Raf and RhoA cooperate to transform intestinal epithelial cells and induce growth resistance to transforming growth factor beta.
Although unregulated activation of the Ras/Raf/mitogen-activated protein kinase kinase/Erk signaling pathway is believed to be a central mechanism by which many cell types undergo oncogenic transformation, recent studies indicate that activation of Raf kinase by oncogenic Ras is not sufficient to cause tumorigenic transformation in intestinal epithelial cells. Thus, identification of signaling proteins and pathways that interact with Raf to transform intestinal epithelial cells may be critical for understanding aberrant growth control in the intestinal epithelium. Functional interactions between Raf and the small GTPase RhoA were studied in RIE-1 cells overexpressing both activated Raf(22W) and activated RhoA(63L). Double transfectants were morphologically transformed, formed colonies in soft agar, grew in nude mice, overexpressed cyclin D1 and cyclooxygenase-2 (COX-2), and were resistant to growth inhibition by transforming growth factor (TGF) beta. RIE-Raf and RIE-RhoA single transfectants showed none of these characteristics. Expression of a dominant-negative RhoA(N19) construct in RIE-Ras(12V) cells was associated with markedly reduced COX-2 mRNA, COX-2 protein, and prostaglandin E2 levels when compared with RIE-Ras(12V) cells transfected with vector alone. However, no change in transformed morphology, growth in soft agar, cyclin D1 expression, TGFalpha expression, or TGFbeta sensitivity was observed. In summary, coexpression of activated Raf and RhoA induces transformation and TGFbeta resistance in intestinal epithelial cells. Although blockade of RhoA signaling reverses certain well-described characteristics of RIE-Ras cells, it is insufficient to reverse the transformed phenotype and restore TGFbeta sensitivity. Blockade of additional Rho family members or alternate Ras effector pathways may be necessary to fully reverse the Ras phenotype.
Du
Jianguo
J
Department of Pediatrics, Center for Cell and Vascular Biology, Columbus Children's Research Institute and Ohio State College of Medicine and Public Health, Columbus, Ohio 43205, USA.
Jiang
Bo
B
Coffey
Robert J
RJ
Barnard
John
J
D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D004789:Enzyme Activation; D051231:Farnesyltranstransferase; D015972:Gene Expression Regulation, Neoplastic; D005799:Genes, Dominant / Q000235:genetics; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D008715:Methionine / Q000031:analogs & derivatives* / Q000494:pharmacology; D051379:Mice; D009154:Mutation / Q000235:genetics; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D051381:Rats; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000494:pharmacology*; D014410:Tumor Stem Cell Assay; D023041:Xenograft Model Antitumor Assays; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
C096856:FTI 277; D016212:Transforming Growth Factor beta; D008715:Methionine; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras); D020742:rhoA GTP-Binding Protein