The chemopreventive agent α‐difluoromethylornithine blocks Ki‐ras–dependent tumor formation and specific gene expression in Caco‐2 cells

Publisher Website
Google Scholar
Abstract
Mutation of the Kirsten‐ras (Ki‐ras) proto‐oncogene occurs frequently in colorectal cancers. α‐Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme, ornithine decarboxylase (ODC), inhibits Ki‐ras transformation and colon tumorigenesis in carcinogen‐treated animal models by mechanisms yet to be elucidated. Caco‐2 cells transfected with an activated Ki‐ras, but not parental cells, formed tumors in severe combined immunodeficient (SCID) mice. DFMO treatment (2% in drinking water) prevented tumor growth. Gene expression profiling was performed to identify Ki‐ras–and DFMO‐dependent patterns of gene expression. Microarray results were validated with real‐time or semi‐quantitative RT‐PCR and/or Western blot analysis. Genes upregulated in Caco‐2 cells expressing an activated Ki‐ras encoded cytoskeletal‐, transport‐, protease‐, and gap junction–associated proteins. These genes are important for normal development and maintenance of colonic epithelial tissue. Caco‐2 cells transfected with an activated Ki‐ras displayed increased expression of the integrin alpha 1 (INGA1) and enhanced cell migration on laminin. These parameters were unaffected by DFMO, but Ki‐ras–dependent migration was inhibited by INGA1 antibodies. Other Ki‐ras–dependent, but DFMO‐independent, genes included transglutaminase (TGase) and kallikrein 6 (KLK6). Ki‐ras–transfected cells also expressed increased levels of connexin43 (Cx43) (RNA and protein), tight junction protein, and endothelin 1. DFMO reversed these increases. The results indicated that the Ki‐ras oncogene caused changes in experimental cell migration and cell‐cell communication genes and that some of these changes could be reversed by DFMO.