Gerhard Wirl | PubMed Health

PubMed2005

Tumor cell invasiveness correlates with changes in integrin expression and localization.

In nontumorigenic mammary epithelial cells (EpH4), transforming growth factor-beta (TGFbeta1) causes cell cycle arrest/apoptosis, but induces epitheliomesenchymal transition (EMT) in Ha-Ras-transformed EpH4 cells (EpRas). EMT is closely correlated with late-stage tumor progression and results in fibroblastic, migratory cells displaying a mesenchymal gene expression program (FibRas). EpRas and FibRas cells showed strongly increased cell substrate adhesion to fibronectin, collagens I/IV and laminin 1. Furthermore, Ras transformation caused enhanced or de-novo expression of the integrin subunits beta1, alpha2 and alpha3, or alpha5 and alpha6, respectively, the latter subunits being even more strongly expressed in FibRas cells. Importantly, polarized EpRas cells expressed integrin subunits beta1 and alpha6 at distinct (apical and lateral) membrane domains, while FibRas cells coexpressed these integrins and alpha5 at the entire plasma membrane. During EMT, EpRas cells formed an alpha5beta1 complex and deposited its ligand fibronectin into the extracellular matrix. Function-blocking alpha5 antibodies attenuated migration, and caused massive apoptosis in EpRas cells undergoing TGFbeta1-induced EMT in collagen gels, but failed to affect EpRas- or FibRas-derived structures. We conclude that functional alpha5beta1 integrin is centrally implicated in EMT induction. Importantly, FibRas cells also failed to deposit the alpha6beta4 ligand laminin 5, suggesting that alpha6beta4 is no longer functional after EMT and replaced by mesenchymal integrins such as alpha5beta1.

Breast↗

PubMed2004

Enhanced tenascin-C expression and matrix deposition during Ras/TGF-beta-induced progression of mammary tumor cells.

Overexpression of tenascin-C (TN-C) in breast carcinomas has been associated with a migratory or even invasive tumor cell phenotype. The mechanisms regulating expression and matrix deposition of TN-C in normal and cancerous breast tissues are, however, little understood. Here, we demonstrate that mouse mammary epithelial cells (EpH4) transformed by oncogenic Ha-Ras (EpRas) overexpress TN-C, which accumulates in the cytoplasm. When EpRas cells undergo epithelial-mesenchymal transition (EMT) in response to TGFbeta1, they secrete TN-C into the culture medium. In EpRas cells undergoing TGFbeta1-induced EMT in three-dimensional (3D)-collagen gel cultures, TN-C was deposited into an extracellular matrix (ECM) already containing fibronectin and perlecan. Under less physiological 2D plastic cultures, EpRas cells undergoing EMT failed to deposit TN-C into an (apparently incomplete) ECM. Ras-downstream signaling was dissected by pharmacological inhibitors and effector-specific Ras mutants (V12S35, V12C40), specifically inhibiting or activating ERK/MAPK or PI3K signaling, respectively. We showed that TN-C overexpression required a hyperactive ERK/MAPK-signaling pathway, while elevated PI3K signaling did not enhance TN-C expression. Similarly, tumors induced by cells exhibiting hyperactive ERK/MAPK signaling showed expression of TN-C in the tumor cells themselves, while only endothelial cells expressed TN-C in tumors caused by the V12C40 mutant (incapable of EMT in vivo). Taken together, our data indicate that hyperactive ERK/MAPK signaling causes enhanced expression of TN-C, while its secretion is induced by TGFbeta1 and both signals cooperate in TN-C matrix deposition. Importantly, both signals also cooperate to induce EMT in vitro and tumor progression/metastasis in vivo.

Animals↗