Research ArticleCancer

The Rho Exchange Factors Vav2 and Vav3 Control a Lung Metastasis–Specific Transcriptional Program in Breast Cancer Cells

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Science Signaling  02 Oct 2012:
Vol. 5, Issue 244, pp. ra71
DOI: 10.1126/scisignal.2002962
  • Fig. 1

    Defective tumorigenesis and lung metastasis of Vav-deficient 4T1 cells. (A and B) Rac1 (A and B) and RhoA (A) activity was calculated in the indicated cell lines by G-LISA (A) and GST-Pak1 pull-down (B) experiments (n = 3 experiments). KD, knockdown cell line. Table S3 contains a description and designation of the cell lines used in these experiments. (C to F) Growth kinetics of tumors induced by the indicated cell lines. Statistics were performed at the 35-day time point. The number of independent samples in (C) was as follows: control, n = 15; KD2(A), n = 4; KD2(B), n = 4; KD3(A), n = 4; KD3(B), n = 4; KD2/3(A), n = 8. In (D) and (E), n = 8; in (F), n = 4. (G and H) Number of metastatic nodules detected in mice harboring tumors induced by the indicated cell lines (n = 5 animals). (I) Images showing the lack of macro- and micrometastasis in the lung of a mouse containing a tumor-derived KD2/3(A) cells. As control, we included a lung section from a mouse harboring a tumor derived from the control cell line. Scale bars, 100 μm (top image of each group) and 200 μm (bottom image of each group).

  • Fig. 2

    Vav family proteins have overlapping, but not identical, roles in primary tumorigenesis. (A and B) Example (A; scale bar, 50 μm) and quantitation (B; n = 3 sections per tumor, four animals) of the proliferation of tumors derived from the indicated cell lines. (C and D) Example (C; scale bar, 100 μm) and quantitation (D; n = 3 sections per tumor, three animals) of angiogenesis in tumors developed from the indicated cell lines. (E and F) Examples (E; scale bar, 100 μm) and quantification (F; n = 2 sections per tumor, three animals) of the vessel permeability of tumors derived from the indicated cell lines. (G and H) Example (G; scale bar, 50 μm) and quantification (H; n = 3 sections per tumor, four animals) of apoptosis in tumors derived from the indicated cell lines.

  • Fig. 3

    Vav proteins function during several metastatic stages. (A) Percentage of circulating cancer cells in mice with tumors derived from the indicated cell lines [n = 8 in experiments with control, KD2/3(A), and KD2/3 + V2/V3 cells, and n = 4 for the rest of the experiments]. a.u., arbitrary units. (B to E) Number of metastases (B and E) and extent of metastasis (C and D) formed in the lung by the indicated intravenously injected cell lines (n = 3 lung sections per mice, four mice in each experimental condition). Scale bar, 100 μm. (F) Lungs from mice intravenously injected with the indicated chromophore-labeled cell lines were imaged by confocal microscopy to visualize cancer cells (green) and the vascular endothelium (red). Scale bars, 100 μm (left image of each group) and 25 μm (right image of each group). (G) Quantification of the number of chromophore-labeled cancer cells in either whole lung sections (left) or the indicated lung locations (right) (n = 6 confocal sections per animal, four mice in each condition).

  • Fig. 4

    Protumorigenic and prometastatic roles of Vav proteins in independent experimental systems. (A and B) Primary tumorigenesis (A and B) and lung metastasis (B) induced by PyMT-transformed wild-type (WT) and Vav2−/−;Vav3−/− primary breast epithelial cells upon implantation in the fat pads of recipient WT female mice (n = 11 animals per genotype). (C) Abundance of Vav2 (left) and Vav3 mRNAs (right) in the indicated cells (n = 3 experiments). (D) Rac1 activity in the indicated cell lines detected with G-LISA assays (n = 3 experiments). (E and F) Growth kinetics of breast tumors formed by the indicated orthotopically transplanted cell lines (E) (n = 6 animals). In (F), the x/y ratio indicates the number (x) of animals that scored positive for metastasis in the indicated tissues compared to the total number (y) of mice analyzed in these experiments. (G) Example (left) and quantification (right) of metastases generated by the indicated intravenously injected cell lines (n = 4 animals). (H and I) In vitro proliferative (H) and invasion (I) properties of the indicated cell lines (n = 3 experiments). OD, optical density.

  • Fig. 5

    Vav family targets play overlapping, but not identical, roles in biological processes linked to primary tumorigenesis. (A) Growth of tumors derived from the indicated orthotopically transplanted cell lines (n = 4 animals). KD, knockdown cell. Clones A and B refer to two knockdown cell clones generated with two independent shRNAs for the indicated target (see also table S3). (B) Metastases found in tissues (top) of mice bearing tumors from the indicated cell clones. Values are given as in Fig. 4F. ND, not determined. (C and D) Example (C; scale bar, 50 μm) and quantitation (D; n = 3 sections per tumor, four animals) of the proliferation of tumors derived from the indicated cell lines. (E and F) Example (E; scale bar, 100 μm) and quantitation (F; n = 3 sections per tumor, three animals) of angiogenesis in tumors derived from the indicated cell lines. (G and H) Example (G; scale bar, 100 μm) and quantitation (H; n = 2 sections per tumor, three animals) of the vessel permeability of tumors derived from the indicated cell lines. (I and J) Example (I; scale bar, 50 μm) and quantitation (J; n = 3 sections per tumor, three animals) of apoptosis in tumors from the indicated cell lines.

