p120 RasGAP and ZO-2 promote the tumor suppressor function of p190A RhoGAP

ARHGAP35 encoding p190A RhoGAP (p190A) was identified by GWAS as a major human cancer gene. Our published studies have determined that p190A is a tumor suppressor that activates the canonical Hippo pathway. To elucidate how p190A signals in this context, we performed mass spectrometry to identify p190A binding proteins. Two interactors were identified significantly more frequently than others: p120 RasGAP (p120) and ZO-1. Originally p190A was cloned via its direct binding to p120, hence validating our approach. In addition, we found that p190A also binds to ZO-2. The interaction of p190A with ZO-2 is dependent on p120, thus suggesting formation of a ternary complex. Next, we determine that both p120 and ZO-2, but not ZO-1, are necessary for p190A to activate LATS kinases and suppress tumorigenesis in a xenograft mouse model. Moreover, the interactions with p120 and ZO-2 are essential for transcriptional modulation by p190A, including the CDH1 gene encoding E-cadherin, which we previously have demonstrated is essential for p190A signaling. Collectively, this work identifies a novel tumor suppressor interactome of p190A, which includes ZO-2, an established constituent of the Hippo pathway, and p120, which in spite of its strong association with Ras signaling, is essential for p190A to activate LATS kinases. H661 cells, which harbor defined ARHGAP35 loss-of-function alterations, were restored with wild-type p190A expression to generate H661-p190A cells. H661 cells were also restored with p190A mutant (Y1087F+Y1105F), we abbreviate this mutant p190A(Y2F). To generate H661-p190A cells with knockout of TJP1 and TJP2, H661-p190A cells were transduced with lentivirus encoding Cas9 and TJP1 or TJP2 sgRNA. Global transcriptomes were profiled by bulk RNA-Seq for H661-control cells, H661-p190A cells, H661-p190A(Y2F) cells, H661-p190A+TJP1-ko cells, H661-p190A+TJP2-ko cells. To investigate the H661 cell line specific Hippo pathway gene sets, we generated H661+p190A+LATS1/2-kd cells as well as H661+p190A as control, for which we have tested their function in our previous Oncogene 2020 paper (doi.org/10.1038/s41388-020-1385-2).