The loss of EBP50 expression potentiates cell transformation

KREIMANN EL; MORALES FC; TAKAHASHI Y; GEORGESCU MM

Anaheim Convention Center. Anaheim, Orange County, CA, USA

Congreso; 96th Annual Meeting of the American Association for Cancer Research, AACR.; 2005

American Association for Cancer Research

EBP50 is an adaptor protein containing two PDZ domains that interacts with the Ezrin-radixin-moesin (ERM) family of proteins. The ERM proteins link membrane proteins to the actin cytoskeleton. They are involved in epithelial cell polarization, cell shape determination, and are important components of transduction pathways triggered by EGF and PDGF. It has been shown that PDGF stimulation induces ruffling of the cell membrane and that these actin-rich protrusions or ruffles are the major driving force for cell movements. Because EBP50 also binds to PDGF receptor and enhances its effect, we believe that EPB50 could be involved in ruffle formation and cell migration. We generated EBP50 deficient mice, in order to define the importance of the interaction between EBP50 and ERM proteins in cell growth and motility, as well as the effect on cell transformation.                  Mouse embryonic fibroblasts (MEFs) from the EBP50 (-/-) and EBP50 (+/+) littermates were isolated from E14 embryos. After individual isolation, the MEFs with identical genotype were pooled and the resulting EBP50 (-/-) and (+/+) populations were immortalized with SV40 t-antigens. We performed migration and proliferation assays to characterized the EBP50 (-/-) and (+/+) MEFs in the parental, and SV40 t-antigen immortalized populations.  We also studied the anchorage-independent growth of the immortalized by culturing these cells in soft agar for 5 weeks.  The cell migration was studied using the wound-healing assay.                 No difference in proliferation between EBP50 (-/-) and (+/+) was apparent. However, we found alterations in the migration pattern of the parental and immortalized EBP50 (-/-) and (+/+) cells. Briefly, the migration of the EBP50 (-/-) MEFs is more spread and there is more cell movements compared with the (+/+); in the immortalized ones the migration of the EBP50 (-/-) MEFs is faster compared to the (+/+). In addition, only the EBP50 (-/-) MEFs were capable of forming colonies in soft agar, while no colonies grew in the EBP50 (+/+) plates. These findings clearly show that the lack of the EBP50 function potentiates cell transformation. Also that the EBP50 gene could act as a tumor suppressor gene in the cell.  The molecular mechanisms underlying these effects are under investigation.