Abstract B04: The role of mechanical force and integrin-ECM signaling in glioblastoma aggression

Abstract

Abstract Glioblastoma multiforme (GBM) is characterized by extensive angiogenesis, inflammation, and edema. These changes are associated with increased extracellular matrix (ECM) deposition and stiffness as well as increased intracranial pressure and tissue compression. Our lab has shown that stiffened ECM leads to enhanced integrin signaling and cell contractility, which promotes tumor cell invasion and progression of breast cancer. We therefore hypothesized that the unique force environment of brain tumors promotes aggressive behavior of GBM cells through similar mechanisms. We began by testing whether a correlation exists between malignancy and the ECM makeup and tensional state of GBM tumors. GBMs have recently been grouped into molecular subclasses, including proneural and mesenchymal, which are predictive of better and worse response to therapy and patient survival, respectively. Using mouse orthotopic xenografts we showed that, compared to proneural, mesenchymal tumors exhibit increased ECM content, integrin clustering, focal adhesions and myosin activity; predictive of a stiffer tumor with greater tumor cell contractility. This data suggests a negative correlation between patient survival and the tension state of the tumor. Whether an increased force environment is a result or a driver of GBM progression is unclear. To test the idea that an increased force environment can drive a proneural-mesenchymal transition we cultured cells on ECM gels of increasing stiffness (ranging from 0.4-40.0kPa, where normal brain is on average less than 1.0kPa and mesenchymal xenografts are ~2.5kPa). While the morphology of mesenchymal cells was similar on all gels, proneural cells plated on gels greater than 2.7kPa adopted a morphology and transcriptional state reminiscent of mesenchymal cells. Quantitative PCR and immunofluorescence staining revealed significant increases in the mesenchymal markers N-cadherin, fibronectin, vimentin, TGFβ, ZEB1, and nuclear (active) YAP. Antibody blocking of beta1 integrin and small molecule inhibition of focal adhesion kinase or ROCK led to partial rescue of proneural morphology and gene expression. Thus integrin-ECM engagement and myosin activity are necessary for this apparent proneural-mesenchymal transition. The results of increased substrate rigidity were mimicked by enhancing beta1 integrin clustering (through expression of the ITGB1V737N mutant developed in our lab). Collectively these findings suggest that mechanical forces, arising through enhanced cell-ECM interactions and transduced via integrin-focal adhesion signaling, act to drive a mesenchymal, malignant state in GBM cells. In vivo experiments interrogating the effect of forced integrin clustering and cell contractility on GBM aggressiveness and progression are ongoing. Citation Format: J. Matthew Barnes, Valerie M. Weaver. The role of mechanical force and integrin-ECM signaling in glioblastoma aggression. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B04.