title: Silencing of the mRNA-binding protein HuR increases the sensitivity of colorectal cancer cells to ionizing radiation through upregulation of caspase-2.
abstract: Increased abundance of the mRNA-binding protein human antigen R (HuR) is a characteristic feature of many cancers and frequently associated with a high grade malignancy and therapy resistance. HuR elicits a broad cell survival program mainly by stabilizing or increasing the translation of mRNAs coding for anti-apoptotic effector proteins. Conversally, we previously identified the pro-apoptotic caspase-2 as a novel HuR target which is mainly regulated at the level of translation. In this study, we investigated whether siRNA-mediated HuR knockdown interferes with cell survival and radiation sensitivity by monitoring apoptosis, DNA repair and three-dimensional (3D) clonogenic survival. We observed a significant elevation in caspase-2 upon HuR depletion and in turn, a sensitization of colorectal DLD-1 and HCT-15 cells to radiation-induced apoptosis as implicated by the dose-dependent elevation of sub-G1phase cell entry and increased caspase-2, -3 and poly ADP-ribose polymerase (PARP)-cleavage, respectively. Coincidentally, HuR deficiency significantly elevated the number of radiation-induced γH2AX/53BP1-positive foci indicating an increase in DNA damage. Accordingly, the irradiation-dependent reduction in clonogenic cell survival was further impaired after knockdown of HuR. Importantly, HuR knockdown remained ineffective to radiation-induced cell responses after additional knockdown of caspase-2. Furthermore, by using RNA-pull down assay we demonstrate that irradiation (6 Gy) robustly increased HuR binding to caspase-2 mRNA. Collectively, sensitization of colon carcinoma cells to radiation-induced cell death and DNA-damage by HuR knockdown critically depends on caspase-2 and may represent a valuable approach to intervene with therapy resistance of colorectal cancer (CRC).
authors.affiliation: pharmazentrum frankfurt/ZAFES, University of Frankfurt, Medical School, Frankfurt/Main, Germany.
authors.forename: Amel
authors.initials: A
authors.lastname: Badawi
authors.affiliation: Department of Radiotherapy and Oncology, University of Frankfurt, Frankfurt/Main, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Hehlgans
authors.affiliation: pharmazentrum frankfurt/ZAFES, University of Frankfurt, Medical School, Frankfurt/Main, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Pfeilschifter
authors.affiliation: Department of Radiotherapy and Oncology, University of Frankfurt, Frankfurt/Main, Germany.
authors.forename: Franz
authors.initials: F
authors.lastname: Rödel
authors.affiliation: pharmazentrum frankfurt/ZAFES, University of Frankfurt, Medical School, Frankfurt/Main, Germany. Electronic address: w.eberhardt@em.uni-frankfurt.de.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Eberhardt
mesh_terms: D020121:5' Untranslated Regions; D017209:Apoptosis / Q000235:genetics / Q000528:radiation effects*; D001665:Binding Sites; D053143:Caspase 2 / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000532:radiotherapy*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D000067780:ELAV-Like Protein 1 / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D034622:RNA Interference; D011836:Radiation Tolerance / Q000235:genetics; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation
chemical_list: D020121:5' Untranslated Regions; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; C490360:CASP2 protein, human; D053143:Caspase 2; D003546:Cysteine Endopeptidases
keywords: Apoptosis; Caspase-2; Colorectal carcinoma cells; HuR; Radiotherapy resistance