KRAS 4B Silent Mutations in NIH/3T3 Cells Lead to a Tumorigenic Phenotype
It has long been assumed that silent mutations do not affect the fate of a particular protein because the amino acid sequence remains unchanged. However, we are learning that redundancies in the genetic code can lead to changes in protein expression and subsequent phenotypic changes. Surprisingly, silent mutations have recently been associated with more than fifty human diseases. KRAS, the most frequently mutated proto-oncogene in human cancers, is often constitutively activated in cancers that are associated with a poor prognosis. While the majority of research on KRAS mutant cancers has focused on missense mutations at G12 (glycine, residue 12), G13 (glycine, residue 13), and Q61 (glutamine, residue 61), there exists a subset of cancer patients with non-random clustering of silent mutations at G12, G13, and G60. The goal of this study was to examine these silent mutations in the human KRAS 4B gene and begin to clarify the role synonymous codons play in conferring tumorigenic phenotypes on mouse NIH/3T3 cells. We present data showing silent mutations in the human KRAS 4B gene at G12, G13, and G60 altered KRAS protein expression levels when expressed in NIH/3T3 cells and affected activation status of RAS-associated signaling pathways. Moreover, NIH/3T3 cells that expressed the human wild-type (WT) KRAS 4B sequence were contact inhibited, whereas some of the cells transfected with silent KRAS mutations resulted in loss of contact inhibition. The KRAS silent mutations also affected proliferation rates, saturation densities, migration, and invasion. This study links silent mutations in the KRAS gene to tumorigenic phenotypes in mammalian cells and uncovers a previously underappreciated aspect of cancer research.
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