Effects of mutant human Ki-rasG12C gene dosage on murine lung tumorigenesis and signaling to its downstream effectors

Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki-ras(G12C) allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 mu g/ml of doxycycline (DOX) allowed for maximal expression of the human Ki-ras(G12c) allele in the lung, and resulted in the development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression Would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 mu g/ml of DOX resulted in close-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 mu g/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 mu g/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki-ras(G12C) expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models. (c) 2008 Elsevier Inc. All rights reserved.