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Summary Tumor progression to invasive carcinoma is associated with activation of SRC family kinase (SRC, YES, FYN) activity and loss of cellular cohesion. The hippo pathway-regulated cofactor YAP1 supports the tumorigenicity of RAS mutations but requires both inactivation of hippo signaling and YES-mediated phosphorylation of YAP1 for oncogenic activity. Exactly how SRC kinases are activated and hippo signaling is lost in sporadic human malignancies remains unknown. Here, we provide evidence that hippo-mediated inhibition of YAP1 is lost upon promoter methylation of the RAS effector and hippo kinase scaffold RASSF1A. We find that RASSF1A promoter methylation reduces YAP phospho-S127, which derepresses YAP1, and actively supports YAP1 activation by switching RASSF1 transcription to the independently transcribed RASSF1C isoform that promotes Tyr kinase activity. Using affinity proteomics, proximity ligation, and real-time molecular visualization, we find that RASSF1C targets SRC/YES to epithelial cell-cell junctions and promotes tyrosine phosphorylation of E-cadherin, β-catenin, and YAP1. RASSF1A restricts SRC activity, preventing motility, invasion, and tumorigenesis in vitro and in vivo, with epigenetic inactivation correlating with increased inhibitory pY527-SRC in breast tumors. These data imply that distinct RASSF1 isoforms have opposing functions, which provide a biomarker for YAP1 activation and explain correlations of RASSF1 methylation with advanced invasive disease in humans. The ablation of epithelial integrity together with subsequent YAP1 nuclear localization allows transcriptional activation of β-catenin/TBX-YAP/TEAD target genes, including Myc, and an invasive phenotype. These findings define gene transcript switching as a tumor suppressor mechanism under epigenetic control., Graphical Abstract, Highlights • Methylation of RASSF1A correlates with loss of hippo-inhibitory phospho-S127-YAP1 • SRC, FYN, and YES are activated by RASSF1C in RASSF1A-methylated cells • RASSF1C promotes E-cadherin internalization and reduces cell junction integrity • RASSF1A loss drives RASSF1C-YAP1/β-catenin-mediated transcription and invasion, In a wide variety of sporadic malignancies, promoter methylation of the RASSF1 gene is associated with tumor invasion and metastasis. Vlahov et al. show that the clinical phenotype is driven by both RASSF1A loss and the independently transcribed RASSF1C isoform, which promotes SRC activation, pseudo-EMT, and β-catenin/YAP1-dependent invasion of tumor cells.

SummaryHypermethylation of CpG islands in the RASSF1 promoter is one of the most frequent events identified in human cancer [1–3]. The epigenetic-driven loss of RASSF1A protein expression is observed more often in tumors of higher grade and correlates with a decreased responsiveness to DNA-damaging therapy [4–6]. Ras association domain-containing family 1A (RASSF1A) promotes apoptosis by signaling through the MST2 and LATS1 kinases, leading to stabilization of the YAP1/p73 transcriptional complex [7]. Here we provide evidence for a new pathway linking DNA damage signaling to RASSF1A via the main sensor of double-strand breaks in cells, ataxia telangiectasia mutated (ATM). We show that, upon DNA damage, RASSF1A is phosphorylated by ATM on Ser131 and is involved in the activation of both MST2 and LATS1, leading to the stabilization of p73. Furthermore, lung and ovarian tumor cell lines that retain RASSF1A expression commonly harbor polymorphisms in the region of Ser131 [8], and our analysis shows that the S131F polymorphism conveys resistance to DNA-damaging agents. Thus, we present a novel DNA damage pathway emanating from ATM that is frequently disabled in tumors via epigenetic silencing of RASSF1 or mutation of an ATM phosphorylation site.