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Increased overall survival for patients with glioma brain tumours is associated with mutations in the metabolic regulator isocitrate dehydrogenase 1 (IDH1). Gliomas develop within a mechanically challenged microenvironment that is characterized by a dense extracellular matrix (ECM) that compromises vascular integrity to induce hypoxia and activate HIF1α. We found that glioma aggression and patient prognosis correlate with HIF1α levels and the stiffness of a tenascin C (TNC)-enriched ECM. Gain- and loss-of-function xenograft manipulations demonstrated that a mutant IDH1 restricts glioma aggression by reducing HIF1α-dependent TNC expression to decrease ECM stiffness and mechanosignalling. Recurrent IDH1-mutant patient gliomas had a stiffer TNC-enriched ECM that our studies attributed to reduced miR-203 suppression of HIF1α and TNC mediated via a tension-dependent positive feedback loop. Thus, our work suggests that elevated ECM stiffness can independently foster glioblastoma aggression and contribute to glioblastoma recurrence via bypassing the protective activity of IDH1 mutational status.

s: AACR Special Conference: Advances in Breast Cancer; October 17-20, 2015; Bellevue, WA Breast cancers frequently develop treatment resistance that leads to recurrence, dissemination and patient mortality. Among the mechanisms that foster treatment resistance is the ability of tumor cells to undergo an epithelial-to-mesenchymal transition (EMT) and exhibit stem-like behavior. Accumulating evidence now supports the concept that the mechanical properties of the extracellular matrix microenvironment can critically influence developmental cell fate and modify several features of tumor progression. Data from our laboratory using preclinical models and clinical samples suggest that tissue tension and elevated mechanosignaling increase prior to and accompany malignant transformation. Therefore, we hypothesize that enhanced tissue mechanical tension and high mechanosignaling foster mammary stemness, EMT and breast cancer aggression. We tested this prediction by generating transgenic mice conditionally expressing a β1-Integrin clustering mutant (V737N) in the mammary epithelium. V737N expression stimulated integrin-mediated mechanosignaling in mammary epithelial cells (MECs), as determined by elevated phosphorylation of FAK and p130Cas, and this heightened mechanosignaling promoted precocious epithelial ductal branching, end bud formation and increased MEC proliferation in the mammary gland. These mammary phenotypes were accompanied by an increase in the ratio of basal/myoepithelial to luminal MECs in V737N mammary glands. Isolated V737N-expressing MECs possessed higher levels of genes associated with EMT and stemness, and Matrigel colony formation and transplantation assays revealed a functional increase in progenitor/stem cell frequency in the V737N-expressing basal/myoepithelial MECs compared to their corresponding controls. To examine the effect of tissue tension on breast cancer progression, we combined the V737N-β1-Integrin together with a mouse model of HER2-positive breast cancer (MMTV-NEU). While V737N expression had no observable effect on primary tumor outgrowth, overall tumor incidence and lung metastasis were significantly augmented. Further histological examination of tumors and gene expression analysis uncovered a phenotypic shift, such that V737N tumors displayed expression patterns resembling an EMT-like basal tumor when compared to control tumors. Thus, tissue tension by way of enhanced mechanosignaling promotes mammary stemness, higher mammary tumor incidence and a more basal-like aggressive tumor character. Potential V737N mechanistic actions currently under investigation involve altered orientation of cell divisions resulting from disrupted MEC polarity, as well as a heightened sensitivity to hormone-induced expansion of stem/progenitor cells. (Supp by: USMRAA Department of Defense-BCRP BC122990 and NIH NCI R01CA192914 to VW and AACR 15-40-01-NORT to JN). Citation Format: Jason J. Northey, Yoshihiro Yui, Janna K. Mouw, Johnathan N. Lakins, Valerie M. Weaver. Tissue tension promotes mammary stemness and breast cancer aggression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr PR04.

Focal adhesions are the conduits through which cells receive and interpret mechanical signals. It is not known if nanoscale protein organization is altered to accommodate changes in mechanical inputs from the cytoskeleton and extracellular matrix components. We hypothesized that the relative position of specific focal adhesion proteins could correspond to the engagement of a physical protein clutch for different adhesion functions. To this end we employed Scanning Angle Interference Microscopy to determine the 3D organization of proteins comprising focal adhesions with a precision of ∼5nm. We found paxillin, FAK, vinculin, talin, and zyxin to be stratified in distinct layers over a vertical range of 60 nm. We then compared nascent versus focal adhesions at the cell leading edge, and found that paxillin localized ∼7nm towards the cell membrane in developing adhesions. We inhibited intracellular contractility to see how adhesion architecture dynamically responds to changes in mechanical input, and observed that paxillin and zyxin, but not vinculin, undergoes a marked increase in height of >15nm. Conversely, vinculin without a force dependent auto-inhibition domain, T12; undergoes dramatic reorganization at the nanoscale after contractility inhibition. Overexpression of vinculinT12 resulted in increased intramolecular forces as seen in a vinculinT12 FRET tension sensor, targeting of vinculin to an architecture that corresponded with talin and actin engagement, but not changes in cellular traction. When we reduced cellular motility through overexpression of a constitutively active Rac1 mutant, adhesions at the lamella-lamellipodia border had different vinculin architecture than other adhesions in the cell. Our results suggest that elimination of vinculin force dependent auto-inhibition can dictate focal adhesion architecture and morphology, but not cellular traction; and that there are specific vinculin architectures that reflect distinct states of cellular contractility and motility.