Abstract 2131: m6A demethylase ALKBH5 promote tumor growth through IGF2BPs' recognition of m6A modified CDKN1A in non-small lung cancer
Authors: Tsuchiya Kazuo , Yoshimura Katsuhiro , Inoue Yusuke , Iwashita Yuji , Ohta Tsutomu , Yamada Hidetaka , Watanabe Hirofumi , Tao Hong , Kawase Akikazu , Tanahashi Masayuki , Ogawa Hiroshi , Funai Kazuhito , Shinmura Kazuya , Suda Takafumi , Sugimura Haruhiko 
Published: July 2021

Abstract
Background: N6-methyladenosine (m6A), the most prevalent and abundant RNA modification on eukaryote mRNA, is regulated by the methylation complex (writer) and demethylase (eraser), subsequently being recognized by RNA-binding protein (reader). As an m6A eraser, ALKBH5 has been shown to promote the development of breast cancer and glioblastoma. In lung cancer, two experiments described contradictory findings that one is a cancer-promoting effect and the other is a cancer-suppressing effect. Accordingly, the carcinogenic mechanisms of ALKBH5 are not completely elucidated in lung cancer. The purpose of this study is to investigate the role of m6A demethylase ALKBH5 in lung cancer.
Method: We investigated the relationship between eraser protein expression level by immunostaining (IHC) and prognosis in 629 patients with non-small cell lung cancer (NSCLC). Small interfering RNA transfection was used to down-regulate ALKBH5 in PC9 and A549 cells. Then, cell function assays (cell proliferation, migration ability, cell cycle, apoptosis) were conducted in cells. In addition, mRNA and m6A expression under the ALKBH5 knockdown were comprehensively analyzed by microarray. The m6A target genes were identified by Methylated RNA immunoprecipitation (MeRIP) assay using m6A antibody. Furthermore, the mRNA stability and protein expression of the m6A target gene was examined.
Result: High expression of ALKBH5 showed a poor prognosis (p&lt;0.001). ALKBH5 knockdown suppressed cell proliferation ability in PC9 and A549. G1 arrest and increased apoptotic cells were observed by ALKBH5 knockdown in cells. In addition, overexpression of ALKBH5 increased the cell proliferation ability in HEK293 and BEAS2B. Comprehensive analysis of microarray revealed up-regulation of CDKN1A, E2F1, GADD45A, TIMP3, and DNMT3B, and down-regulation of CASP14 and CCNG2 by ALKBH5 knockdown. m6A microarray showed up-regulation of m6A modification in 22 genes by ALKBH5 knockdown. MeRIP qPCR with fragmented mRNA showed m6A level in 3'UTR regions of CDKN1A, TIMP3, E2F1, DNMT3B, and CCNG2 were increased by ALKBH5 knockdown. The CDKN1A mRNA was stabilized by ALKBH5 knockdown, and the increased expression of CDKN1A was down-regulated by IGF2BPs knockdown. The protein expression of p21, MFAP5, and TIMP3 was up-regulated by ALKBH5 knockdown. The up-regulated expression of p21 was p53-independent.
Conclusion: High expression of ALKBH5 has an unfavorable prognostic value in NSCLC. ALKBH5 knockdown increases m6A of 3'UTR of CDKN1A, which is recognized by IGF2BPs and enhances mRNA stability. The increased expression of p21 leads to the regulation of the cell cycle, regulating cell proliferation ability. Taken together, ALKBH5 plays a cancer-promoting role in non-small cell lung cancer.
Citation Format: Kazuo Tsuchiya, Katsuhiro Yoshimura, Yusuke Inoue, Yuji Iwashita, Tsutomu Ohta, Hidetaka Yamada, Hirofumi Watanabe, Hong Tao, Akikazu Kawase, Masayuki Tanahashi, Hiroshi Ogawa, Kazuhito Funai, Kazuya Shinmura, Takafumi Suda, Haruhiko Sugimura. m6A demethylase ALKBH5 promote tumor growth through IGF2BPs' recognition of m6A modified CDKN1A in non-small lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2131.