Expression and novel function of CD43 in colon cancer cells

Abstract

CD43 is a transmembrane protein containing an extracellular domain that is extensively O-glycosylated, and a relatively large intracellular domain with a conserved sequence between human, rat and mouse. It is frequently expressed in haematopoietic cell lines where it functions as a regulator of cell adhesiveness, a signal transducer and a cytoskeleton-interacting protein. Previous findings by our group have shown that CD43 can also be expressed in colon adenomas and carcinomas, which led us to further investigate the function of CD43 in these types of cells. Different monoclonal antibodies against the intracellular domain of CD43 were raised and characterized. Using these antibodies, the CD43 expression was studied in several non-haematopoietic cell lines including different types of cancer cell lines. By examining the CD43 expression using immunohistochemistry, flow cytometry, RT-PCR, immunoprecipitation and western blot, we concluded that CD43 was ubiquitously expressed in all the cell lines studied, albeit at varying levels. A more detailed analysis of the CD43 expression in colon cancer cells revealed that the cytoplasmic tail of CD43 could translocate to the cell nucleus. A potential nuclear localization signal (NLS) was found in the intracellular domain of CD43, which was confirmed to be functional by mutagenesis studies. In the nucleus, co-immunoprecipitation of CD43 revealed that it could interact with the tumor-associated molecule b-catenin. b-Catenin is known to cause proliferation in colon carcinomas by escaping from the degradation pathway, entering the nucleus, binding to transcription factors and activating different oncogenes (e.g. c-MYC and Cyclin D1). Using a reporter system, the intracellular domain of CD43 was found to increase the transactivational properties of the b-catenin/TCF-4 complex and to cause an elevated level of c-MYC and Cyclin D1 protein expression. The proteolytic processing of CD43 was also studied. We found that the previously observed proteolytic release of the extracellular domain of CD43 was followed by a second intramembrane cleavage, releasing the intracellular domain of CD43. The intramembrane cleavage was found to be carried out by two separate proteolytic pathways; either the cleavage was dependent of g-secretase activity or it could occur in a g-secretase independent manner. In summary, this thesis presents results suggesting a novel function of CD43 in colon cancer cells. The results imply that CD43 can be involved in colon cancer development by entering the cell nucleus and participate in the activation of certain oncogenes.