Abstract 2529: TTF-1/NKX2-1 induced miR-532-5p targets KRAS and MKL2 oncogenes and causes apoptosis in lung adenocarcinoma cells

Abstract

Abstract Our group and others have previously identified TTF-1/NKX2-1 as a lineage survival oncogene in lung adenocarcinoma. Subsequent studies revealed double-edged sword characteristics of TTF-1 in the development of lung adenocarcinoma. TTF-1 elicits lineage-survival signaling by inducing transcriptional targets such as ROR1, while TTF-1 also inhibits tumor progression by decreasing cell motility through direct activation of genes such as MYBPH. It is thus clear that a more comprehensive picture of this still enigmatic lineage-survival oncogene needs to be elucidated. Whereas most of the previous studies on TTF-1 have focused on its regulation of protein-coding genes, very little is known about regulation of microRNAs by TTF-1. In the present study, we combined in vivo patient data with in vitro data from lung adenocarcinoma cell lines, aiming at identifying miRNAs, which are transcriptionally regulated by TTF-1. Our integrative approach identified miR-532-5p as a microRNA regulated by TTF-1. Expression of miR-532-5p decreased, when cell lines with a high endogenous level of TTF-1 were transfected with siRNA against TTF-1, while it was increased by overexpression of exogenous TTF-1 in TTF-1-negative cell lines. By ChIP and luciferase assays, TTF-1 was found to induce miR-532-5p expression through its binding to the MIR532 promoter region, identifying miR-532-5p as a direct target of TTF-1. We next employed microarray analysis and target prediction using TARGETSCAN to search for potential targets of miR-532-5p. We consequently identified KRAS and MKL2 as candidates and confirmed this by qRT-PCR, Western blot and luciferase analyses. Overexpression of miR-532-5p induced apoptosis and inhibited colony formation in lung adenocarcinoma cell lines, while depletion of MKL2 phenocopied the effects of miR-532-5p introduction, similarly exhibiting occurrence of marked apoptosis. In addition, miR-532-5p significantly decreased tumor formations in vivo in a mouse xenograft model. It was also of note that miR-532-5p inhibited lung adenocarcinoma cell lines regardless of the presence or absence of KRAS mutations. In summary, we have found that TTF-1 transactivates miR-532-5p, which potently induces apoptosis in lung adenocarcinoma cells. MiR-532-5p directly targets MKL2, which we have also uncovered to be crucially involved in lung adenocarcinoma survival. In addition, while previous reports have shown that TTF-1 inhibits KRAS-driven tumorigenesis, this study identifies KRAS as a target of miR-532-5p, providing their potential mechanistic link. Our results thus shed light on how TTF-1 plays a tumor-suppressive role in the process of lung carcinogenesis, which might ultimately lead to development of a novel therapeutic strategy for this devastating disease. Citation Format: Sebastian Griesing, Taisuke Kajino, Mei Chee Tai, Zhuoran Liu, Masahiro Nakatochi, Motoshi Suzuki, Takashi Takahashi. TTF-1/NKX2-1 induced miR-532-5p targets KRAS and MKL2 oncogenes and causes apoptosis in lung adenocarcinoma cells [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 2529. doi:10.1158/1538-7445.AM2017-2529