Microenvironmental Stiffness Enhances Glioma Cell Proliferation by Stimulating Epidermal Growth Factor Receptor Signaling

Abstract

The aggressive and rapidly lethal brain tumor glioblastoma (GBM) is associated with profound tissue stiffening and genomic lesions in key members of the epidermal growth factor receptor (EGFR) pathway. Previous studies from our laboratory have shown that increasing microenvironmental stiffness in culture can strongly enhance glioma cell behaviors relevant to tumor progression, including proliferation, yet it has remained unclear whether stiffness and EGFR regulate proliferation through common or independent signaling mechanisms. Here we test the hypothesis that microenvironmental stiffness regulates cell cycle progression and proliferation in GBM tumor cells by altering EGFR-dependent signaling. We began by performing an unbiased reverse phase protein array screen, which revealed that stiffness modulates expression and phosphorylation of a broad range of signals relevant to proliferation, including members of the EGFR pathway. We subsequently found that culturing human GBM tumor cells on progressively stiffer culture substrates both dramatically increases proliferation and facilitates passage through the G1/S checkpoint of the cell cycle, consistent with an EGFR-dependent process. Western Blots showed that increasing microenvironmental stiffness enhances the expression and phosphorylation of EGFR and its downstream effector Akt. Pharmacological loss-of-function studies revealed that the stiffness-sensitivity of proliferation is strongly blunted by inhibition of EGFR, Akt, or PI3 kinase. Finally, we observed that stiffness strongly regulates EGFR clustering, with phosphorylated EGFR condensing into vinculin-positive focal adhesions on stiff substrates and dispersing as microenvironmental stiffness falls to physiological levels. Our findings collectively support a model in which tissue stiffening promotes GBM proliferation by spatially and biochemically amplifying EGFR signaling.

Tissue stiffness regulates serine/arginine-rich protein-mediated splicing of the extra domain B-fibronectin isoform in tumors

“…Interestingly, AKT signaling has previously been implicated in SRp40-mediated splicing of the PKC family (14,16), suggesting that AKT is likely to be involved in a similar way in both stiffness-mediated PKC and EDB-FN splicing. In addition, the regulation of PI3K/AKT pathway by matrix stiffness is increasingly well documented in a number of different experimental tumor models (17)(18)(19)44). Taken together, our data uncover a fundamental mechanism regulating EDB-FN expression in ECs.…”

Periodic Mechanical Stress Activates PKCδ-Dependent EGFR Mitogenic Signals in Rat Chondrocytes via PI3K-Akt and ERK1/2

“…EGFR, as a nonintegrin mechanosensor, could mediate mechanical responses, and the reports that EGF-EGFR induced chondrocyte proliferation prompted investigation to examine the involvement of EGFR in the periodic mechanical stress-induced chondrocytic mitogenic effects [34,35]. The present study showed that the specific EGFR inhibitor AG1478 and shRNA targeted to EGFR blocked chondrocyte proliferation in response to periodic mechanical stress.…”

Transient mechanical strain promotes the maturation of invadopodia and enhances cancer cell invasion in vitro

“…Much of the early focus has been on the obvious changes in stiffness, since the stroma surrounding most tumors often becomes more rigid and dense due to an enrichment of collagen type I and fibronectin (Miles and Sikes, 2014;Pickup et al, 2014). This increase in rigidity leads to an increase in tumor cell proliferation, migration and invasion (Alexander et al, 2008;Charras and Sahai, 2014;Jerrell and Parekh, 2014;Kostic et al, 2009;Parekh and Weaver, 2009;Tilghman et al, 2010;Ulrich et al, 2009;Umesh et al, 2014).…”