Abstract 3147: Low oxygen-driven expression of KrasV12 controls a switch between lung tumor growth and migration

Abstract

Abstract Cancer cell migration in vivo occurs at the single cell level or as a chain of motile cells leaving the primary tumor site. Mutation of Kras to a constituitively active form of Kras, is a common alteration in lung cancer. Kras controls many cellular processes such as cell migration and proliferation. Being constantly active results in a hyper activation of downstream signaling cascades, which lead to enhanced tumorigenicity and high metastatic potential. How constitutive Kras activation translates into these cell functions, however, has remained unclear. Using a lung cancer cell transformation model, which expresses oncogenic KrasV12, we discovered that Kras expression got substantially upregulated under long-term culture in 2% O2. This was accompanied by a significant increase in growth of tumor-like aggregates compared to the same cells cultured under atmospheric 21% O2 conditions. Up-regulation of KrasV12 also correlated with a morphological change from adherent single-cells into sphere-like aggregates. 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. The expression of Kras in these two morphological different types of clones correlated with migration behavior. Low Kras expressers were mobile whereas high Kras expressers remained stationary in aggregates. In agreement, protein analysis of these cells forced to form spheres by culture in low adhesion dishes dramatically increased their Kras content. Most interestingly, this increase was reversible by stimulation of adhesion and migration on collagen coated substrates. Under these conditions, cells at the periphery of the aggregate migrate onto the surrounding extracellular matrix (ECM). Curiously, they often stayed attached to a second cell that did not actively migrate. Using immunofluorescence imaging we could identify that in such pairs the actively migrating cell had a low expression of Kras whereas the passive ‘cargo cell’ retained high Kras levels. As both cells originated from the same aggregate of high Kras expressers we conclude that interaction of these lung cancer cells with the ECM negatively regulates Kras expression. This new form of combined migration was also observed in a 3D collagen matrix. By co-culturing high- and low-Kras clones we showed that low Kras expressers actively haul non-motile, high Kras expressers through a collagen scaffold. Based on these findings we propose a novel mechanism for controlling cancer cell migration by a low O2-driven, intrinsic Kras switch. Cells expressing high levels of Kras show a more sphere-like, non-motile phenotype and can give rise to motile cells upon down-regulation of Kras in contact with ECM. These cells carry high Kras expressers with high tumorigenic potential as cargo generating a dangerous team able to migrate out, spread, and regrow elsewhere. Citation Format: Anette C. Schafer, Jerry W. Shay, Gaudenz Danuser. Low oxygen-driven expression of KrasV12 controls a switch between lung tumor growth and migration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3147. doi:10.1158/1538-7445.AM2014-3147