Summary Tumors are characterized by extracellular matrix (ECM) remodeling and stiffening. The importance of ECM remodeling to cancer is appreciated; the relevance of stiffening is less clear. We found that breast tumorigenesis is accompanied by collagen crosslinking, ECM stiffening and increased focal adhesions. Inducing collagen crosslinking stiffened the ECM, promoted focal adhesions, enhanced PI3 Kinase (PI3K) activity, and induced the invasion of an oncogene-initiated epithelium. Inhibiting integrin signaling repressed the invasion of a premalignant epithelium into a stiffened, crosslinked ECM, and forced integrin clustering promoted focal adhesions, enhanced PI3K signaling and induced the invasion of a premalignant epithelium. Consistently, reducing lysyl oxidase-mediated collagen crosslinking prevented MMTV-Neu-induced fibrosis, decreased focal adhesions and PI3K activity, impeded malignancy and lowered tumor incidence. These data show how collagen crosslinking can modulate tissue fibrosis and stiffness to force focal adhesions, growth factor signaling and breast malignancy.

SUMMARY: A fundamental feature of cellular plasma membranes (PM) is asymmetric lipid distribution between the bilayer leaflets. However, neither the detailed, comprehensive compositions of individual PM leaflets, nor how these contribute to structural membrane asymmetries have been defined. We report the distinct lipidomes and biophysical properties of both monolayers in living mammalian PMs. Phospholipid unsaturation is dramatically asymmetric, with the cytoplasmic leaflet being ~2-fold more unsaturated than the exoplasmic. Atomistic simulations and spectroscopy of leaflet-selective fluorescent probes reveal that the outer PM leaflet is more packed and less diffusive than the inner leaflet, with this biophysical asymmetry maintained in the endocytic system. The structural asymmetry of the PM is reflected in asymmetric structures of protein transmembrane domains (TMD). These structural asymmetries are conserved throughout Eukaryota, suggesting fundamental cellular design principles.

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Eukaryotic plasma membranes are compartmentalized into functional lateral domains, including lipid-driven membrane rafts. Rafts are involved in most plasma membrane functions by selective recruitment and retention of specific proteins. However, the structural determinants of transmembrane protein partitioning to raft domains are not fully understood. Hypothesizing that protein transmembrane domains (TMDs) determine raft association, here we directly quantify raft affinity for dozens of TMDs. We identify three physical features that independently affect raft partitioning, namely TMD surface area, length, and palmitoylation. We rationalize these findings into a mechanistic, physical model that predicts raft affinity from the protein sequence. Application of these concepts to the human proteome reveals that plasma membrane proteins have higher raft affinity than those of intracellular membranes, consistent with raft-mediated plasma membrane sorting. Overall, our experimental observations and physical model establish general rules for raft partitioning of TMDs and support the central role of rafts in membrane traffic.

Tissue mechanics regulate development and homeostasis and are consistently modified in tumor progression. Nevertheless, the fundamental molecular mechanisms through which altered mechanics regulate tissue behavior and the clinical relevance of these changes remain unclear. We demonstrate that increased matrix stiffness modulates microRNA expression to drive tumor progression through integrin activation of β-catenin and MYC. Specifically, in human and mouse tissue, increased matrix stiffness induced miR-18a to reduce levels of the tumor suppressor PTEN, both directly and indirectly by decreasing levels of HOXA9. Clinically, extracellular matrix stiffness correlated significantly with miR-18a in human breast tumor biopsies. miR-18a expression was highest in basal-like breast cancers in which PTEN and HOXA9 levels were lowest and predicted for poor prognosis in patients with luminal breast cancers. Our findings identify a mechanically-regulated microRNA circuit that can promote malignancy and suggest potential prognostic roles for HOXA9 and miR-18a levels in stratifying patients with luminal breast cancers.