J Biol Chem. 2009 Dec 4;284(49):34145-56. Epub 2009 Oct 12.

p130Cas is required for mammary tumor growth and transforming growth factor-beta-mediated metastasis through regulation of Smad2/3 activity.

Source

Department of Pharmacology, University of Colorado, Denver, Anschutz Medical Campus, Aurora, Colorado 80045, USA.

Abstract

During breast cancer progression, transforming growth factor-beta (TGF-beta) switches from a tumor suppressor to a pro-metastatic molecule. Several recent studies suggest that this conversion in TGF-beta function depends upon fundamental changes in the TGF-beta signaling system. We show here that these changes in TGF-beta signaling are concomitant with aberrant expression of the focal adhesion protein, p130Cas. Indeed, elevating expression of either the full-length (FL) or just the carboxyl terminus (CT) of p130Cas in mammary epithelial cells (MECs) diminished the ability of TGF-beta1 to activate Smad2/3, but increased its coupling to p38 MAPK. This shift in TGF-beta signaling evoked (i) resistance to TGF-beta-induced growth arrest, and (ii) acinar filling upon three-dimensional organotypic cultures of p130Cas-FL or -CT expressing MECs. Furthermore, rendering metastatic MECs deficient in p130Cas enhanced TGF-beta-stimulated Smad2/3 activity, which restored TGF-beta-induced growth inhibition both in vitro and in mammary tumors produced in mice. Additionally, whereas elevating TbetaR-II expression in metastatic MECs had no affect on their phosphorylation of Smad2/3, this event markedly enhanced their activation of p38 MAPK, leading to increased MEC invasion and metastasis. Importantly, depleting p130Cas expression in TbetaR-II-expressing metastatic MECs significantly increased their activation of Smad2/3, which (i) reestablished the physiologic balance between canonical and noncanonical TGF-beta signaling, and (ii) reversed cellular invasion and early mammary tumor cell dissemination stimulated by TGF-beta. Collectively, our findings identify p130Cas as a molecular rheostat that regulates the delicate balance between canonical and noncanonical TGF-beta signaling, a balance that is critical to maintaining the tumor suppressor function of TGF-beta during breast cancer progression.

PMID:: 19822523; [PubMed - indexed for MEDLINE]
PMCID:: PMC2797185

Free PMC Article

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FIGURE 2.

Overexpression of p130Cas inhibits Smad2/3 activity and alters normal mammary epithelial acinar formation. A, NMuMG cells were transduced with retroviral particles containing vector control (cntrl), full-length p130Cas (Cas-FL), or the carboxyl terminus of p130Cas (Cas-CT). Stable transgene expression was assessed by immunoblotting for phosphorylated (p-p130Cas) and total p130Cas (t-p130Cas). β-Actin (Actin) is shown as a loading control. B, the p130Cas-manipulated cell lines as described in A were stimulated with TGF-β1 (5 ng/ml) for the indicated times, and subsequently analyzed for the phosphorylation of Smad2 (psmad2), Smad3 (psmad3), and p38 MAPK (p-p38). Membranes were stripped and reprobed for total Smad2/3 (tsmad2/3) and p38 MAPK (t-p38) as loading controls. Data are from representative experiments that were performed at least three times with identical results. C, control (cntrl) and p130Cas-expressing (Cas-CT and Cas-FL) NMuMG cells were transiently co-transfected with pSBE-luciferase and pCMV-β-gal plasmids, and subsequently stimulated overnight with TGF-β1 (5 ng/ml) prior to measuring luciferase and β-gal activities. NS, no stimulation. Data are the mean ± S.E. of SBE/CMV activity ratios observed in three independent experiments completed in triplicate. ***, p < 0.001). D, control (cntrl) and p130Cas-expressing (Cas-CT and Cas-FL) NMuMG cells were stimulated with increasing concentrations of TGF-β1 (0–5 ng/ml) for 48 h, and subsequently assayed for [³H]thymidine incorporation into cellular DNA. Data are the mean ± S.E. quantities of incorporated [³H]thymidine normalized to unstimulated controls observed in three independent experiments completed in triplicate (*, p < 0.05 between Cntrl and Cas-FL; #, p < 0.05 between Cntrl and Cas-CT). E, the p130Cas-manipulated NMuMG cells described in A were grown in three-dimensional organotypic cultures for 10 days, at which point the percentage of hollowed acini were quantified by phase-contrast microscopy (**, p < 0.001; ***, p < 0.0001). Representative acini are shown.

p130Cas Is Required for Mammary Tumor Growth and Transforming Growth Factor-β-mediated Metastasis through Regulation of Smad2/3 Activity

J Biol Chem. J Biol Chem;284(49):34145-34156.