co-localization of E2F5 with pSMAD3L in the nuclei of tumor and PC3 cells indicated a functional interface between the proteins. Downregulation of E2F5 and p38 in PC3 cells resulted in marked reduction of phosphorylation of SMAD3 and perturbation of cell cycle with an arrest of cells in G1 . Our findings unearthed that E2F5/p38 axis played a cardinal role in uncontrolled cellular proliferation in prostate cancer through pSMAD3L activation. It also underscores a strong potential for E2F5 to be incorporated as a tool in early detection of prostate cancer. J. Cell. Physiol. 231: 2482-2492, 2016. © 2016 Wiley Periodicals, Inc.

Pathway-based expression profiling of benign prostatic hyperplasia and prostate cancer delineates an immunophilin molecule associated with cancer progression

“…Cells were passaged regularly when they reached 80–85% confluency. Cells were detached by standard trypsinization with Trypsin-EDTA solution 47 .…”

“…Nuclear-emitted fluorescence was measured with a BD FACSVerse TM (BD Biosciences). 10,000 events were analyzed from each run 47 . The percentages of cells distributed in the G 1 , S, G 2 /M and sub-G 1 phases of the cell cycle were determined by analysis with the BD FACSuite™ software, version 1.0.5.…”

“…The cells were counterstained with hematoxylin, dehydrated in ethanol and mounted in DPX. For negative controls, cells were incubated in the absence of the primary antibody 47 . For localization and co-localization studies of proteins, immunofluorescence was performed on LNCaP cells grown on cover-slips.…”

Effects of miR‑129‑3p on biological functions of prostate cancer cells through targeted regulation of Smad3

“…As a multifunctional cytokine, transforming growth factor-β (TGF-β) plays a certain role in cells in autocrine and paracrine manner. Smad3 is an important member of the Smad protein family and a major component of TGF-β/Smad signaling pathway (18,19). We regulated the expression level of Smad3 and found that the downregulation of the expression level significantly inhibited the proliferation and invasion of prostate cancer cells, but significantly increased the apoptotic rate of the cells.…”

Long Non­coding RNA SNHG16 Functions as Tumor Activator by Sponging hsa-miR-373-3p to Regulate TGFBR2/SMAD Pathway in Prostate Cancer

“…E2F4 were overexpressed in PCa epithelial cells [24], and regulated cell cycle by forming complexes with P130 [25]. E2F5 overexpression induced uncontrolled cellular proliferation by up-regulating phosphorylation of SMAD3 and p38 in PCa [26]. The endogenous SMAD2/3 interacted with PKCε to cause SMAD3 to bind to the promoter of glycolysis genes, induced the expression of glycolysis genes HIF-1α, HKII, PFKP and MCT4 and promoted aerobic glycolysis, thereby promoting PCa cell proliferation [27].…”