Exploring the involvement of E6AP in prostate cancer: a combined transcriptomic and proteomic approach

© 2017 Dr. Twishi GulatiIn Australia, one in seven men are at a risk of being diagnosed with and one in 30 men at the risk of dying from prostate cancer (PC) by the age of 85 [1]. Limited treatment possibilities for castration resistant prostate cancer (CRPC) highlight the need for novel therapeutics. Restoring tumour suppressors in PC by proteasomal inhibition has demonstrated promising results in clinical trials, albeit with significant side effects [2]. Developing more specific and effective treatments for PC is therefore warranted. E6-Associated Protein (E6AP) is an E3 ubiquitin ligase and a transcription cofactor. Recent work from our laboratory and colleagues has demonstrated elevated expression of E6AP in a subset of PC patients [3]. Moreover, genetic manipulations of E6AP in PC cells exposed a role of E6AP in promoting growth and survival of PC cells in vitro and in vivo experimental systems [4]. Previous work from our laboratory unravelled that E6AP mediates this impact on PC cells via tumour suppressors such as promyelocytic leukaemia protein (PML; [4]) and p27 [5]. However, the effect of E6AP on PC cells is broad and it cannot be explained fully by these two tumour suppressors. To identify additional players that mediate E6AP phenotype, we utilised a combined transcriptomic (next generation RNA sequencing) and proteomic (SILAC) approaches. We assessed changes in the total transcriptome and proteome upon E6AP knockdown in CRPC cell line, DU145. We identified 16,130 transcripts using RNA-seq and 7,209 proteins using SILAC. A total of 2,763 transcripts and 308 proteins were considered significantly altered (± 1.5-fold, p-value < 0.05). A comparison of the omics data revealed candidates were either regulated transcriptionally alone, post-transcriptionally alone, or altered at both mRNA and protein level. Pathway analyses supported the known phenotypic effect of E6AP knockdown in PC cells as well as exposed novel links of E6AP with cancer metabolism, DNA damage repair and immune response. In parallel, we explored proteins regulated by E6AP in different PC cell lines, PC3 and LNCaP in addition to DU145 cells. We identified 4,814, 4,827 and 4,819 proteins inDU145, PC3 and LNCaP cells, respectively, upon E6AP knockdown using LC/MS-MS and data-independent acquisition in combination with label-free quantitation. A total of 225, 107 and 139 proteins were considered significantly altered (± 1.5-fold, p-value < 0.05) in DU145, PC3 and LNCaP cells, respectively, following knockdown of E6AP. Metabolism was the most deregulated biological pathway in all three PC cell lines upon E6AP knockdown, substantiating the novel link between E6AP and cancer metabolism. The significantly altered proteins and biological processes in various cell lines warrant further validation and investigation. Of the top ranked candidates, clusterin was pursued further. Clusterin is a stress-induced chaperone protein that modulates apoptosis, lipid transport, DNA repair and cell migration [6]. Clusterin is commonly deregulated in cancer, including prostate cancer. Our results demonstrate that E6AP negatively regulates clusterin at mRNA and protein level. Concomitant knockdown of E6AP and clusterin demonstrates that growth inhibition induced following knockdown of E6AP is in part restored by clusterin. Therefore, restoration of clusterin in the presence of low levels of E6AP can lead to tumour suppression. Overall, the results from this thesis identify novel candidates regulated by E6AP and this sheds new light on potential mechanisms by which E6AP promotes PC

Molecular basis of intervertebral disc regeneration by recombinant human bone morphogenetic protein 13

Low back pain (LBP) is a major health problem and one of the main sources of LBP is intervertebral disc (IVD) degeneration. IVD degeneration is characterised by loss of disc cells and increased catabolism of extracellular matrix (ECM). The use of anabolic growth factors to regenerate the ECM of the IVD continues to be evaluated in preclinical studies. Bone morphogenetic protein 13 (BMP-13) stimulates proteoglycan production in chondrocyte-like cells and could be a potential therapeutic agent in regenerating the degenerate disc. However, the molecular effects of recombinant protein (rh) BMP-13 on IVD cells are not known. The effect of different doses of rhBMP-13 on nucleus pulposus, annulus fibrosus and end plate cells derived from degenerate human intervertebral discs cultured in alginate beads was evaluated by studying the changes in cell viability, cell proliferation and proteoglycan accumulation. Temporal responses were also evaluated by cellular proliferation and proteoglycan accumulation. Furthermore, the effect of rhBMP-13 stimulation on the expression of selected chondrogenic and osteogenic genes and proteins by IVD cells was determined. The findings show that rhBMP-13 induced significant proteoglycan accumulation in nucleus pulposus, annulus fibrosus and end plate cells at a concentration of 400ng/ml after 7 days. In nucleus pulposus cells, rhBMP-13 may have an anti-osteogenic effect, by potentially increasing sox9 gene expression and repressing runx2 and osteocalcin gene expression. In annulus fibrosus cells, rhBMP-13 increased collagen I gene and protein expression, enhancing the fibrocytic phenotype of the cells. In end plate cells, rhBMP-13 may have a chondrocytic effect, by increasing aggrecan, collagen II and sox9 gene expression. There was, however, an increase in runx2 and osteocalcin gene expression in both annulus fibrosus and end plate cells suggests a potential for bone formation and warrants further investigation. RhBMP-13 stimulation had a limited effect on the cellular viability and proliferation of IVD cells in vitro. The findings from the present study suggest that rhBMP-13 may enhance the expression of critical ECM genes that are characteristic of the cells in each region of the disc. Therefore, further exploration of BMP-13 as a potential therapeutic agent to ameliorate IVD degeneration is warranted

Obesity Drives STAT-1-Dependent NASH and STAT-3-Dependent HCC

Obesity is a major driver of cancer, especially hepatocellular carcinoma (HCC). The prevailing view is that non-alcoholic steatohepatitis (NASH) and fibrosis or cirrhosis are required for HCC in obesity. Here, we report that NASH and fibrosis and HCC in obesity can be dissociated. We show that the oxidative hepatic environment in obesity inactivates the STAT-1 and STAT-3 phosphatase T cell protein tyrosine phosphatase (TCPTP) and increases STAT-1 and STAT-3 signaling. TCPTP deletion in hepatocytes promoted T cell recruitment and ensuing NASH and fibrosis as well as HCC in obese C57BL/6 mice that normally do not develop NASH and fibrosis or HCC. Attenuating the enhanced STAT-1 signaling prevented T cell recruitment and NASH and fibrosis but did not prevent HCC. By contrast, correcting STAT-3 signaling prevented HCC without affecting NASH and fibrosis. TCPTP-deletion in hepatocytes also markedly accelerated HCC in mice treated with a chemical carcinogen that promotes HCC without NASH and fibrosis. Our studies reveal how obesity-associated hepatic oxidative stress can independently contribute to the pathogenesis of NASH, fibrosis, and HCC.info:eu-repo/semantics/publishe