Inhibition of IRES-dependent translation of caspase-2 by HuR confers chemotherapeutic drug resistance in colon carcinoma cells
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Amel Badawi1,*, Abhiruchi Biyanee1,3,*, Usman Nasrullah1, Sofia Winslow2, Tobias Schmid2, Josef Pfeilschifter1 and Wolfgang Eberhardt1
1Pharmazentrum Frankfurt/ZAFES, Medical School, Goethe-University Frankfurt, Frankfurt/Main, Germany
2Institute of Biochemistry I, Goethe-University Frankfurt, Frankfurt/Main, Germany
3Present address: Institut für Biochemie, Westfälische Wilhelms-Universität Münster, Münster, Germany
*These authors contributed equally to this work
Wolfgang Eberhardt, email: [email protected]
Keywords: caspase-2; colon carcinoma cells; chemotherapeutic drug resistance; HuR; IRES translation
Received: July 08, 2017 Accepted: February 23, 2018 Published: April 06, 2018
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 5ʹuntranslated region (5ʹUTR) 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-2-5ʹUTR. 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-2-5ʹUTR by EMSA revealed a critical HuR-binding site residing between nucleotides 111 and 241 of caspase-2-5ʹUTR. 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-2-5ʹUTR-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.
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