Endogenous oncogenic KRAS expression increases cell proliferation and motility in near-diploid hTERT RPE-1 cells

Abstract

AbstractAbout 18% of all human cancers carry a mutation in theKRASgene making it among the most sought-after anti-cancer targets. However, mutant KRas protein has proved remarkably undruggable. The recent approval of the first generation of RAS inhibitors therefore marks a seminal milestone in the history of cancer research. Inevitably though, it also raises the predictable challenges of limited drug efficacies and acquired resistance. Hence, new approaches that improve our understanding of the tumorigenic mechanisms of oncogenic RAS within more physiological settings continue to be essential. Here, we have employed the near-diploid human hTERT RPE-1 cells to generate isogenic cell lines in which one of the endogenousKRASalleles carries an oncogenicKRASmutation at glycine 12. Cells with aKRASG12V/+,KRASG12C/+, orKRASG12D/+genotype, together with wild-typeKRASG12G(WT)/+cells, reveal that oncogenicKRAS.G12Xmutations increase cell proliferation rate, while further analyses showed thatKRASG12V/+cells had increased cell motility and reduced focal adhesions. EGF-induced ERK phosphorylation was marginally increased inKRASG12V/+cells, while EGF-induced AKT phosphorylation was comparable betweenKRASG12V/+andKRASG12G(WT)/+cells. Interestingly, theKRASG12V/+cells were more sensitive to hydroxyurea and a MEK inhibitor, U0126, but more resistant to a PI3K inhibitor, PIK-90, than theKRASG12G(WT)/+cells. A combination of low doses of hydroxyurea and U0126 showed an additive inhibition on growth rate that was greater inKRASG12V/+than wild-type cells. Collectively, these cell lines will be a valuable resource for studying oncogenic RAS signalling and developing effective anti-KRAS reagents with minimum cytotoxicity on wild-type cells.