Abstract 3810: ARID1A is a novel HuR target: Implications in breast cancer radiotherapy
Authors: Andrade Daniel , Griffith James , Mehta Meghna , Muralidharan Ranganayaki , Berry William , Gorospe Myriam , Ramesh Rajagopal , Munshi Anupama 
Published: July 2016

Abstract
ARID1A is part of the chromatin remodeling complex SWI/SNF, which can modulate transcription positively or negatively. The role of ARID1A in cancer is controversial. It is described as a tumor suppressor since its expression is found lost in several types of cancer. However, recent investigations suggest that ARID1A could also be an important target to sensitize cancer cells to chemotherapy and radiation. HuR, an important RNA binding protein, post-transcriptionally regulates specific mRNAs by binding to the 3’ untranslated region (UTR) of its target mRNAs. Through this action, HuR regulates the expression of its target genes by facilitating either RNA stability or protein translation, thereby affecting several signaling pathways including cell-cycle, DNA damage response and apoptosis.
In order to identify novel mRNAs regulated by HuR that could be targeted for breast cancer therapy, we performed a ribonucleoprotein immunoprecipitation (RNP-IP) microarray using an HuR antibody on irradiated and non-irradiated samples from triple negative breast cancer cell lines. This assay showed ARID1A to be potentially regulated by HuR. Accordingly, analysis of the AU-rich elements (ARE) database of the University of Vienna identifies three potential HuR binding sites on ARID1A 3’UTR, two of which are highly conserved in 100 vertebrate species. To demonstrate that HuR post-transcriptionally regulates ARID1A, we first evaluated the effect of HuR expression on ARID1A mRNA and protein levels in breast cancer cell lines. Genetic or pharmacological inhibition of HuR led to a decrease in ARID1A mRNA and protein levels. Conversely, HuR forced expression led to an increase in ARID1A levels both at the mRNA and protein levels. We also evaluated the effect of HuR on the stability of ARID1A mRNA. In cells where HuR is genetically inhibited, ARID1A mRNA half-life is dramatically reduced compared to the control. These results demonstrate that HuR positively regulates ARID1A by promoting the stability of ARID1A mRNA. To test whether this regulation is exerted by direct interaction of HuR on ARID1A mRNA, we performed an RNP-IP assay, where we pulldown HuR and analyzed the presence of ARID1A mRNA by RT-PCR. Consistent with our previous results, HuR was found to physically interact with ARID1A, thereby promoting its stabilization. Most importantly, through clonogenic assays, we found that genetic inhibition of either HuR or ARID1A results in radiation sensitivity of TNBC cell lines. Conversely, forced expression of ARID1A promotes radiation resistance.
Our results show that (i) ARID1A is a novel HuR target and that (ii) it can dictate radio resistance in HuR overexpressing cancer cells. ARID1A is known to regulate cell cycle and to promote DNA damage repair, which can contribute to resistance to therapy. Altogether, our findings further support an oncogenic role for ARID1A making it an important target for breast cancer treatment.
Citation Format: Daniel Andrade, James Griffith, Meghna Mehta, Ranganayaki Muralidharan, William Berry, Myriam Gorospe, Rajagopal Ramesh, Anupama Munshi. ARID1A is a novel HuR target: Implications in breast cancer radiotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3810.