UC San Diego

Tumor suppressor RASSF2 (Ras association domain family member 2) is a key regulator of the mammalian Hippo pathway, which controls the cell cycle by balancing cell proliferation and apoptosis. RASSF2 expression is often suppressed in human cancers at both the mRNA and protein levels, however little is known about its regulation. In the present study, we found that RASSF2 protein is rapidly degraded by ubiquitin-mediated proteasomal degradation. Interestingly, RASSF2 protein with a deletion of the SARAH domain (Sav, RASSF2, Hpo) is more stable than the wildtype RASSF2, suggesting that the SARAH domain may be a target for negative regulation. Therefore, we hypothesized that one or more ubiquitin E3 ligase(s) specifically target the SARAH domain of RASSF2 to promote its degradation. We employed a shRNA-mediated approach to knockdown E3 ligases that are potentially specific to the SARAH domain of RASSF2 and monitored the changes in RASSF2 protein stability. As a result, we have identified E3 ligase ITCH as a negative regulator of RASSF2 protein stability. Knockdown of ITCH in the Kasumi-1 cell line which normally harbors low RASSF2 expression, successfully increased RASSF2 protein levels. We further show that ITCH overexpression negatively modulates the stability of both RASSF2 and an additional Hippo pathway member, MST1, in a manner dependent on the E3-ligase enzymatic activity. Our results suggest that small molecule inhibitors targeting ITCH enzyme activity may provide a novel approach for cancer therapy through the ability to restore expression of RASSF2 and other Hippo signaling pathway proteins to normal levels in cancer cells.