Abstract 3532: Hypoxia is a master regulator of Drosha- and Dicer-dependent miRNA biogenesis in cancer
Authors: Rupaimoole Rajesha , Ivan Cristina , Pecot Chad , Wu Sherry , Pradeep Sunila , Zand Behrouz , Nagaraja Archana , Gharpure Kshipra , Dalton Heather , Sadaoui Nouara , Zhang Wei , Lopez-Berestein Gabriel , Sood Anil 
Published: May 2015

Abstract
Background: Drosha and Dicer are 2 key enzymes involved in miRNA biogenesis. Individual regulators of Dicer (e.g let7) have been implicated, but the underlying mechanisms are currently poorly understood. Here, we identified new mechanisms of deregulation of miRNA biogenesis in tumor cells, whereby hypoxia causes reduction in Drosha and Dicer. Methods and Results: Differential levels of Drosha and Dicer was found between tumor samples and their expression correlated negatively with CA9, a hypoxia marker (Drosha vs. CA9 r=-0.65, Dicer vs. CA9 r=-0.69; p&lt;0.05). We further found that Drosha and Dicer are down-regulated by &gt;70% in hypoxic compared to normoxic conditions across a panel of ovarian and breast cancer cell lines and microdisected tumor tissues. Analysis of TCGA data showed that the expressions of Drosha and Dicer or hypoxia are independent predictors of poor patient survival. Furthermore, RNA-sequencing and mature miRNA array analyses both revealed a significant disruption in miRNA maturation when cells were exposed to hypoxia. Through ChIP assays and promoter reporter studies, we found that ETS1 and ELK1 are responsible for transcriptionally repressing Drosha expression under hypoxia by binding to its promoter region. We further show that ETS1/ELK1 recruit ARID5B and HDAC1 to methylate Drosha promoter. In contrast to Drosha, Dicer level is altered post-transcriptionally under hypoxia. Several miRNAs that are up-regulated under hypoxia are predicted to target Dicer. By transfecting tumor cells with miRNA mimics or anti-miR constructs along with 3′ UTR luciferase assays, we showed that miR-630 is a strong regulator of Dicer in hypoxic conditions. Bioinformatics analysis of downregulated miRNAs and their gene targets from RNA-seq data identified novel miRNA (let7a, miR-30c, miR-135a, miR-146a)-mRNA (RHOB, TAGLN, JUN, CTGF, SERTAD, TXNIP, JAG1) interactions aiding metastasis under hypoxia. Given the potential for anti-VEGF therapies in inducing significant hypoxia in tumors, we examined the biological effects of anti-miR-630 or siETS1/ELK1 in orthotopic ovarian and breast cancer mouse models. We first demonstrated that in vivo tumor samples treated with anti-VEGF therapy results in significant Drosha and Dicer down-regulation. ETS1+ELK1siRNA or anti-miR630 DOPC nanoparticles in vivo rescued Drosha and Dicer expression under anti-VEGF therapy, resulting in a significant reduction in cancer metastasis (p&lt;0.05). Conclusion: In summary, these findings provide new insights into the mechanisms underlying Drosha and Dicer downregulation following hypoxia and resultant effects on cancer metastasis.
Citation Format: Rajesha Rupaimoole, Cristina Ivan, Chad Pecot, Sherry Wu, Sunila Pradeep, Behrouz Zand, Archana Nagaraja, Kshipra Gharpure, Heather Dalton, Nouara Sadaoui, Wei Zhang, Gabriel Lopez-Berestein, Anil Sood. Hypoxia is a master regulator of Drosha- and Dicer-dependent miRNA biogenesis in cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3532. doi:10.1158/1538-7445.AM2014-3532