Attenuation of the ELAV1-like protein HuR sensitizes adenocarcinoma cells to the intrinsic apoptotic pathway by increasing the translation of caspase-2L

Abstract

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.

HuR Contributes to TRAIL Resistance by Restricting Death Receptor 4 Expression in Pancreatic Cancer Cells

“…In Panc-1 cells pretreated with control or HuR siRNA, apoptosis was triggered with sTRAIL or staurosporine, an established inducer of intrinsic apoptosis and HuR engagement (25, 38). HuR knockdown caused a significant increase in TRAIL-induced caspase-3 cleavage, but not in staurosporine-treated cells (Fig.…”

Identification of TRIM25 as a Negative Regulator of Caspase-2 Expression Reveals a Novel Target for Sensitizing Colon Carcinoma Cells to Intrinsic Apoptosis

“…Cells were lysed by using a method described previously [6]. In brief, the cells were lysed in cold lysis buffer (137 mM NaCl, 20 mM Tris-HCl pH 8.0, 5 mM EDTA pH 8.0, 10% glycerol, and 1% Triton X-100) supplemented with a protease inhibitor mix from Roche (Mannheim, Germany).…”

“…As a consequence, therapy-resistant tumor cells are characterized by impaired activation of caspases, a family of cysteine-aspartate proteases that mediate the proteolytic processing of diverse downstream substrates in response to disruptive insults (for a review, see [2,5]). In addition to elevations in IAP proteins, we previously identified direct inhibition of caspase-2 translation by the ubiquitous mRNA-binding protein human antigen R (HuR) as a novel path of therapy resistance in colon carcinoma cells [6][7][8] (for a review, see [9]). In contrast to other members of the caspase family, the defined role of caspase-2 in apoptosis is still debated.…”

Interaction between C2ORF68 and HuR in human colorectal cancer

“…HuR modulates posttranscriptional processing of target premRNAs or mRNA stabilization and translation through interaction with AU-rich elements (ARE) within 3'-untranslated regions (UTRs) of the target mRNAs to transformation (24). Furthermore, it has been shown that HuR stabilizes mRNAs that encode p53 and WEE1 (25), activates ATF2 (26), Jun D (27) and XIAP (28) and enhances the translation of mRNAs that encode c-Myc (29), ICH-1 (30) and IL-1β (31). Many of these transcripts are reported to participate in certain key cellular processes including cell proliferation, cell apoptosis, angiogenesis, immune response and metastasis.…”