Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Jul;8(7):939-51.
doi: 10.1158/1541-7786.MCR-09-0523. Epub 2010 Jun 22.

Tissue inhibitor of metalloproteinase-2 regulates matrix metalloproteinase-2-mediated endothelial barrier dysfunction and breast cancer cell transmigration through lung microvascular endothelial cells

Qiang Shen  1 , Eugene S Lee, Robert L Pitts, Mack H Wu, Sarah Y Yuan
Affiliations

Tissue inhibitor of metalloproteinase-2 regulates matrix metalloproteinase-2-mediated endothelial barrier dysfunction and breast cancer cell transmigration through lung microvascular endothelial cells

Qiang Shen et al. Mol Cancer Res. 2010 Jul.

Abstract

Matrix metalloproteinases (MMP) have been implicated in multiple stages of cancer metastasis. Tissue inhibitor of metalloproteinase-2 (TIMP-2) plays an important role in regulating MMP-2 activity. By forming a ternary complex with pro-MMP-2 and its activator MMP-14 on the cell surface, TIMP-2 can either initiate or restrain the cleavage and subsequent activation of MMP-2. Our recent work has shown that breast cancer cell adhesion to vascular endothelial cells activates endothelial MMP-2, promoting tumor cell transendothelial migration (TEM(E)). However, the mechanism of MMP-2 regulation during TEM(E) remains unclear. In the current study, we present evidence that MMP-14 is expressed in both invasive breast cancer cells (MDA-MB-231 and MDA-MB-436) and lung microvascular endothelial cells (HBMVEC-L), whereas TIMP-2 is exclusively expressed and released from the cancer cells. The tumor cell-derived TIMP-2 was further identified as a major determinant of endothelial MMP-2 activity during tumor cell transmigration in the presence of MMP-14. This response was associated with endothelial barrier dysfunction because coculture of MDA-MB-231 or MDA-MB-436 with HBMVEC-L caused a significant decrease in transendothelial electrical resistance concomitantly with endothelial cell-cell junction disruption and tumor cell transmigration. Knockdown of TIMP-2 or inhibition of TIMP-2/MMP-14 attenuated MMP-2-dependent transendothelial electrical resistance response and TEM(E). These findings suggest a novel interactive role of breast cancer cells and vascular endothelial cells in regulating the TIMP-2/MMP-14/MMP-2 pathway during tumor metastasis.

