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

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 formation1. We have previously investigated stromelysin-1/matrix metalloproteinase-3 (MMP-3), a stromal enzyme upregulated in many breast tumours2, and found that MMP-3 can cause epithelial–mesenchymal transition (EMT) and malignant transformation in cultured cells3,4,5, and genomically unstable mammary carcinomas in transgenic mice3. 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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Figure 1: MMP-3 induces EMT through Rac1b.
Figure 2: MMP-3/Rac1b stimulate mitochondrial production of ROS.
Figure 3: MMP-3-induced EMT is dependent on ROS.
Figure 4: MMP-3-induced ROS cause DNA damage and genomic instability.

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Acknowledgements

We thank G. Stark for advice with the PALA assay; M. LaBarge, M. Adriance and other members of the Bissell laboratory for discussions; J. Campisi for critical reading of the manuscript; and C. Chen for technical assistance. This work was supported by grants from the OBER office of the Department of Energy and an Innovator award from the Department of Defense (to M.J.B.) and from the National Institutes of Health (to M.J.B. and Z.W.), and by fellowships from the American Cancer Society (D.C.R.), the National Cancer Institute (L.E.L.), the Department of Defense (H.L. and C.M.N.) and the California Breast Cancer Research Program (J.E.F).

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Correspondence to Derek C. Radisky or Mina J. Bissell.

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Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figure S1

Properties of MMP-3-induced EMT (PDF 245 kb)

Supplementary Figure S2

Dependence of MMP-3-induced EMT on Rac1 activity (PDF 143 kb)

Supplementary Figure S3

Selective knockdown of cotransfected constructs by siRNA (PDF 3121 kb)

Supplementary Figure S4

Induction of EMT by proteolytic activity of MMP-3 (PDF 570 kb)

Supplementary Figure S5

Validation of Rac1b antibody (PDF 109 kb)

Supplementary Figure S6

Effect of YFP-fused Rac1 constructs on cell morphology (PDF 4359 kb)

Supplementary Figure S7

Activity assay of YFP-fused mouse Rac1b and Rac1V12 (PDF 95 kb)

Supplementary Figure Legends

Legends to accompany Supplementary Figures S1-S7 (DOC 28 kb)

Supplementary Methods

Genomic instability assays (DOC 26 kb)

Supplementary Notes

Statistical significance of data for Supplementary Figures S1-S4 (DOC 25 kb)

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Radisky, D., Levy, D., Littlepage, L. et al. Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability. Nature 436, 123–127 (2005). https://doi.org/10.1038/nature03688

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