Regulation of Cripto-1 signaling and biological activity by caveolin-1 in mammary epithelial cells

Abstract

Human and mouse Cripto-1 (CR-1/Cr-1) proteins play an important role in mammary gland development and tumorigenesis. In this study, we examined the relationship between Cripto-1 and caveolin-1 (Cav-1), a membrane protein that acts as a tumor suppressor in the mammary gland. Cripto-1 was found to interact with Cav-1 in COS7 cells and mammary epithelial cells. Using EpH4 mouse mammary epithelial cells expressing Cr-1 (EpH4 Cr-1) or Cr-1 and Cav-1 (EpH4 Cr-1/Cav-1), we demonstrate that Cav-1 expression markedly reduced the ability of Cr-1 to enhance migration, invasion, and formation of branching structures in EpH4 Cr-1/Cav-1 cells as compared to EpH4 Cr-1 cells. Furthermore, coexpression of Cav-1 together with Cr-1 in EpH4 Cr-1/Cav-1 cells inhibited Cr-1-mediated activation of c-src and mitogen-activated protein kinase signaling pathways. Conversely, primary mammary epithelial cells isolated from Cav-1 null(-/-)/mouse mammary tumor virus-CR-1 transgenic animals showed enhanced motility and activation of mitogen-activated protein kinase and c-src as compared to Cav-1(+/-)/CR-1 mammary cells. Finally, mammary tumors derived from mouse mammary tumor virus-CR-1 mice showed a dramatic reduction of Cav-1 expression as compared to mammary tissue from normal FVB/N mice, suggesting that in vivo Cav-1 is down-regulated during the process of CR-1-mediated mammary tumorigenesis.

Figure 1

Colocalization of human CR-1 and Cav-1 in low-density sucrose fractions in 293 and COS7 cells. Western blot analysis on 5% to 40% sucrose gradient fractions isolated from 293 cells transiently transfected with expression plasmids encoding human CR-1 and Alk4-Flag (A) or from COS7 cells transiently transfected with a human CR-1 expression vector (B). Western blot analysis was performed using anti-Flag monoclonal antibody to detect Alk4-Flag fusion protein, anti-CR-1 mouse monoclonal antibody, anti-Cav-1 mouse monoclonal antibody, and anti-β-actin mouse monoclonal antibody. The lipid raft marker GM1 was detected with horseradish peroxidase-conjugated CT-B by dot blot. Low-density sucrose fractions: 4, 5, and 6.

Figure 2

Binding of Cripto-1 to Cav-1 in coimmunoprecipitation experiments in COS7 cells and in FVB/N and MCF-10A mammary epithelial cells. A: COS7 cells were transiently transfected with Cav-1-myc/His, CR-1 and c-src expression vectors either alone or in various combinations, and 48 hours after transfection the cells were lysed and proteins were immunoprecipitated with anti-myc monoclonal antibody agarose beads. The myc-tagged immunoprecipitated proteins were then analyzed by Western blot analysis using anti-CR-1 rabbit polyclonal antibody, anti-c-src mouse monoclonal antibody or anti-His mouse monoclonal antibody to detect Cav-1-myc/His fusion protein. B: COS7 cells were transiently transfected with Cav-1-myc/His, CR-1 and glypican-1-Fc expression vectors either alone or in various combinations and 48 hours after transfection the cells were lysed and protein G-agarose beads were used to immunoprecipitate glypican-1-Fc fusion protein. The bound proteins were analyzed by Western blotting with anti-CR-1 rabbit polyclonal antibody, anti-His mouse monoclonal antibody, or anti-human IgG horseradish peroxidase-linked whole antibody to detect glypican-1-Fc fusion protein. C and D: Western blot analysis for CR-1, Cav-1, c-src, and glypican-1 on cell lysates of COS7 cells transiently transfected with various expression vectors as described above. E: FVB/N primary mouse mammary epithelial cells and MCF-10A human mammary epithelial cells were lysed and immunoprecipitated with an anti-CR-1 rabbit polyclonal antibody. The immunoprecipitated proteins were then analyzed by Western blotting using an anti-Cav-1 rabbit polyclonal antibody and anti-CR-1 rabbit polyclonal antibody. IP, immunoprecipitation; WB, Western blot.

Figure 3

Stable Cav-1 expression in EpH4 mouse mammary epithelial cells. Cell lysates from EpH4 WT, EpH4 Cav-1, EpH4 Cr-1, and EpH4 Cr-1/Cav-1 cells were analyzed by Western blotting to confirm gene expression using anti-CR-1 mouse monoclonal antibody, anti-V5 mouse monoclonal antibody to detect Cav-1-V5/His fusion protein, or anti-β-actin monoclonal antibody to ensure equal loading of the protein samples. WB, Western blot.