  • Fig. 6

    Role of Vav-dependent targets in the metastatic steps of breast cancer cells. (A) Percentage of circulating cancer cells present in mice harboring tumors derived from the indicated cell lines (n = 4 animals). The description of each cell line can be found in table S3. (B and C) Visualization (B) and quantification of the extravasation rates (C) of the indicated chromophore-labeled cell lines after intravenous injection into mice. Scale bars in (B), 100 μm (left image of each group) and 25 μm (right image of each group) (n = 6 confocal sections per animal, four mice in each condition). (D to F) Number (D) and extent (E and F) of the metastatic nodules formed by the indicated intravenously injected cell lines (n = 3 independent lung sections per mice, four mice in each condition). Scale bar, 100 μm.

  • Fig. 7

    The Vav pathway is involved in the survival of cancer cells inside the lung. (A and B) Visualization (A; green color; scale bar, 100 μm) and quantification (B; n = 6 confocal sections per animal, four mice in each condition) of the extravasation of the indicated chromophore-labeled cell lines after intravenous injection in monocrotaline-treated (+MCT) and untreated (−MCT) mice. Endothelial cells are labeled in red in (A). (C and D) Visualization (C) and quantification (D; n = 4 mice) of the metastatic nodules induced by the indicated cell lines after intravenous injection in MCT-treated (+MCT) or untreated (−MCT) mice. (E) Area of the metastatic nodules generated by the indicated cell lines in the lung parenchyma (n = 3 sections per mice, four mice per experimental condition). (F) Summary of our results. The importance of a protein in the indicated process is proportional to the thickness and darkness of horizontal bars. Rac1-dependent and Rac1-independent routes are depicted in black and gray arrows, respectively. We cannot rule out the possibility that Ilk abundance is regulated by Vav3-mediated stimulation of another Rho GTPase. However, Vav2 contributes to this response in a GEF-independent manner.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/5/244/ra71/DC1

    Fig. S1. Human tumors show differential abundance of Rho GEF transcripts.

    Fig. S2. Abundance of Vav family mRNAs and proteins in cell lines used in this study.

    Fig. S3. Vav proteins control a large transcriptomal program in breast cancer cells.

    Fig. S4. Regulation of the breast cancer cell transcriptome by Vav proteins.

    Fig. S5. Vav proteins control the breast cancer cell transcriptome through common, synergistic, and Vav family member–specific routes.

    Fig. S6. Generation of knockdown cell lines for Vav family–dependent targets.

    Fig. S7. Vav-dependent mRNA signatures have prognostic value in breast cancer.

    Table S1. Differential abundance of Rho GEFs in human breast cancer subtypes.

    Table S2. Abundance of Vav2 and Vav3 mRNAs in the breast cancer luminal A and B subtypes.

    Table S3. Cell lines used in this study.

    Table S4. Metastasis of 4T1 knockdown cell clones in recipient mice.

    Table S5. Transcriptomal changes in 4T1 cells lacking Vav2 and Vav3 (Excel).

    Table S6. The Vav2/Vav3-dependent transcriptome associated to the metastatic potential of the 4T1 cells used in this study (Excel).

    Table S7. Overexpressed and repressed genes from table S5 associated with human cancer clinical outcome (Excel).

    Table S8. Genes belonging to the Vav2/Vav3 lung metastasis signature (Excel).

  • Supplementary Materials for:

    The Rho Exchange Factors Vav2 and Vav3 Control a Lung Metastasis–Specific Transcriptional Program in Breast Cancer Cells

    Carmen Citterio, Mauricio Menacho-Márquez, Ramón García-Escudero, Romain M. Larive, Olga Barreiro, Francisco Sánchez-Madrid, Jesús M. Paramio, Xosé R. Bustelo*

    *To whom correspondence should be addressed. E-mail: xbustelo{at}usal.es

    This PDF file includes:

    • Fig. S1. Human tumors show differential abundance of Rho GEF transcripts.
    • Fig. S2. Abundance of Vav family mRNAs and proteins in cell lines used in this study.
    • Fig. S3. Vav proteins control a large transcriptomal program in breast cancer cells.
    • Fig. S4. Regulation of the breast cancer cell transcriptome by Vav proteins.
    • Fig. S5. Vav proteins control the breast cancer cell transcriptome through common, synergistic, and Vav family member–specific routes.
    • Fig. S6. Generation of knockdown cell lines for Vav family–dependent targets.
    • Fig. S7. Vav-dependent mRNA signatures have prognostic value in breast cancer.
    • Table S1. Differential abundance of Rho GEFs in human breast cancer subtypes.
    • Table S2. Abundance of Vav2 and Vav3 mRNAs in the breast cancer luminal A and B subtypes.
    • Table S3. Cell lines used in this study.
    • Table S4. Metastasis of 4T1 knockdown cell clones in recipient mice.

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.45 MB

    Other Supplementary Material for this manuscript includes the following:

    • Table S5 (Microsoft Excel format). Transcriptomal changes in 4T1 cells lacking Vav2 and Vav3.
    • Table S6 (Microsoft Excel format). The Vav2/Vav3-dependent transcriptome associated to the metastatic potential of the 4T1 cells used in this study.
    • Table S7 (Microsoft Excel format). Overexpressed and repressed genes from table S5 associated with human cancer clinical outcome.
    • Table S8 (Microsoft Excel format). Genes belonging to the Vav2/Vav3 lung metastasis signature.

    [Download Tables S5 to S8]

    Technical Details

    Format: Microsoft Excel

    Size: 1.29 MB


    Citation: C. Citterio, M. Menacho-Márquez, R. García-Escudero, R. M. Larive, O. Barreiro, F. Sánchez-Madrid, J. M. Paramio, X. R. Bustelo, The Rho Exchange Factors Vav2 and Vav3 Control a Lung Metastasis–Specific Transcriptional Program in Breast Cancer Cells. Sci. Signal. 5, ra71 (2012).

    © 2012 American Association for the Advancement of Science

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