Oncotarget

Research Papers:

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

Amel Badawi, Abhiruchi Biyanee, Usman Nasrullah, Sofia Winslow, Tobias Schmid, Josef Pfeilschifter and Wolfgang Eberhardt _

PDF  |  HTML  |  Supplementary Files  |  How to cite

Oncotarget. 2018; 9:18367-18385. https://doi.org/10.18632/oncotarget.24840

Metrics: PDF 1716 views  |   HTML 2497 views  |   ?  


Abstract

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

Correspondence to:

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

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 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.


Creative Commons License All site content, except where otherwise noted, is licensed under a Creative Commons Attribution 4.0 License.
PII: 24840