Alternate RASSF1 Transcripts Control SRC Activity, E-Cadherin Contacts, and YAP-Mediated Invasion

Abstract

SummaryTumor 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.

TGF-β Targets the Hippo Pathway Scaffold RASSF1A to Facilitate YAP/SMAD2 Nuclear Translocation

“…Similarly, the YAP/TEAD/Smad3/p300 complex was found to drive CTGF expression in malignant mesothelioma, resulting in increased tumor growth (Fujii et al, 2012). We recently showed that RASSF1A also inhibits YAP1-mediated cell invasion in an SRC kinase-dependent manner (Vlahov et al, 2015), which is in line with clinical correlations with increased invasiveness and metastasis in RASSF1-methylated tumors (Bilgrami et al, 2014;Fackler et al, 2003;Korah et al, 2013). Here we provide evidence that RASSF1A eliminates TGF-b-induced invasion, in keeping with increasing reports supporting a role for RASSF1A in invasion (Dubois et al, 2016;Lee et al, 2016), offering further mechanistic insights into how RASSF1A elicits its tumor-suppressive function ( Figure 4D).…”

“…Interestingly, the TGF-b gene set showed significance across all four analyses where RASSF1A gene expression is affected (p = 0.0001). We have recently demonstrated that loss of RASSF1A replicates a partial epithelial to mesenchymal transition (EMT) (Vlahov et al, 2015), a tumor progression phenotype also promoted by TGF-b signaling. Because TGFb signaling had also been linked to RASSF1A stability (Bhaskaran and Souchelnytskyi, 2008), we reasoned that TGF-b may regulate endogenous RASSF1A and that the absence of expression in methylated tumors may affect TGF-b transcriptional output.…”

Ras signaling through RASSF proteins

“…However, it may also promote growth [27]. This may involve the activation of β-catenin [28] and src [29]. RASSF1C is less studied than RASSF1A and there is little information describing the role of RASSF1C in Ras signaling.…”

Phosphorylation of Tyr188 in the WW domain of YAP1 plays an essential role in YAP1-induced cellular transformation

“…17 Most recently, using advanced tools of proteomics and proximity ligation, the O'Neill laboratory documented that Ras association domain family 1C (RASSF1C) was able to target Src and Yes kinases to epithelial cell to cell junctions to promote tyrosine phosphorylation of YAP along with b-catenin and E-cadherin and thereby promoting YAP-mediated invasion of carcinomas. 16 All these studies implicate tyrosine phosphorylation in the function of YAP. In this report we explore one mechanistic facet of tyrosine-phosphorylated YAP in breast cancer biology.…”

“…9,10 Although YAP1 was originally identified as a binding partner for the Src family member, Yes protein-tyrosine kinase, 11,12 only recent studies raised the possibility that YAP1 could be phosphorylated at tyrosine sites in certain signaling scenarios. 13,14,15,16 In terms of context-dependent signaling it is important to note that the immunoprecipitated endogenous YAP1 protein from primary chicken embryo fibroblasts, which were labeled with radioactive orthophosphate, showed that only Serine was decorated by phosphate in the phospho-amino acid analysis. 12 The Stein laboratory was the first to report that tyrosine phosphorylation of YAP is necessary for its interaction with the Runx2 transcription factor, as well as for its subsequent nuclear trafficking.…”