The data presented here identify fibronectin as an important effector of acinar morphogenesis. Down-regulation of fibronectin expression by MCF-10A cells is necessary for formation of spheric hollow acini, as inclusion of excess fibronectin stimulated cell growth and reversed established growth arrest of late-stage cultures. We also found that excess fibronectin induced MCF-10A cells to produce more fibronectin in a process that is affected by substrate rigidity. Thus, excess fibronectin perturbs both the acquisition and maintenance of acinar architecture by inducing cell proliferation, and this effect is likely to be exacerbated by increased production of fibronectin. These findings suggest that misregulation of fibronectin expression by transformed mammary epithelial cells in vivo may contribute to breast cancer progression by stimulating aberrant proliferation and loss of tissue architecture.
Previous work investigating the role of fibronectin in the mammary gland has correlated its expression with proliferation of epithelial cells. In vivo
fibronectin and α5β1 expression and localization in epithelial cells of the intact gland correlate with periods of cell growth (8
). Additionally, expression of a transgenic dominant-negative β1 integrin disrupted mammary gland development by decreasing proliferation and increasing apoptosis (37
). In vitro
mammary epithelial cells have been shown to respond to regulatory hormones when plated on fibronectin but not on laminin (38
). Our results show that, during the initial stages of acinar differentiation on Matrigel, excess fibronectin increased proliferation. Early growth stimulatory effects have been linked to expression of a variety of signaling molecules, including human papillomavirus (HPV) E7 oncoprotein, ErbB2, cyclin D, and activated Akt (22
). Whereas HPV E7 and ErbB2 each induce sustained growth stimulation throughout morphogenesis, activated Akt, like fibronectin, has its primary proliferative effect in the early growth stage (23
). Continuous activation of ErbB2 has a more dramatic effect than the others, causing significant overproliferation of MCF-10A cells into multiacinar structures with filled lumens (22
). Together, these results show the differential effects on acinar morphogenesis of signaling through growth factor receptors, cell cycle pathways, and cell adhesion and indicate that control of cell growth and growth arrest depends on the integration of multiple signals.
Signals from other matrix molecules are also essential because laminin-1 is needed to establish cell polarity and growth arrest (24
). Laminin and laminin-binding integrins are well-known mediators of morphogenesis. Blocking β4 or β1 integrin function with antibodies perturbs acinar differentiation with effects including interruption of morphogenesis, inhibition of cell growth, and induction of apoptosis (21
). Certainly, some of these effects are due to loss of laminin binding; however, our results suggest a reduction of interactions between α5β1 and fibronectin by anti-β1 function-blocking antibodies may also contribute. The dynamic nature of fibronectin production and its regulation by exogenous fibronectin during morphogenesis may provide new insights into mammary differentiation, development, and cancer progression. It is also interesting to speculate on the role fibronectin plays in branching morphogenesis in the breast as it is highly associated with epithelial cells during that process, and fibronectin has already been identified as an early signal inducing branching morphogenesis in the salivary gland (39
During tumorigenesis in the breast, primary tumors are characterized by increased deposition of fibronectin (12
), increased tissue rigidity (17
), loss of epithelial cell polarity, increased proliferation, and luminal filling (1
). Our data show that addition of excess fibronectin to differentiated cultures resulted in a reversal of growth arrest and failure to maintain the acinar structure. The reinitiation of cell growth and luminal filling seen here seem analogous to the changes seen with the emergence of breast cancer in vivo
. Therefore, fibronectin may serve a key mitogenic role for mammary epithelial cells and promote oncogenic progression by stimulating aberrant proliferation.
Our results point toward a mechanism by which stiffening of the matrix affects proper acinar morphogenesis by modulating fibronectin expression. Conditions closest to those found in the normal mammary gland, laminin-rich with a low elastic modulus, favored down-regulation of fibronectin gene transcription, resulting in reduced protein production. In contrast, conditions similar to those found in mammary tumors, fibronectin-rich with a high elastic modulus, favored up-regulation of fibronectin mRNA and protein levels. Previous work has shown that progressively increasing the stiffness of the matrix prevents cells from differentiating into acini (17
). Small increases in rigidity (0.1–4 kPa) resulted in increased proliferation and failure to form a hollow lumen, a phenotype similar to that seen for acini in the presence of excess fibronectin where epithelial cells are expressing high levels of fibronectin. This phenocopy and the increased fibronectin production on substrates with elastic moduli above that of Matrigel and normal mammary tissue may indicate that fibronectin contributes to the aberrant cell behaviors noted on stiff substrates.
Thus, in our model for fibronectin function during acinar morphogenesis, MCF-10A cells initially express fibronectin during the growth phase of differentiation. Over time, signals from Matrigel, including laminin-1 and low substrate rigidity, induce down-regulation of fibronectin, such that levels are negligible in fully differentiated, growth-arrested acini. This pattern is similar to that found in vivo
in both mammary gland and lung (8
). However, when the matrix is abnormal due to addition of exogenous fibronectin, a positive feedback loop is initiated, whereby fibronectin levels during the proliferative phase of morphogenesis are not reduced even after the onset of growth arrest, possibly contributing to the failure of acini in these circumstances to form a hollow lumen.
Modulation of fibronectin expression by tissue stiffness and the effects of increased fibronectin on cell growth may together have profound implications for breast cancer progression. It is understood that dense breast tissue, which is more rigid than normal tissue, is also prone to development of hyperplasias and dysplasias (16
). Should the altered stiffness of the mammary tissue promote increased fibronectin production by mammary epithelial cells, this would further stimulate proliferation. Indeed, a direct correlation was observed between mammographic density, deposition of the ECM proteoglycans lumican and decorin, and the presence of hyperplastic lesions (41
). The propensity for hyperplastic growths to form in dense breast tissue may be in part due to increased deposition of fibronectin by mammary epithelial cells, which our results show can stimulate further fibronectin production and proliferation.
These data lend themselves to a model for the promotion of breast cancer progression by fibronectin (Supplementary Fig. S4
). Oncogenic mutations in the mammary epithelium often result in a failure to maintain the basement membrane. This failure could then initiate a desmoplastic response, characterized by increased and aberrant deposition of ECM molecules (42
) which would increase the elastic modulus of the tissue. Decreased contact with basement membrane laminin-1, increased contact with stromal fibronectin, and higher substrate rigidity may then stimulate mammary epithelial cells to produce their own fibronectin. Activation of this positive feedback loop would stimulate cell proliferation, leading to luminal filling and loss of normal tissue architecture. Thus, fibronectin may promote primary tumor formation by modifying the ECM to provide a substratum permissive for aberrant cell growth, thereby encouraging the acquisition of further oncogenic mutations.