Abstract B31: HuR cytoplasmic translocation and doxorubicin: how phosphorylation is involved in chemoresistance

Abstract

Abstract Introduction: HuR, an RNA binding protein involved in the post-transcriptional regulation of a wide spectrum of mRNAs, has been demonstrated to be a determinant of carcinogenesis and tumor aggressiveness in several cancer types. In this study, we identified HuR as a key protein in the mechanisms leading to the insurgence of chemoresistance induced by the widely used anticancer drug doxorubicin in human breast cancer cells (MCF-7) and investigated the signaling pathways responsible of its activation. Material and methods: We challenged a small library of about 90 chemical compounds with a high-content screening assay to quantitavely measure HuR translocation. Hits pointed to PKCδ, Rho kinase and ERK as potential HuR regulators. Results and discussion: We showed that HuR acts in the early phase of cell response to doxorubicin, being induced to translocate into the cytoplasm upon phosphorylation. Reducing HuR levels diminished the apoptotic response to doxorubicin. We identified HA1004, AG494, U0126, AG490 and Erbstatin compounds that could inhibit HuR cytoplasmic accumulation directly hampering HuR phosphorylation or indirectly blocking kinase pathways. Among the hits rottlerin showed to be the most effective in blocking HuR nuclear export and in having correspondingly antagonistic effects with doxorubicin on cell toxicity. Co-immunoprecipitation of PKCδ and HuR upon doxorubicin confirmed the validity of HCS indications. In in vitro selected doxorubicin resistant MCF-7 cells (MCF-7/doxoR) overexpressing the multidrug resistance (MDR) related ABCG2 transporter, we observed a significant HuR downregulation that was paralleled by a corresponding downregulation of HuR targets as TOP2A and by loss of rottlerin toxicity. Restoration of HuR expression in these cells resensitized MCF-7/doxoR cells to doxorubicin, reactivating the apoptotic response. Conclusions: The present study shows that HuR is necessary to elicit the apoptotic cell response to doxorubicin, that restoration of HuR expression in resistant cells resensitizes them to the action of this drug. Moreover we suggest a novel mechanism of pharmacoresistance based on the interplay among the doxorubicin target TOP2A, its post-transcriptionally regulator HuR and the signaling control of PKCδ.