The RASSF8 candidate tumor suppressor inhibits cell growth and regulates the Wnt and NF-κB signaling pathways

The Ras-assocation domain family (RASSF) of tumor suppressor proteins until recently contained six proteins named RASSF1–6. Recently, four novel family members, RASSF7–10, have been identified by homology searches for RA-domain-containing proteins. These additional RASSF members are divergent and structurally distinct from RASSF1–6, containing an N-terminal RA domain and lacking the Sav/RASSF/Hpo (SARAH) domain. Here, we show that RASSF8 is ubiquitously expressed throughout the murine embryo and in normal human adult tissues. Functionally, RNAi-mediated knockdown of RASSF8 in non-small-cell lung cancer (NSCLC) cell lines, increased anchorage-independent growth in soft agar and enhanced tumor growth in severe combined immunodeficiency (SCID) mice. Furthermore, EdU staining of RASSF8-depleted cells showed growth suppression in a manner dependent on contact inhibition. We show that endogenous RASSF8 is not only found in the nucleus, but is also membrane associated at sites of cell–cell adhesion, co-localizing with the adherens junction (AJ) component β- catenin and binding to E-cadherin. Following RASSF8 depletion in two different lung cancer cell lines using alternative small interfering RNA (siRNA) sequences, we show that AJs are destabilized and Ecadherin is lost from the cell membrane. The AJ components β-catenin and p65 are also lost from sites of cell–cell contact and are relocalized to the nucleus with a concomitant increase in β-catenindependent and nuclear factor-κB (NF-κB)-dependent signaling following RASSF8 depletion. RASSF8 may also be required to maintain actin -cytoskeletal organization since immunofluorescence analysis shows a striking disorganization of the actin- cytoskeleton following RASSF8 depletion. Accordingly, scratch wound healing studies show increased cellular migration in RASSF8-deficient cells. These results implicate RASSF8 as a tumor suppressor gene that is essential for maintaining AJs function in epithelial cells and have a role in epithelial cell migration.