In a number of human
cancer cells, K-RAS is frequently mutated and activated constitutively, culminating in the induction of continuous cell growth, a hallmark of
cancer cells. It is still unclear, however, how the mutated K-RAS induces morphological abnormalities in cancerous tissues. To investigate the mechanism underlying the K-RAS-induced morphological changes, we utilized an
auxin-dependent
protein expression system, which enabled us to rapidly induce and evaluate constitutively active K-Ras in MDCK (Madin-Darby canine kidney)
cysts, a model for polarized epithelial structure. Cells carrying the constitutively active KRasV12 gene were morphologically indistinguishable from normal cells in two-dimensional culture. However, in a gel of extracellular matrix, KRasV12-expressing cells failed to form a spherical
cyst. When KRasV12 induction was delayed until after
cyst formation, some cells in the
cyst wall lost polarity and were extruded into and accumulated in the
luminal space. With effector-specific mutants of KRasV12 and inhibitors for
MEK and
PI3-kinase, we found that both the Raf-
MEK-ERK and
PI3-kinase axes are necessary and sufficient for this phenotype. Live cell imaging with cell cycle indicators showed that KRasV12 expression promoted cell cycle progression, which was prevented by either
MEK or
PI3-kinase inhibitors. From these results, we provide a model wherein active-Ras induces cell cycle progression leading to apical cell extrusion through Raf and
PI3-kinase in a cooperative manner. The system developed here can be applied to
drug screening for various
cancers originating from epithelial cells.