Abstract B53: LKB1 kinase-dependent and -independent defects disrupt polarity and adhesion signaling to create a uniquely invasive amoeboid cancer cell

Abstract

Abstract The tumor suppressor LKB1 is a serine/threonine kinase and the 3rd most commonly mutated gene in lung adenocarcinoma. The majority of LKB1 mutations are truncations that predictably disrupt its kinase activity and remove its C-terminal domain (CTD). Since LKB1 inactivation drives cancer metastasis in mice and leads to aberrant cell invasion in vitro, we sought to determine how domain-specific LKB1 inactivation impacts lung cancer cell adhesion and polarity during 3D invasion. We generated lung cancer cell lines stably expressing empty GFP or GFP-tagged LKB1: wildtype, a farnesylation motif mutant, a kinase dead mutant, CTD (lacking kinase activity) and the CTD containing the farnesylation motif mutant, and then embedded spheroids of these cells in a 3D collagen matrix. Using confocal immunofluorescence and live cell imaging, we show cells re-expressing either wildtype LKB1 or the LKB1 CTD alone exhibited a mesenchymal polarity with strong directional persistence, which is completely abrogated upon loss of farnesylation. Given that the CTD lacks kinase activity, these data highlight a farnesylation-dependent but kinase-independent regulation of mesenchymal polarity and directional persistence during invasion. We next examined Rho-GTPase activity in response to LKB1 farnesylation and show that the LKB1 CTD alone is sufficient to activate RhoA. This activation is again completely abrogated upon loss of LKB1 farnesylation, highlighting another farnesylation-dependent but kinase-independent regulation. Given that farnesylation regulates both mesenchymal polarity and RhoA activity, we next probed the relationship between mesenchymal polarity and RhoA by creating cell lines re-expressing either constitutively active RhoA or cdc42. We show that restoring RhoA activity, but not cdc42, in farnesylation-compromised cells restores mesenchymal polarization and directional persistence during invasion. Importantly, restoring RhoA activity in the absence of LKB1 fails to restore mesenchymal polarity, indicating that a region of LKB1 must be necessary for the regulation of polarity through RhoA. We next examined the role of LKB1 in regulating focal adhesion kinase (FAK) activity during invasion. While farnesylation does not affect FAK activity, LKB1 signals to MARK1 in a kinase-dependent manner to repress FAK, which then represses collagen remodeling during 3D invasion. Since LKB1 frequently undergoes truncating mutations that predictably affect both farnesylation and kinase activity, cancer cells with LKB1 loss would exhibit a unique amoeboid morphology with both hyperactive FAK and the ability to remodel collagen. Together, these data suggest that it is a combination of kinase-dependent and -independent defects that create a uniquely invasive cell upon LKB1 inactivation. Citation Format: Scott Wilkinson, Jessica Konen, Adam I. Marcus. LKB1 kinase-dependent and -independent defects disrupt polarity and adhesion signaling to create a uniquely invasive amoeboid cancer cell. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B53.