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Link to original content: https://pubmed.ncbi.nlm.nih.gov/16001073/
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. 2005 Jul 7;436(7047):123-7.
doi: 10.1038/nature03688.

Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability

Derek C Radisky  1 , Dinah D Levy, Laurie E Littlepage, Hong Liu, Celeste M Nelson, Jimmie E Fata, Devin Leake, Elizabeth L Godden, Donna G Albertson, M Angela Nieto, Zena Werb, Mina J Bissell
Affiliations

Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability

Derek C Radisky et al. Nature. .

Abstract

The tumour microenvironment can be a potent carcinogen, not only by facilitating cancer progression and activating dormant cancer cells, but also by stimulating tumour formation. We have previously investigated stromelysin-1/matrix metalloproteinase-3 (MMP-3), a stromal enzyme upregulated in many breast tumours, and found that MMP-3 can cause epithelial-mesenchymal transition (EMT) and malignant transformation in cultured cells, and genomically unstable mammary carcinomas in transgenic mice. Here we explain the molecular pathways by which MMP-3 exerts these effects: exposure of mouse mammary epithelial cells to MMP-3 induces the expression of an alternatively spliced form of Rac1, which causes an increase in cellular reactive oxygen species (ROS). The ROS stimulate the expression of the transcription factor Snail and EMT, and cause oxidative damage to DNA and genomic instability. These findings identify a previously undescribed pathway in which a component of the breast tumour microenvironment alters cellular structure in culture and tissue structure in vivo, leading to malignant transformation.

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Figures

Figure 1
Figure 1. MMP-3 induces EMT through Rac1b
a, MMP-3-induced alterations in actin cytoskeleton. Scale bar, 25μm. b, Analysis of active and total levels of Rac. c, RT–PCR of Rac1 and Rac1b. d, Rac1b protein expression. e, Rac1b transcript levels in response to MMP-3 treatment (days 1–4) and washout (days 5–6); blue circles, treated; red squares, untreated. f, Cell motility assessed by scratch assay. dn, dominant-negative. g, Quantification of knockdown of endogenous gene expression. h, Selective knockdown of Rac1b inhibits MMP-3-induced cell scattering. Scale bar, 25μm. For all graphs, error bars represent s.e.m.
Figure 2
Figure 2. MMP-3/Rac1b stimulate mitochondrial production of ROS
a, Cellular ROS levels assessed by DCFDA. dn, dominant-negative. Error bars represent s.e.m. b, Mitochondrial pattern of DCFDA fluorescence. Scale bar, 25μm. c, Precipitation of nitroblue tetrazolium. Scale bar, 15μm. d, Mitochondrial depolarization assessed with JC-1. Scale bar, 50μm. e–g, Cells co-transfected with EYFP and catalase (CAT; e), superoxide dismutase 1 (SOD1; f) or superoxide dismutase 2 (SOD2; g) and then cultured in the absence (top) or presence (middle) of MMP-3 for 6 days. Green, EYFP fluorescence; red, nuclei. Graphs at bottom show gene transcript levels in transfected cell populations. Scale bar, 100μm.
Figure 3
Figure 3. MMP-3-induced EMT is dependent on ROS
a, NAC inhibits MMP-3-induced downregulation of epithelial cytokeratin protein levels. b, Induction of Snail by MMP-3, and dependence on ROS. c, Snail transcript levels in response to MMP-3 treatment (days 1–4) and washout (days 5–6); blue diamonds, treated; red squares, untreated. d, e, Exogenous expression of Snail in SCp2 cells reduces E-cadherin transcript (d) and protein levels (e). f, Cell scattering induced by treatment with MMP-3 or H2O2, or by exogenous expression of Snail. Scale bar, 50μm. g, h, ROS and Snail dependence of vimentin (g) and Rac1b (h) expression. For all graphs, error bars represent s.e.m.
Figure 4
Figure 4. MMP-3-induced ROS cause DNA damage and genomic instability
a, b, 8-Oxoguanosine-induced treatment with MMP-3 (a; scale bar, 50μm) and quantification of increased nuclear staining relative to untreated (b; error bars, 95% confidence intervals). c, Induction of PALA resistance by MMP-3 (blue diamonds, MMP-3; red squares, untreated). d, Fluorescence in situ hybridization of the CAD gene locus (red). e, ROS and oxygen dependence of PALA resistance induced by 14 days of treatment with MMP-3. f, Frequency plots of comparative genomic hybridization analyses of cells grown in the absence (top) or presence (bottom) of MMP-3, and then selected with PALA. Green, amplifications; red, deletions. For all graphs, error bars represent s.e.m.
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