The RASSF8 candidate tumor suppressor inhibits cell growth and regulates the Wnt and NF-kappaB 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 beta-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 beta-catenin and p65 are also lost from sites of cell-cell contact and are relocalized to the nucleus with a concomitant increase in beta-catenin-dependent and nuclear factor-kappaB (NF-kappaB)-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.
Department of Medical and Molecular Genetics, Institute of Biomedical Research, University of Birmingham, Birmingham, UK.
F E
FE
Lock
N
N
Underhill-Day
T
T
Dunwell
D
D
Matallanas
W
W
Cooper
L
L
Hesson
A
A
Recino
A
A
Ward
T
T
Pavlova
E
E
Zabarovsky
M M
MM
Grant
E R
ER
Maher
A D
AD
Chalmers
W
W
Kolch
F
F
Latif
D022005:Adherens Junctions / Q000502:physiology; D000818:Animals; D015820:Cadherins / Q000502:physiology; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D003599:Cytoskeleton / Q000737:chemistry; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D016328:NF-kappa B / Q000235:genetics / Q000502:physiology*; D011401:Promoter Regions, Genetic; D015398:Signal Transduction / Q000502:physiology*; D051996:Transcription Factor RelA / Q000032:analysis; D025521:Tumor Suppressor Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D051153:Wnt Proteins / Q000502:physiology*; D014982:Xenopus laevis
D015820:Cadherins; D016328:NF-kappa B; C512680:RASSF8 protein, human; D051996:Transcription Factor RelA; D025521:Tumor Suppressor Proteins; D051153:Wnt Proteins