Figure 4

Colocalization of Cripto-1 and Cav-1 in COS7 cells expressing Cr-1 and Cav-1, in EpH4 Cr-1/Cav-1, EpH4 Cr-1, and FVB/N primary mammary epithelial cells. A: Immunofluorescence staining for Cr-1 and Cav-1 in COS7 cells transiently transfected with Cr-1 and Cav-1 expression vectors. B: Immunofluorescence staining for Cr-1, Cav-1, and GM-1 in EpH4 Cr-1/Cav-1 and EpH4 Cr-1 cells and immunofluorescence staining for Cr-1 and Cav-1 in primary FVB/N mouse mammary epithelial cells. C: Negative controls were obtained by replacing the primary antibody with irrelevant control isotype Ig. Fluorescence images were acquired by laser scanner confocal microscopy.

Figure 5

Cav-1 inhibits Cr-1 enhanced migration, invasion, and branching of EpH4 mouse mammary epithelial cells. A: Migration or invasion assay (B) of EpH4 WT, EpH4 Cav-1, EpH4 Cr-1, and EpH4 Cr-1/Cav-1 cells. OD, optical density. *P < 0.05 compared to EpH4 WT cells; **P < 0.05 compared to EpH4 Cr-1 cells. C–E: Growth in Matrigel of EpH4 WT, EpH4 Cav-1, EpH4 Cr-1, and EpH4 Cr-1/Cav-1 cells. After 1 week, colonies with branching structures were photographed (C) and counted (E). D: Magnification of EpH4 Cr-1 branching structures. *P < 0.0001 compared to EpH4 WT cells; **P < 0.0001 compared to EpH4 Cr-1 cells.

Figure 6

Cavtratin inhibits migration and invasion of EpH4 Cr-1 cells. Migration (A) and invasion (B) assay of EpH4 Cr-1 cells incubated in the presence of cavtratin. OD, optical density. *P < 0.05, compared to control, 5 μmol/L; **P < 0.05, compared to control, 10 μmol/L. Cav-1 interferes with the ability of Cr-1 to activate MAPK and c-src in EpH4 mouse mammary epithelial cells. Serum-starved EpH4 WT, EpH4 Cav-1, EpH4 Cr-1, and EpH4 Cr-1/Cav-1 cells were lysed and analyzed by Western blot analysis using phospho- and nonphospho-specific anti-MAPK (C) and anti-c-src antibodies (D). Serum-starved EpH4 WT and EpH4 Cr-1 cells were treated with cavtratin (10 μmol/L) or a control peptide (10 μmol/L) and analyzed by Western blotting using phospho- and nonphospho-specific anti-MAPK (E) and anti-c-src (F) antibodies. WB, Western blot.

Figure 7

Loss of Cav-1 enhances migration and invasion and induces hyperactivation of MAPK and c-src signaling molecules in primary mammary epithelial cells derived from Cav-1 null/MMTV-CR-1 double transgenic mice. A: Western blot analysis for Cav-1, CR-1, and β-actin in cell lysates derived from primary mammary epithelial cells isolated from FVB/N, Cav-1^+/−/CR-1, and Cav-1^−/−/CR-1 mice. Migration (B) and invasion (C) assay of FVB/N, Cav-1^+/−/CR-1, and Cav-1^−/−/CR-1 primary mammary epithelial cells. Cav-1^−/−/CR-1 primary mammary epithelial cells were also treated with cavtratin (10 μmol/L) or with a control peptide (10 μmol/L). *P < 0.05 compared to FVB/N cells; **P < 0.05 compared to Cav-1^+/−/CR-1 cells; ***P < 0.05 compared to control 10 μmol/L treated Cav-1^−/−/CR-1 cells. D: Protein lysates from FVB/N, Cav-1^+/−/CR-1, and Cav-1^−/−/CR-1 primary mammary epithelial cells were subjected to immunoblotting with antibodies directed against phospho- and total-MAPK and phospho- and total-c-src.

Figure 8

Cav-1 expression is reduced in mammary tumors of MMTV-CR-1 transgenic mice. A: Western blot analysis for Cav-1 in tissues from one normal FVB/N or two MMTV-CR-1 virgin mice and from mammary tumors found in four MMTV-CR-1 multiparous mice (T1, T2, T3 and T4). For equal loading, the blot was reprobed with anti-β-actin antibody. B: Immunohistochemistry for Cav-1 and CR-1 in mammary tissue serial sections derived from FVB/N virgin or multiparous mice and in mammary tumor of MMTV-CR-1 mice. These sections were incubated with an anti-Cav-1 or with anti-CR-1 antibodies as described in Materials and Methods. Magnification, ×400. Negative controls are shown in the insets. WB, Western blot.