Mammary epithelial cell: influence of extracellular matrix composition and organization during development and tumorigenesis

Abstract

Stromal-epithelial interactions regulate mammary gland development and are critical for the maintenance of tissue homeostasis. The extracellular matrix, which is a proteinaceous component of the stroma, regulates mammary epithelial growth, survival, migration and differentiation through a repertoire of transmembrane receptors, of which integrins are the best characterized. Integrins modulate cell fate by reciprocally transducing biochemical and biophysical cues between the cell and the extracellular matrix, facilitating processes such as embryonic branching morphogenesis and lactation in the mammary gland. During breast development and cancer progression, the extracellular matrix is dynamically altered such that its composition, turnover, processing and orientation change dramatically. These modifications influence mammary epithelial cell shape, and modulate growth factor and hormonal responses to regulate processes including branching morphogenesis and alveolar differentiation. Malignant transformation of the breast is also associated with significant matrix remodeling and a progressive stiffening of the stroma that can enhance mammary epithelial cell growth, perturb breast tissue organization, and promote cell invasion and survival. In this review, we discuss the role of stromal-epithelial interactions in normal and malignant mammary epithelial cell behavior. We specifically focus on how dynamic modulation of the biochemical and biophysical properties of the extracellular matrix elicit a dialogue with the mammary epithelium through transmembrane integrin receptors to influence tissue morphogenesis, homeostasis and malignant transformation.

Fig. 1

Mammary epithelial growth and morphogenesis is regulated by matrix stiffness. (A) 3D cultures of normal mammary epithelial cells within collagen gels of different concentration. Stiffening the ECM through an incremental increase in collagen concentration (soft gels: 1 mg/ml Collagen I, 140 Pa; stiff gels 3.6 mg/ml Collagen I, 1200 Pa) results in the progressive perturbation of morphogenesis, and the increased growth and modulated survival of MECs. Altered mammary acini morphology is illustrated by the destabilization of cell–cell adherens junctions and disruption of basal tissue polarity indicated by the gradual loss of cell–cell localized β-catenin (green) and disorganized β4 integrin (red) visualized through immunofluorescence and confocal imaging. On the right side of the panel is an illustration of a mammary ductal structure (luminal and myoepithelial cells surrounded by BM), and depicting MEC morphogenesis in soft and stiff gels. Scale bars represent 25 μm. (B) Confocal immunofluorescence images of MEC colonies on soft and stiff gels (140 vs. >5000 Pa) stained for β-catenin (red) and E-cadherin (green), and counterstained with DAPI (blue) after triton X-100 extraction. β-catenin could be extracted from the sites of cell–cell interaction in MEC colonies formed on a stiff but not on a soft gel, indicating that adherens junctions are less stable in MEC structures formed on stiff gels. White arrowheads indicate diffuse staining patterns of β-catenin and E-cadherin. Modified from Paszek et al. (2005).

Fig. 2

Malignant transformation of mammary epithelial cells is regulated by matrix stiffness. Breast transformation ensues through progressive acquisition of genetic alterations in the luminal epithelial cells residing within the mammary ducts. The tissue stroma responds to these epithelial alterations by initiating a desmoplastic response that is characterized by activation and transdifferentiation of fibroblasts, infiltration of immune cells, increased secretion of growth factors and cytokines, and elevated matrix synthesis and remodeling that manifests as matrix stiffening. (A) Cartoon depicting the stages of breast tumorigenesis (from left to right; normal ducts, ductal carcinoma in situ and invasive phenotype), highlighting key desmoplastic changes within the tissue stroma. (B) Force-dependent focal adhesion maturation mediated by elevated tumor matrix stiffness. Integrins are bidirectional mechanosensors that integrate biochemical and biophysical cues from the matrix and the actin cytoskeleton and transduce cell-generated force to the surrounding microenvironment. Activated integrins bind to ECM proteins via cooperative interactions between their alpha and beta extracellular domains and form nascent highly dynamic adhesion signaling complexes. In response to external mechanical force or elevated cell-generated contractility integrin clustering is enhanced and the recruitment of multiple integrin adhesion plaque proteins including talin and vinculin is favored. These, in turn, associate with the actin cytoskeleton and multiple signaling proteins including focal adhesion kinase (FAK), Src family kinases, and integrin-linked kinase, to promote cell growth, survival, migration and differentiation. Matrix stiffening, which reflects elevated matrix deposition, linearization and cross-linking, can co-operate with oncogenic signaling to enhance cell-generated contractility to foster integrin associations and focal adhesion maturation. Maturation of focal adhesions promotes cell generated forces by enhancing Rho GTPase and ERK-mediated acto-myosin contractility—which feed forward to further promote integrin clustering and focal adhesion assembly and transmit acto-myosin-generated cellular forces to the ECM, as outlined in Paszek et al. (2005). Inset: representative traction maps showing the typical force distribution in fibroblasts on soft (450 Pa) and on stiff fibronectin gels (5600 Pa). These maps allow the measurement of the forces generated by the cell, which are dependent on the stiffness of the substrate. Modified from Paszek et al. (2005).