A disintegrin and metalloproteinase 17 (ADAM17) is a metalloprotease that is overexpressed in many cancer types, including renal cancers. However, the regulatory mechanisms of ADAM17 in cancer development and progression are poorly understood. In the present work, we provide evidence using overexpression and inhibition of microRNA 145 (miR-145) that miR-145 negatively regulates ADAM17 expression. Furthermore, we show that ADAM17 negatively regulates miR-145 through tumor necrosis factor-α, resulting in a reciprocal negative feedback loop. In this study, the expression of ADAM17 and miR-145 correlated negatively in renal cancer tumor tissues and cell lines, suggesting an important regulatory mechanism. Additionally, we showed that the regulation of ADAM17 is partly involved in the effects of miR-145 on proliferation and migration, whereas no involvement in chemosensitivity was observed. Importantly, in the healthy kidney, miR-145 was detected in different cell types including tubular cells, which are considered the origin of renal cancer. In renal cancer cell lines, miR-145 expression was strongly suppressed by methylation. In summary, miR-145 is downregulated in renal cancer patients, which leads to the up-regulation of ADAM17 in renal cancer. Importantly, miR-145 and ADAM17 are regulated in a reciprocal negative feedback loop.

Caspase-2 represents the most conserved member of the caspase family, which exhibits features of both initiator and effector caspases. Using ribonucleoprotein (RNP)-immunoprecipitation assay, we identified the proapoptotic caspase-2L encoding mRNA as a novel target of the ubiquitous RNA-binding protein HuR in DLD-1 colon carcinoma cells. Unexpectedly, crosslinking-RNP and RNA probe pull-down experiments revealed that HuR binds exclusively to the caspase-2-5′ untranslated region (UTR) despite that the 3′ UTR of the mRNA bears several adenylate- and uridylate-rich elements representing the prototypical HuR binding sites. By using RNAi-mediated loss-of-function approach, we observed that HuR regulates the mRNA and in turn the protein levels of caspase-2 in a negative manner. Silencing of HuR did not affect the stability of caspase-2 mRNA but resulted in an increased redistribution of caspase-2 transcripts from RNP particles to translational active polysomes implicating that HuR exerts a direct repressive effect on caspase-2 translation. Consistently, in vitro translation of a luciferase reporter gene under the control of an upstream caspase-2-5′UTR was strongly impaired after the addition of recombinant HuR, whereas translation of caspase-2 coding region without the 5′UTR is not affected by HuR confirming the functional role of the caspase-2-5′UTR. Functionally, an elevation in caspase-2 level by HuR knockdown correlated with an increased sensitivity of cells to apoptosis induced by staurosporine- and pore-forming toxins as implicated by their significant accumulation in the sub G1 phase and an increase in caspase-2, -3 and poly ADP-ribose polymerase cleavage, respectively. Importantly, HuR knockdown cells remained insensitive toward STS-induced apoptosis if cells were additionally transfected with caspase-2-specific siRNAs. Collectively, our findings support the hypothesis that HuR by acting as an endogenous inhibitor of caspase-2-driven apoptosis may essentially contribute to the antiapoptotic program of adenocarcinoma cells by HuR.

Edited by Beat ImhofKeywords: AU-rich element Hepatocellular carcinoma Lymphotoxin a mRNA-stability a b s t r a c tThe role of the RNA-binding protein human antigen R (HuR) in hepatocarcinogenesis is still elusive. By employing short hairpin (sh)RNA-dependent knockdown approach, we demonstrate that lymphotoxin a (LTa) is a target of posttranscriptional gene regulation by HuR in hepatocellular carcinoma (HepG2) cells. Consequently, the increased mRNA decay upon HuR depletion significantly affects lymphotoxin expression at both, the mRNA and protein level. Biotin-pulldown assay showed that HuR specifically interacts with the 3 0 -untranslated region (3 0 -UTR) of the LTa mRNA. Furthermore, electrophoretic mobility shift assay (EMSA) implicates that the RNA-binding critically depends on the RNA recognition motif 2 (RRM2) and the hinge region of HuR.