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Inhibition of IRES-dependent translation of caspase-2 by HuR confers chemotherapeutic drug resistance in colon carcinoma cells.

ABSTRACT: HuR plays an important role in tumor cell survival mainly through posttranscriptional upregulation of prominent anti-apoptotic genes. In addition, HuR can inhibit the translation of pro-apoptotic factors as we could previously report for caspase-2. Here, we investigated the mechanisms of caspase-2 suppression by HuR and its contribution to chemotherapeutic drug resistance of colon carcinoma cells. In accordance with the significant drug-induced increase in cytoplasmic HuR abundance, doxorubicin and paclitaxel increased the interaction of cytoplasmic HuR with the 5'untranslated region (5'UTR) of caspase-2 as shown by RNA pull down assay. Experiments with bicistronic reporter genes furthermore indicate the presence of an internal ribosome entry site (IRES) within the caspase-2-5'UTR. Luciferase activity was suppressed either by chemotherapeutic drugs or ectopic expression of HuR. IRES-driven luciferase activity was significantly increased upon siRNA-mediated knockdown of HuR implicating an inhibitory effect of HuR on caspase-2 translation which is further reinforced by chemotherapeutic drugs. Comparison of RNA-binding affinities of recombinant HuR to two fragments of the caspase-2-5'UTR by EMSA revealed a critical HuR-binding site residing between nucleotides 111 and 241 of caspase-2-5'UTR. Mapping of critical RNA binding domains within HuR revealed that a fusion of RNA recognition motif 2 (RRM2) plus the hinge region confers a full caspase-2-5'UTR-binding. Functionally, knockdown of HuR significantly increased the sensitivity of colon cancer cells to drug-induced apoptosis. Importantly, the apoptosis sensitizing effects by HuR knockdown were rescued after silencing of caspase-2. The negative caspase-2 regulation by HuR offers a novel therapeutic target for sensitizing colon carcinoma cells to drug-induced apoptosis.

SUBMITTER: Badawi A

PROVIDER: S-EPMC5915078 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Inhibition of IRES-dependent translation of caspase-2 by HuR confers chemotherapeutic drug resistance in colon carcinoma cells.

Badawi Amel A Biyanee Abhiruchi A Nasrullah Usman U Winslow Sofia S Schmid Tobias T Pfeilschifter Josef J Eberhardt Wolfgang W

Oncotarget 20180406 26

HuR plays an important role in tumor cell survival mainly through posttranscriptional upregulation of prominent anti-apoptotic genes. In addition, HuR can inhibit the translation of pro-apoptotic factors as we could previously report for caspase-2. Here, we investigated the mechanisms of caspase-2 suppression by HuR and its contribution to chemotherapeutic drug resistance of colon carcinoma cells. In accordance with the significant drug-induced increase in cytoplasmic HuR abundance, doxorubicin ...[more]

PMID: 29719611

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Project description:BackgroundTargeted therapies for Primary liver cancer (HCC) is limited to the multi-kinase inhibitors, and not fully effective due to the resistance to these agents because of the heterogeneous molecular nature of HCC developed during chronic liver disease stages and cirrhosis. Although combinatorial therapy can increase the efficiency of targeted therapies through synergistic activities, isoform specific effects of the inhibitors are usually ignored. This study concentrated on PI3K/Akt/mTOR pathway and the differential combinatory bioactivities of isoform specific PI3K-α inhibitor (PIK-75) or PI3K-β inhibitor (TGX-221) with Sorafenib dependent on PTEN context.MethodsThe bioactivities of inhibitors on PTEN adequate Huh7 and deficient Mahlavu cells were investigated with real time cell growth, cell cycle and cell migration assays. Differentially expressed genes from RNA-Seq were identified by edgeR tool. Systems level network analysis of treatment specific pathways were performed with Prize Collecting Steiner Tree (PCST) on human interactome and enriched networks were visualized with Cytoscape platform.ResultsOur data from combinatory treatment of Sorafenib and PIK-75 and TGX-221 showed opposite effects; while PIK-75 displays synergistic effects on Huh7 cells leading to apoptotic cell death, Sorafenib with TGX-221 display antagonistic effects and significantly promotes cell growth in PTEN deficient Mahlavu cells. Signaling pathways were reconstructed and analyzed in-depth from RNA-Seq data to understand mechanism of differential synergistic or antagonistic effects of PI3K-α (PIK-75) and PI3K-β (TGX-221) inhibitors with Sorafenib. PCST allowed as to identify AOX1 and AGER as targets in PI3K/Akt/mTOR pathway for this combinatory effect. The siRNA knockdown of AOX1 and AGER significantly reduced cell proliferation in HCC cells.ConclusionsSimultaneously constructed and analyzed differentially expressed cellular networks presented in this study, revealed distinct consequences of isoform specific PI3K inhibition in PTEN adequate and deficient liver cancer cells. We demonstrated the importance of context dependent and isoform specific PI3K/Akt/mTOR signaling inhibition in drug resistance during combination therapies. ( https://github.com/cansyl/Isoform-spesific-PI3K-inhibitor-analysis ).

Project description: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.

Dataset Information

Inhibition of IRES-dependent translation of caspase-2 by HuR confers chemotherapeutic drug resistance in colon carcinoma cells.

Publications

Inhibition of IRES-dependent translation of caspase-2 by HuR confers chemotherapeutic drug resistance in colon carcinoma cells.

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