PubMed Disclaimer

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

FIGURE 1
FIGURE 1
Breast cancer cells (MDA-MB-231 and MDA-MB-436) activate human lung microvascular endothelial cells (HBMVEC-L) MMP-2. A, zymographic analysis of conditioned media collected from HBMVEC-L, MDA-MB-231, or MDA-MB-436 cell monoculture and coculture at various time points. HBMVEC-L cells produce latent MMP-2 that can be activated when cocultured with breast cancer MDA-MB-231 or MDA-MB-436 cells. B, quantitative analysis of active MMP-2 in conditioned media of HBMVEC-L/MDA-MB-231 coculture. C, quantitative analysis of active MMP-2 in conditioned media of HBMVEC-L/MDA-MB-436 coculture. Data were normalized by the levels of active MMP-2 at 24 h and presented as mean ± SE (n = 3). *, P < 0.01.
FIGURE 2
FIGURE 2
Expression of MMP-14, TIMP-4, and TIMP-2 in lung microvascular endothelial cells and breast cancer cells (MDA-MB-231 and MDA-MB-436). A, Western blot analysis showing the protein levels of MMP-14, TIMP-4, and TIMP-2 in HBMVEC-L, MDA-MB-231, and MDA-MB-436 cells. Tubulin is used as a loading control. B, ELISA assays for TIMP-2 and TIMP-4 levels in conditioned media from MDA-MB-231 monoculture or MDA-MB-231/HBMVEC-L coculture. C, TIMP-2 and TIMP-4 levels in conditioned media of MDA-MB-436 monoculture or MDA-MB-436/HBMVEC-L coculture. Data presented as mean ± SE (n = 3). NS, not statistically significant; #, P < 0.05.
FIGURE 3
FIGURE 3
Tumor cell–derived TIMP-2 and MMP-14 are required for MMP-2 activation. A, top, TIMP-2 levels in MDA-MB-231 and MDA-MB-436 cells 48 h after transfection of TIMP-2 siRNA or mock transfection. Bottom, TIMP-2 levels in conditioned media (12 h) of MDA-MB-231 and MDA-MB-436 cells transfected with TIMP-2 siRNA. Mock transfectants were used as controls. B, top, representative zymography showing the levels of latent and active MMP-2 in conditioned media of HBMVEC-L cocultured with MDA-MB-231 cells that were transfected with TIMP-2 siRNA or in the presence of a blocking antibody against TIMP-2 or MMP-14. Mock transfectants were used as controls for TIMP-2-knockdown cells. Normal IgG is used as an isotype control for antibody blockage assays. Middle, quantitative analysis of active MMP-2 in HBMVEC-L/MDA-MB-231 coculture conditioned media. For TIMP-2-knockdown MDA-MB-231 cells, data were normalized to the levels of active MMP-2 in coculture with mock transfectants. For the antibody blockage assays, data were normalized to the levels of active MMP-2 in the presence of control antibodies. Bottom, normalized levels of active MMP-2 in HBMVEC-L/MDA-MB-436 coculture conditioned media. Data presented as mean ± SE (n = 3). *, P < 0.01.
FIGURE 4
FIGURE 4
TIMP-2 promotes transmigration of breast cancer cells (MDA-MB-231 and MDA-MB-436) through HBMVEC-L monolayers. A, representative images of transmigrated MDA-MB-231 cells: a, mock-transfected MDA-MB-231 cells; b, TIMP-2-knockdown MDA-MB-231 cells. MDA-MB-231 cells were identified by the fluorescence of cell-loaded calcein-AM. B, quantitative analysis of transmigrated MDA-MB-231 and MDA-MB-436 cells (number/mm2) that are transfected with mock or TIMP-2 siRNA. Untreated MDA-MB-231 and MDA-MB-436 cells were used as controls. C, quantitative analysis of transmigrated MDA-MB-231 and MDA-MB-436 cells (number/mm2) in the presence of isotype control antibody or anti–TIMP-2, anti–MMP-2, or anti–MMP-14 antibody. Data presented as mean ± SE (n = 6). #, P < 0.05; *, P < 0.01. Bar, 50 µm.
FIGURE 5
FIGURE 5
TER indicative of barrier function is reduced in HBMVEC-L monolayers during coculture with MDA-MB-231. A, TER dynamics in the HBMVEC-L monolayers cultured with serum-free EBM medium, MDA-MB-231 cells (103 or 105 cells/cm2), and formaldehyde-fixed MDA-MB-231 cells (105 cells/cm2). B, concentration-dependent effects of MDA-MB-231 cells on the TER of HBMVEC-L monolayer after 12 h of coculture. The data are presented as mean ± SE (n = 6). *, P < 0.01.
FIGURE 6
FIGURE 6
TIMP-2 induces endothelial barrier dysfunction in HBMVEC-L cell monolayers. A, effects of knocking down TIMP-2 in MDA-MB-231 and MDA-MB-436 cells on the TER of HBMVEC-L monolayers after 12 h of coculture. B, effects of neutralizing TIMP-2, MMP-2, and MMP-4 on MDA-MB-231– and MDA-MB-436–induced TER decreases after 12 h of coculture. Normal IgG was used as isotype control. C, representative image of HBMVEC-L monolayer (red) after a 12-h coincubation with MDA-MB-231 cells (green) in the presence of normal IgG. Insets show higher magnifications of the designated area under individual channel, where HBMVEC-L cells were identified by VE-cadherin (red), MDA-MB-231 cells were identified by cell-loaded calcein-AM (green), and nuclei by Hoechst 33342 (blue). Note the MDA-MB-231–occupied intercellular gaps, where VE-cadherin was sequestrated from the periphery of HBMVEC-L (white arrows). D, representative images of HBMVEC-L monolayers (red) after a 12-h coincubation with MDA-MB-231 cells (green) in the presence of a blocking antibody against TIMP-2, MMP-14, or MMP-2. Insets show higher magnifications of the designated areas in anti–TIMP-2. Compared with the images in C, interendothelial gaps were less frequent and more VE-cadherin (red) was localized to the cell periphery; there were fewer MDA-MB-231 cells (green cells indicated by white arrow) transmigrating to endothelial tricellular corner. Data presented as mean ± SE (n = 6). #, P < 0.05; *, P < 0.01. Bar, 50 µm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kamby C, Ejlertsen B, Andersen J, et al. The pattern of metastases in human breast cancer. Influence of systemic adjuvant therapy and impact on survival. Acta Oncol. 1988;27:715–9. - PubMed
    1. Patanaphan V, Salazar OM, Risco R. Breast cancer: metastatic patterns and their prognosis. South Med J. 1988;81:1109–12. - PubMed
    1. Pagani O, Senkus E, Wood W, et al. International guidelines for management of metastatic breast cancer: can metastatic breast cancer be cured? J Natl Cancer Inst. 2010;102:456–63. - PMC - PubMed
    1. Murphy G, Stanton H, Cowell S, et al. Mechanisms for pro matrix metalloproteinase activation. Apmis. 1999;107:38–44. - PubMed
    1. Ellerbroek SM, Stack MS. Membrane associated matrix metalloproteinases in metastasis. Bioessays. 1999;21:940–9. - PubMed

Publication types

MeSH terms

Substances