Abstract 2337: miR-6883 and family miRNAs induce G1-arrest in colon cancer cells by targeting CDK4/6

Abstract

Abstract Dysregulation of the CDK4/6-Rb pathway is a hallmark of various tumor types. Cancer cells subvert different cell cycle checkpoints to continue unchecked growth and proliferation. Some common mechanisms of overcoming cell cycle checkpoints involve loss of tumor-suppressor proteins such as p16 INK4A or Retinoblastoma (Rb). Alternately, cancer cells can also increase expression of oncogenes like cyclin D1, other CDKs by gene amplification or gene translocation. Thus, targeting different oncogenes in the cell cycle pathway, particularly CDKs has been considered an important therapeutic strategy. Events of overexpression of cell cycle oncogenes and suppression of tumor suppressors are mutually exclusive and tumor-type specific. In colorectal cancer (CRC), it has been shown that there is overexpression of CDK4/6 . We conducted TCGA analysis of 50 matched tumor and normal CRC tumors, and validated that CDK4 and CDK6 are significantly overexpressed in patient tumor samples by RNA-seq (p= 1.55 e-09 and p= 4.439 e-05). Thus, ongoing clinical trials for CRC patients with advanced disease are currently evaluating the efficacy of CDK4/6 inhibitors as single agents and in combination with chemotherapy (J Clin Oncol 33, 2015 ,suppl 3; abstr 626; Zhang 2016). We evaluated the efficacy of miRNAs targeting CDK4/6 as novel therapeutics in CRC. We performed in silico analysis using TargetScan to identify miRNAs that can target the 3’UTR regions of CDK4/6. We have identified a novel family of miRNAs (based on seed sequence similarity) that can potently target CDK4/6 mRNA and thereby reduce both the RNA and protein levels of CDK4/6 in cancer cells. Of the four miRNAs in the family, we chose to characterize miR-6883-5p and miR-149* in a panel of CRC, melanoma and pancreatic cancer cell lines. Our preliminary TCGA analysis in 11 matched T/N samples shows miR-149* expression is significantly lost (p= 0.0049) in CRC samples and this further positively correlates with the stage of the tumor. Ongoing in vitro studies using miRNA mimics in CRC cell lines indicate that both miR-6883-5p and miR-149* have anti-proliferative effects as assayed by CellTiterGlo (CTG) and MTT. Further, both miRNAs induce G1-arrest as a consequence of downregulation of CDK4 and CDK6 in all cell lines and we also observe induction of apoptosis in a subset of CRC cell lines. Knockdown of CDK4 and CDK6 mimics the phenotypes observed with restoring expression of both miR-6883 and miR-149* in CRC cell lines. Combination of miR-6883 with FDA-approved drugs Irinotecan and 5-FU showed strong synergy and led to apoptosis as assayed by CTG and PARP cleavage. Our ongoing work is further looking into the mechanisms of synergy and identifying additional target genes in CRC cell lines. Thus, our novel miRNA based strategy to target CDK4 has potential to translate as both a single agent/combinatorial therapy and to identify biomarkers of response, which is critical for understanding the clinical results seen with CDK inhibitors. Citation Format: Amriti R. Lulla, Margret B. Einarson, Yan Zhou, Michael Slifker, David T. Dicker, Wafik S. El-Deiry. miR-6883 and family miRNAs induce G1-arrest in colon cancer cells by targeting CDK4/6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2337. doi:10.1158/1538-7445.AM2017-2337