Abstract 1897: P53-induced microRNA-30e suppresses colorectal cancer cell migration and invasiveness by regulating integrins

Abstract

Abstract Alterations in miRNA expression have been implicated in the pathogenesis of colorectal cancer (CRC). Importantly, the p53 tumor suppressor is not only one of the most commonly altered genes in CRC, but it also regulates the expression of several protein and non-protein coding genes. In this study, we investigated a potential function of p53 in regulating miRNAs that mediate human CRC progression. MiRNA microarrays were performed on human isogenic CRC cell line pairs (p53 wt and ko, a generous gift by Bert Vogelstein) to identify deregulated miRNAs attributable to p53 loss. The analysis revealed members of the miR-30 family, miR-30e particularly, as the most significantly downregulated group in the p53 knock-out cells compared to the wild type. To elucidate the impact of p53 loss on the expression of miR-30e, we also performed p53 overexpression and silencing in CRC cells harboring wt p53. Indeed, p53 silencing resulted in a reduction of miR-30e expression, whereas an increase of mature miR-30e was observed in CRC cells treated with Nutlin-3a, a specific MDM2 antagonist which is known to increase p53 expression or activity. Next, we determined the miR-30e gene promoter region and found putative TP53 transcription factor binding motifs approximately 1 kb upstream the miR-30e genomic sequence by using in silico tools. The regions containing the predicted TF binding sites were cloned (pGL3-30e) and luciferase reporter gene assays were performed to determine if p53 indeed regulates the miR-30e promoter. Interestingly, promoter reporter activity of pGL3-30e was significantly transactivated in cell lines carrying a wt p53, and this response was further enhanced in the presence of Nutlin 3a. However, the activity of pGL3-30e was not affected in p53 ko cells. Our observations indicate that the tumor suppressor p53 is able to induce the expression of miR-30e in colorectal cancer cell lines. In addition, we identified two promising candidate targets of miR-30e within the integrin family. We demonstrated that miR-30e directly interacts with the 3’ UTRs of the mRNAs of these two candidates and downregulates their expression at both the mRNA and protein level. Functionally, we found that the forced expression of miR-30e led to an integrin-mediated reduction of cell motility, invasion, cell proliferation, and adhesion to the extracellular matrix (ECM). These results were corroborated by an inverse correlation of miR-30e and the identified integrins in a panel of resected CRC tissues. Taken together, our findings indicate that miR-30e is a p53 induced miRNA whose loss/downregulation in CRC orchestrates an integrin driven enhancement of in vitro tumor migration, invasion, proliferation and adhesion to the ECM. Ongoing experiments seek to recapitulate the above findings in vivo and also explore the impact of the p53/miR-30e/ITGs axis on metastasis. Citation Format: Sara Laudato, Nitin Patil, Jörg H. Leupold, Mohammed Abba, Heike Allgayer. P53-induced microRNA-30e suppresses colorectal cancer cell migration and invasiveness by regulating integrins. [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 1897.