Matrix metalloproteinase-9 functions as a tumor suppressor in colitis-associated cancer

Abstract

There is a well-documented association of matrix metalloproteinase-9 (MMP-9) and receptor Notch-1 overexpression in colon cancer. We recently showed that MMP-9 is also upregulated in colitis, where it modulates tissue damage and goblet cell differentiation via proteolytic cleavage of Notch-1. In this study, we investigated whether MMP-9 is critical for colitis-associated colon cancer (CAC). Mice that are wild type (WT) or MMP-9 nullizygous (MMP-9(-/-)) were used for in vivo studies and the human enterocyte cell line Caco2-BBE was used for in vitro studies. CAC was induced in mice using an established carcinogenesis protocol that involves exposure to azoxymethane followed by treatment with dextran sodium sulfate. MMP-9(-/-) mice exhibited increased susceptibility to CAC relative to WT mice. Elevations in tumor multiplicity, size, and mortality were associated with increased proliferation and decreased apoptosis. Tumors formed in MMP-9(-/-) mice exhibited expression of p21(WAF1/Cip1) and increased expression of beta-catenin relative to WT mice. In vitro studies of MMP-9 overexpression showed increased Notch-1 activation with a reciprocal decrease in beta-catenin. Notch and beta-catenin/Wnt signaling have crucial roles in determining differentiation and carcinogenesis in gut epithelia. Despite being a mediator of proinflammatory responses in colitis, MMP-9 plays a protective role and acts as a tumor suppressor in CAC by modulating Notch-1 activation, thereby resulting in activation of p21(WAF1/Cip1) and suppression of beta-catenin.

Figure 1

MMP-9 is highly expressed in CAC and MMP-9^-/- mice show increased susceptibility to CAC. WT and MMP-9^-/- mice (mice, n= 6 each group) were treated once with AOM and twice with 3% DSS to induce CAC. Mice were weighed once a week and were sacrificed after 56 days. 1A: western blot of proteins from the mucosal stripping of the colons of WT and MMP-9^-/- mice with and without CAC probed with anti-MMP-9. Each lane shows protein (30μg/lane) from an individual mouse with or without CAC induction. Western blots were quantitated by scanning densitometry. Values are representative of three experiments, each bar represents mean ± S.E., *p<0.05. 1B: change in body weight of WT and MMP-9^-/- mice (mice, n= 10 each group). 1C: inflammatory score, 1D(i): polyps count and 1D(ii): size of the polyp measured in diameter as described in the Methods section. Each bar represents mean ± S.E., *p< 0.05.

Figure 2

MMP-9^-/- mice show increased susceptibility to CAC and mortality. Mortality studies were done as described in the Methods section. 2A: shows the mortality among WT and MMP-9^-/- mice induced with CAC and followed for a total of 140 days or were sacrificed if they developed rectal prolapse and/or ≥20% body weight loss. 2B: polyps count and 2C: size of the polyp measured in diameter as described in the Methods section. Each bar represents mean ± S.E., *p< 0.05. 2D: representative gross anatomy of the colon of the MMP-9^-/- mice and WT mice reflecting the number of polyps among them.

Figure 3

CAC in MMP-9^-/- is associated with increased proliferation and decreased apoptosis. WT and MMP-9^-/- mice (mice, n= 6 each group) with induced CAC and sacrificed after 56 days. Colon of the mice was processed for immunohistochemistry using anti-Ki67 antibody as described in the Methods section. 3A: representative sections of MMP-9^-/- and WT mice with induced CAC. Proliferating cells are indicated by brown nuclei of the crypts with a counter staining of hematoxylin. 3B: graphs representing the number of nuclei positive for Ki67 staining per crypt. Each bar represents mean ± S.E., *p<0.05. 3C: shows the western blot of protein from the mucosal stripping of the colons of WT and MMP-9^-/- with and without induction of CAC probed with anti-pCNA. Each lane shows protein (30μg/lane) from an individual mouse. Western blots were quantified by scanning densitometry and densities of MMP-9^-/- mice relative to WT mice both induced with CAC are graphed adjacently.

Figure 4

A: TUNEL and DAPI staining and apoptotic cells in the right panel. 4B: represents the western blot of whole tissue lysates of the colons of WT and MMP-9^-/- mice with and without CAC induction probed with anti-caspase-3. Each lane shows protein (30μg/lane) from an individual mouse. Adjacent graph shows the quantification of Western blots by scanning densitometry and plotting the densities of MMP-9^-/- mice relative to WT mice both induced with CAC. Values are representative of three experiments, mean ± SE; *p<0.05.

Figure 5

CAC in MMP-9^-/- mice showed altered protein expressions of p21^WAF1/Cip1, β-catenin, NF-κB, COX-2 and i-NOS. WT and MMP-9^-/- mice (mice, n= 6 each group) induced with CAC were sacrificed after 56 days. Each lane shows protein (30μg/lane) from an individual mouse with or without induction of CAC. 5A(i-iii): western blots of protein from the mucosal stripping of the colons of WT and MMP-9^-/- mice with and without CAC induction probed with anti-p21^WAF1/Cip1, anti- β-catenin and anti- NF-κB respectively. Adjacent graphs show the quantification by scanning densitometry. 5B(i-ii): western blots of protein from the mucosal stripping of the colons of WT and MMP-9^-/- mice with and without induction of CAC probed with anti-COX-2 and anti-i-NOS respectively. Western blots were quantitated by scanning densitometry and graphed adjacently. Values are representative of three experiments, each bar represents mean ± S.E., *p<0.05.

Figure 6

MMP-9 overexpression results in altered levels of NICD, β-catenin, COX-2 and i-NOS. Caco2-BBE cells stably transfected with a pEGFP plasmid, with or without the MMP-9 gene were plated on 6 well filters at confluency and cell lysates were collected and immunoblotted for 6A(i): MMP-9, 6A(ii): NICD, 6A(iii): β-catenin, 6B(i): COX-2 and 6B(ii): i-NOS. β-tubulin was used as the loading control. Western blots were quantitated by scanning densitometry and graphed adjacently. Each lane shows protein (30μg/lane). Values are representative of three experiments, mean ± SE; *p<0.05.