Smad ubiquitination regulatory factor 2 promotes metastasis of breast cancer cells by enhancing migration and invasiveness - PubMed (original) (raw)

Smad ubiquitination regulatory factor 2 promotes metastasis of breast cancer cells by enhancing migration and invasiveness

Chaoyang Jin et al. Cancer Res. 2009.

Abstract

Controlled protein degradation mediated by ubiquitin/proteasome system (UPS) plays a crucial role in modulating a broad range of cellular responses. Dysregulation of the UPS often accompanies tumorigenesis and progression. Here, we report that Smad ubiquitination regulatory factor 2 (Smurf2), a HECT-domain containing E3 ubiquitin ligase, is up-regulated in certain breast cancer tissues and cells. We show that reduction of Smurf2 expression with specific short interfering RNA in metastatic breast cancer cells induces cell rounding and reorganization of the actin cytoskeleton, which are associated with a less motile and invasive phenotype. Overexpression of Smurf2 promotes metastasis in a nude mouse model and increases migration and invasion of breast cancer cells. Moreover, expression of Smurf2CG, an E3 ligase-defective mutant of Smurf2, suppresses the above metastatic behaviors. These results establish an important role for Smurf2 in breast cancer progression and indicate that Smurf2 is a novel regulator of breast cancer cell migration and invasion.

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Figures

Figure 1

Up-regulation of Smurf2 in breast cancers. A, immunohistochemical staining of tumor (A3, D5, E9) and matching normal (A4, D6, E10) tissues in a breast cancer tissue array with anti-Smurf1 or anti-Smurf2 antibody. Three pairs of representative images are shown here and coordinates of each sample in the array are given in the top right corner. Scale bar, 4 μm. B, expression of Smurfs, Smads, and TβRI receptor in various cancer and normal control cells. *, a nonspecific band above the Smurf2 band. C, normal activation of Smads in MCF-10A and MDA-MB-231 cells. D, attenuated Smad expression induced by prolonged exposure (2 d) to TGF-β is resistant to RNAi-mediated knockdown of Smurf1 and/or Smurf2 in MDA-MB-231 cells. Left, protein levels in normal MCF-10A cells.

Figure 2

Requirement of elevated Smurf expression for the migration and invasion of MDA-MB-231 cells. Transient transfection of siRNAs was used to attenuate Smurfs expression in MDA-MD-231 cells. A, immunofluorescent staining of F-actin showing disruption of cell protrusion as the result of interfering with Smurfs expression. Scale bar, 5 μm. B and C, cell migration and invasion assays in transwell chambers. Scale bar, 10 μm.

Figure 3

Elevated Smurf2 expression promotes breast cancer cell metastasis. A, comparable endogenous Smurf1 and Smurf2 levels between normal MCF-10A and metastatic MCF10Ca1a cells. B, elevated Smurf2 and Smurf2CG expression in the MCF10Ca1a cell–derived stable lines. C, percentage of animals with lung metastases (n = 25), and (D) number of histologically confirmed lung metastasis foci per lung in mice inoculated with stable MCF10Ca1a cells bearing pBabe vector, pBabe-Smurf2, or pBabe-Smurf2CG (n = 25). Columns, mean; bars, SD. Statistically significant differences (P) between Smurf2 or Smurf2CG-expressing cells versus pBabe-puro vector control cells are indicated.

Figure 4

Enhanced N-cadherin expression, migration, and invasiveness of MCF10Ca1a cells expressing elevated Smurf2. A, Western analyses of stable MCF10Cala cells for Smads and cadherin expression. B, thymidine incorporation assay for TGF-β–induced growth inhibition. C to D, transwell migration and invasion assays of stable MCF10Ca1a cells.

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Smad ubiquitination regulatory factor 2 promotes metastasis of breast cancer cells by enhancing migration and invasiveness - PubMed (original) (raw)