7 At the transcriptional level, nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) can activate HuR transcription downstream of the phosphoinositide 3-kinase

(PI3K)-AKT-signaling pathway.8 At the post-transcriptional level, the ubiquitin (Ub)-proteasome pathway has been implicated in HuR function, whereas the machinery involved in enhancing HuR protein stability remains obscure.9 The Ub-like molecule, NEDD8, is a key regulator of cell growth, viability, and development. NEDD8 is ubiquitously expressed Selleckchem MK0683 and highly conserved among most eukaryotes. At the molecular level, only members of the cullin family of proteins are well-characterized substrates for NEDDylation.10 However, through direct biological approaches or proteomic techniques, it is evident that the NEDD8 proteome is widely diverse. Recent studies have shown that ribonucleoproteins (RBNs) are targets for NEDD8 conjugation, which protect them from destabilization.11, 12 Conversely, cysteine protease (NEDP1) removes NEDD8 molecules from conjugated substrates.13 Murine double minute 2 (Mdm2) acts as an E3 NEDD8 ligase, promoting p53 NEDDylation and stabilization,14 and, of importance, Mdm2 is Trametinib in vitro overexpressed in many human tumors, at least in part because of gene amplification.15 In this study, we showed that HuR is overexpressed in proliferative HCC and colon cancer cells

and biopsies through Mdm2-mediated NEDDylation. Our data suggest that HuR NEDDylation at K313 and K326 residues stabilizes HuR. Mechanistically, Mdm2-mediated NEDDylation of HuR controls the nuclear localization Branched chain aminotransferase of HuR

and protects it from degradation. To our knowledge, this is the first report to implicate NEDDylation in the regulation of HuR stability and localization. Our findings show that NEDDylation is a novel mechanism for HuR regulation that might represent a useful tool for new therapeutic strategies for HCC and colon cancer. ASH, alcoholic steatohepatitis; CHX, cycloheximide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HCC, hepatocellular carcinoma; HuR, Hu antigen R; IgG, immunoglobulin G; IP, immunoprecipitation; Mdm2, murine double minute 2; mRNA, messenger RNA; NASH, nonalcoholic steatohepatitis; NF-κB, nuclear factor kappa light-chain enhancer of activated B cells; Ni-NTA, nickel-nitrilotriacetic acid; NLS, nuclear localization signal; NPTII, neomycin phosphotransferase 2; PI3K, phosphoinositide 3-kinase; PTMA, prothymosin alpha; PCR, polymerase chain reaction; RNP-IP, ribonucleoprotein immunoprecipitation; RRM, RNA-binding domain; siRNA, small interfering RNA; Ub, ubiquitin; UVC, ultraviolet light C; WT, wild type. Surgically resected specimens of 61 patients with metastatic colon cancer to the liver included in two tissue microarrays and 22 HCC (10 hepatitis C, 10 alcoholic steatohepatitis [ASH], and 2 nonalcoholic steatohepatitis [NASH]) patients were examined.