The role of Mob-5 in the participation of ras-mediated RIE-1 cell transformation is the major focus of this revised new application. Dr. Liang hypothesizes that Mob-5 is a signaling molecule downstream of oncogenic ras. He proposes to functionally characterize the gene by identifying Mob-5-interacting protein(s) or receptor(s) and to assess its role in ras-mediated cell transformation, using a combination of methodologies including retroviral gene transfer, antisense RNA, and antibody neutralization.
The specific aims are 1) to determine the role of Mob-5 in Ras transformation of immortalized rat intestinal epithelial cells (RIE-1), and 2) to determine whether Mob-5 plays a role in human pancreatic and colorectal cancer.
The first aim will be accomplished by a) overexpressing Mob-5 protein and preparing Ab, b) surveying Mob-5 expression in rat tissues and determining subcellular location, c) determining kinetics of Mob-5 expression following stable transfection of mutant K-ras into RIE-1 cells, d) functionally characterizing Mob-5 expression following retrovirus mediated transformation of RIE-1 cells, e) determining the effect of Mob-5 antisense or Mob-5 neutralizing Ab's on ras-transformed RIE-1 cells, f) identifying proteins interacting with Mob-5 by coimmunoprecipitation, and g) determining the presence of a possible cell surface receptor for Mob-5 in transformed cells. Studies in Specific Aim 2 are similar to those proposed for the rat gene with, in addition, an evaluation of a large series of tissue samples and an attempt to develop an Ab based clinical test using serum from cancer patients.
