Abstract 2188: The N6-methyladenosine reader YTHDC1 is essential for TGF-beta-mediated metastasis of triple negative breast cancer

Abstract Metastatic triple negative breast cancer (TNBC) patients have a 5-year survival rate of just 12%. Therefore, there is an urgent need to discover novel molecular mechanisms that contribute to TNBC metastasis such as N6-methyladenosine (m6A) RNA modification. To do this, we conducted a selective CRISPR-cas9 screen of 14 m6A-regulators and identified an m6A reader protein, YTHDC1, as an essential gene for the survival of MDA-MB-231 cells. The Cancer Genome Atlas (TCGA) dataset showed that patients with poorer prognosis had elevated YTHDC1 expression, which supports a tumor-promoting role of YTHDC1. In vitro, genetic depletion of YTHDC1 significantly impaired Transwell migration and invasion of human TNBC (MDA-MB-231 and SUM159) cells. In vivo, YTHDC1 overexpressing TNBC cells engrafted into the mammary fat pad of NSG mice formed greater numbers and larger sizes of lung metastatic nodules compared to control. Conversely, YTHDC1 knockout (KO) cells produced fewer and smaller lung metastatic nodules compared to control. Together, our in vitro and in vivo data demonstrate YTHDC1’s role in promoting TNBC metastasis. To identify high confidence YTHDC1 targets, we conducted three layers of transcriptome-wide sequencing with MDA-MB-231 cells: RNA-seq (profiling differential gene expression and nuclear export), m6A-seq (profiling m6A methylation in mRNA), and RIP-seq (profiling YTHDC1 binding sites in mRNA). Pathway analysis of differentially expressed genes in MDA-MB-231 YTHDC1 KO cells identified pathways related to metastasis and TGF-β signaling, which is consistent with our phenotypic observations. Overlapping the three sequencing datasets identified SMAD3 as a YTHDC1 target mRNA. Mechanistically, YTHDC1 KO impairs the nuclear export of SMAD3 mRNA, resulting in reduced protein levels of this critical effector of TGF-β signaling. YTHDC1 KO significantly reduced Transwell migration and invasion of TGF-β induced TNBC cells and the expression of TGF-β responsive genes such as SNAIL, IL11, FN, and CTGF. Overexpression of SMAD3 was able to rescue the impaired migration and invasion of YTHDC1 KO TNBC cells and restore the expression of TGF-β responsive genes following TGF-β treatment. We also demonstrated that the oncogenic role of YTHDC1 is mediated through its recognition of m6A, as m6A-binding defective mutants of YTHDC1 were unable to rescue impaired Transwell migration and invasion of YTHDC1 KO TNBC cells. In summary, our study reveals that TNBC metastasis is mediated at least in part through YTHDC1’s function in promoting SMAD3 expression to augment the TGF-β signaling cascade. Citation Format: Brandon Tan, Wei Liu, Keren Zhou, Emily Prince, Lu Yang, Yangchan Li, Ying Qing, Li Han, Zhicong Zhao, Rui Su, Chun-Wei Chen, Jianjun Chen. The N6-methyladenosine reader YTHDC1 is essential for TGF-beta-mediated metastasis of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2188.