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

Abstract

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 b-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 E-cadherin is lost from the cell membrane. The AJ components b-catenin and p65 are also lost from sites of cell-cell contact and are relocalized to the nucleus with a concomitant increase in b-catenin-dependent and nuclear factorjB (NF-jB)-dependent signaling following RASSF8 depletion. RASSF8 may also be required to maintain actin -cytoskeletal organization since immunofluorescence analysis shows a striking disorganization of the actincytoskeleton 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.

Epigenetic inactivation of the RASSF10 candidate tumor suppressor gene is a frequent and an early event in gliomagenesis

“…There is some evidence that RASSF7 expression is upregulated in certain cancers, hence it may promote cancer development, whereas the majority of the classical RASSF genes show loss of gene expression in tumors and exhibit tumor suppressor properties. There is increasing evidence that RASSF8 may act as a tumor suppressor gene in lung cancer; we have demonstrated that RASSF8 is essential for maintaining adherens junction function in epithelial cells and has a role in epithelial cell migration (Lock et al, 2010). Drosophila RASSF8 (dRASSF8) has been shown to interact with Drosophila ASSP (dASSP) and this interaction is conserved in mammals.…”

RASSF tumor suppressor gene family: Biological functions and regulation

“…Collectively, these findings support the notion that RASSF8 regulates tumor development. Additional functions of RASSF8 include cell-cell adhesion due to its association with adherens junction linked to b-catenin/E-cadherin function [4]. Wound healing assays exhibited increased cell migration in cells lacking RASSF8 expression, suggesting that loss of RASSF8 may contribute to tumor aggressiveness [4].…”

“…These proteins were named due to the presence of a Ras association (RA) domain in their N-terminus or C-terminus. The RA domain potentially interacts with the Ras GTPase family of proteins [2] that control a number of cellular processes including membrane trafficking, apoptosis, and proliferation [1][2][3][4][5]. Direct association with K-Ras has been only observed for RASSF2, 4, 5A, 6 and 9 [6][7][8].…”

BCLAF1 and its splicing regulator SRSF10 regulate the tumorigenic potential of colon cancer cells

“…27) and RASSF8 (ref. 28), which are often downregulated in human colon cancers. However, for the known targets reported for BCLAF1, such as CCND1 and TP53, there were no significant changes observed after BCLAF1-L knockdown ( Supplementary Fig.…”