RACK1 down-regulates levels of the pro-apoptotic protein Fem1b in apoptosis-resistant colon cancer cells

Abstract

Evasion of apoptosis plays an important role in colon cancer progression. Following loss of the Apc tumor suppressor gene in mice, the gene encoding Fem1b is up-regulated early in neoplastic intestinal epithelium. Fem1b is a pro-apoptotic protein that interacts with Fas, TNFR1, and Apaf-1, and increased expression of Fem1b induces apoptosis of cancer cells. Fem1b is a homolog of FEM-1, a protein in Caenorhabditis elegans that is negatively regulated by ubiquitination and proteasomal degradation. To study Fem1b regulation in colon cancer progression, we used apoptotis-sensitive SW480 cells, derived from a primary colon cancer, and their isogenic, apoptosis-resistant counterparts SW620 cells, derived from a subsequent metastatic lesion in the same patient. Treatment with proteasome inhibitor increased Fem1b protein levels in SW620 cells, but not in SW480 cells. In SW620 cells we found that endogenous Fem1b co-immunoprecipitates in complexes with RACK1, a protein known to mediate ubiquitination and proteasomal degradation of other pro-apoptotic proteins and to be up-regulated in colon cancer. Full-length Fem1b, or the N-terminal region of Fem1b, associated with RACK1 when co-expressed in HEK293T cells, and RACK1 stimulated ubiquitination of Fem1b. RACK1 over-expression in SW620 cells led to down-regulation of Fem1b protein levels. Conversely, down-regulation of RACK1 led to up-regulation of Fem1b protein levels, associated with induction of apoptosis, and this apoptosis was inhibited by blocking Fem1b protein up-regulation. In conclusion, RACK1 down-regulates levels of the pro-apoptotic protein Fem1b in metastatic, apoptosis-resistant colon cancer cells, which may promote apoptosis-resistance during progression of colon cancer.