Extracellular matrix stiffness augments integrin and growth factor receptor signaling to drive malignant transformation

Predicting which benign and early stage breast lesions will transform is a major therapeutic challenge for breast cancer clinicians. Malignant transformation is associated with profound changes in the tissue microenvironment which can promote malignant transformation and modulate the aggressive behavior of tumors; yet, the molecular mechanisms whereby alterations in the mammary gland stroma modulate tumorigenesis remain poorly understood. Biochemical and biophysical cues generated by the extracellular matrix (ECM) have long been appreciated as critical regulators of mammary epithelial cell (MEC) growth, survival, migration, and differentiation. Integrins are the primary receptors for the ECM, and their expression, activity, and signaling change dramatically in breast tissues in vivo and mammary tumor cells in vitro. With respect to human breast cancer, enhanced PI3 kinase (PI3K) signaling has been strongly implicated in tumor progression and was shown to be essential for ErbB2-mediated tumorigenesis. Because integrin-dependent signaling, including PI3K, can be modulated by altered biophysical cues, we hypothesized that mammary gland stiffness, induced by changes in ECM organization and composition could promote malignant progression of oncogenically-altered MECs by augmenting integrin and growth factor-associated signaling and crosstalk through PI3K. Here, I investigated how alterations in the physical properties of the ECM could modulate mammary tumor progression through the use of MMTV-neu (ErbB2) mice and three-dimensional (3D) MEC models. Using compression and rheometric analysis, I showed that malignant transformation is preceded by and associated with a progressive stiffening of the tissue. Because I found that gland stiffness and tumor invasiveness were functionally-linked to altered collagen morphology, I examined the relevance and molecular mechanisms whereby ECM stiffness might influence normal breast epithelia and breast tumor behavior. Orthotopic tumor implant and 3D MEC studies illustrated that elevated tissue stiffness, through increased collagen crosslinking, synergistically promoted tumor invasion in cooperation with elevated oncogene expression. Consistent with a functional role for these molecular changes, I found that pharmacological inhibition of collagen crosslinking repressed mammary gland transformation in MMTV-neu mice. I also showed that matrix stiffness promoted breast tumorigenesis by enhancing integrin adhesion maturation and oligomerization and increased epidermal growth factor receptor-dependent PI3K signaling through repression of PTEN expression and/or activity