Abstract 5102: Differential levels of mutated Kras drive lung cancer cell motility and morphology via a hypoxia-sensitive FAK/Erk/Myosin II signaling pathway
Authors: Schafer Anette C. , Discoll Meghan , Mohan Ashwathi , Ludlow Andrew , Burford Wesley , Shay Jerry W. , Danuser Gaudenz 
Published: August 2015

Abstract
Kras controls many cellular processes such as cell migration and survival. Mutation of Kras to a constitutively active form is a common alteration in lung cancer. Being constantly active results in a hyper activation of downstream signaling cascades, which leads to enhanced tumorigenicity and high metastatic potential. How constitutive Kras activation translates into these different functions, however, remains unclear. Using patient-derived lung cancer cell lines expressing oncogenic KrasV12 we discovered that Kras expression, not transcription, gets up-regulated under long-term culture in 1% O2. We confirmed the in vivo relevance of this finding, staining mouse lung tumors for Kras and Hif1α, as an indicator for hypoxic areas, and found heterogeneous Kras levels throughout the tumors with hot-spots that overlaid with Hif1α expression. Up-regulation of KrasV12 also correlated with a significant increase in growth of tumor-like aggregates in soft-agar assays and a morphological change from adherent single-cells into sphere-like aggregates in cell culture. This morphology was reversible upon knock-down of Kras. Separation of single clones confirmed the relation between adherent and sphere-like morphologies and the level of KrasV12 expression. Interestingly, the expression level of Kras correlated with clonal migration behavior. Low Kras expressers were highly motile whereas high Kras expressers remained stationary in aggregates. In agreement, exogenous expression of KrasV12 in motile, low Kras expressers converted their morphology and migration behavior to sphere-like, stationary aggregates. Using Western blot analysis we found that these changes are due to differences in Erk activation down-stream of Kras. In high Kras expressers Erk is highly phosphorylated which leads to higher Myosin II activity and thus to a contractile, sphere-like morphology with low mobility. Low Kras levels result in less Erk activation and therefore lower Myosin II activity which leads to an adherent, motile phenotype. Using a specific Erk inhibitor, we could convert high Kras expressers into adherent, motile low Kras clones. At the same time we found an adhesion-dependent feedback mechanism which regulates cellular Kras levels. Protein analysis of cells cultured in low adhesion dishes formed spheres and dramatically increased their Kras content. Most interestingly, this increase was reversible by stimulation of adhesion. Additionally, blocking FAK activation in low Kras expressers resulted in up-regulation of Kras and Erk phosphorylation which went along with a switch to sphere-like morphology. Based on these findings we propose a novel mechanism for controlling cancer cell migration by a hypoxia-sensitive, adhesion-dependent Kras switch which makes the cell very adaptable to different environments during the whole process of metastasis.
Citation Format: Anette C. Schafer, Meghan Discoll, Ashwathi Mohan, Andrew Ludlow, Wesley Burford, Jerry W. Shay, Gaudenz Danuser. Differential levels of mutated Kras drive lung cancer cell motility and morphology via a hypoxia-sensitive FAK/Erk/Myosin II signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5102. doi:10.1158/1538-7445.AM2015-5102