Abstract 3091: A novel mechanism of hypoxia-mediated cell cycle dysregulation: miR-134 targets CDKN2A.
Authors: Rupaimoole Rajesha , Pradeep Sunila , Unruh Anna , Nagaraja Archana S. , Pecot Chad V. , Wu Sherry Y. , Zand Behrouz , Dalton Heather J. , Ivan Cristina , Zhang Wei , Sood Anil K. 
Published: August 2014

Background: Mechanistic understanding of the biological interplay between the stromal framework and malignant regions of the tumor remain an unmet need. In this study, we examined the molecular networks of interplay between hypoxia and miRNA deregulation. Study methods and results: We profiled the expression of miRNAs in high-grade ovarian cancer cells upon exposure to hypoxia (1% O2) using a microRNA microarray (Agilent v14). In A2780 and Skov3 cell lines, using a statistical cutoff p&lt;0.05 and fold change &gt;1.5, normoxia vs. short term (48 hrs) and long-term (6 days) hypoxia exposure, we found that 23 MicroRNAs were commonly down regulated, whereas 11 were upregulated. Among these, miR-134 was validated to be overexpressed under hypoxic conditions by 3-4 fold for both time-points. Upon extensive bioinformatic analyses, miR-134 was predicted to target CDKN2A (Miranda, microT, PITA, RNA22, TargetScan). CDKN2A is a protein involved in cell cycle control, precisely regulating cell cycle G1 control through complexing with CDK4. CDKN2A was down-regulated under hypoxia by 55% and was inversely correlated with miR-134 expression in the cell lines tested. Quantitative PCR analysis of pri-miR-134 under hypoxia exposure showed a more than 100-fold increase in expression compared to normoxia, suggesting a transcriptional regulatory event leading to high miR-134 across the panel of cell lines tested. Upon transfection of A2780 cells with miR-134 mimics, we observed consistent down-regulation of CDKN2A by &gt;40%. To understand its functional relevance, migration, invasion, proliferation and viability assays were performed. We observed increases in cell proliferation (from 12% to 41%) in miR-134 transfected cells compared to control in the cell lines tested (p&lt;0.05). Orthotopic mouse ovarian tumors treated with an anti-VEGF antibody were found to be substantially hypoxic and showed an inverse correlation between CDKN2A and miR-134. In tumor samples from patients with ovarian cancer, we observed strong negative correlation (r = -0.63, p&lt;0.01) between CDKN2A and miR-134, validating the study at a human level. Discussion: This study provides new insights into the role of miRNA-134 in regulating cell cycle in the context of hypoxia mediated signaling. These findings may have implications for new approaches targeted at curbing the deleterious effects of hypoxia on tumor growth.

Citation Format: Rajesha Rupaimoole, Sunila Pradeep, Anna Unruh, Archana S. Nagaraja, Chad V. Pecot, Sherry Y. Wu, Behrouz Zand, Heather J. Dalton, Cristina Ivan, Wei Zhang, Anil K. Sood. A novel mechanism of hypoxia-mediated cell cycle dysregulation: miR-134 targets CDKN2A. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3091. doi:10.1158/1538-7445.AM2013-3091