Notch1 regulates the effects of matrix metalloproteinase-9 on colitis-associated cancer in mice

Abstract

Background & aims: Inflammatory bowel disease increases the risks of colon cancer and colitis-associated cancer (CAC). Epithelial cell-derived matrix metalloproteinase (MMP)-9 mediates inflammation during acute colitis and the cleavage and activation of the transcription factor Notch1, which prevents differentiation of progenitor cells into goblet cells. However, MMP-9 also protects against the development of CAC and acts as a tumor suppressor. We investigated the mechanisms by which MMP-9 protects against CAC in mice.

Methods: C57/B6 wild-type mice were given a single dose of azoxymethane and 2 cycles of dextran sulfate sodium (DSS). Mice were also given the γ-secretase inhibitor difluorophenacetyl-l-alanyl-S-phenylglycine t-butyl ester (DAPT) or dimethyl sulfoxide (control) during each DSS cycle; they were killed on day 56. We analyzed embryonic fibroblasts isolated from wild-type and MMP-9-/- mice and HCT116 cells that were stably transfected with MMP-9.

Results: Wild-type mice were more susceptible to CAC following inhibition of Notch1 by DAPT, shown by increased numbers of tumors and level of dysplasia compared with controls. Inhibition of Notch1 signaling significantly reduced protein levels of active Notch1, p53, p21WAF1/Cip1, Bax-1, active caspase-3, as well as apoptosis, compared with controls. Similar results were observed in transgenic HCT116 cells and embryonic fibroblasts from MMP-9-/- mice on γ-radiation-induced damage of DNA.

Conclusions: MMP-9 mediates Notch1 signaling via p53 to regulate apoptosis, cell cycle arrest, and inflammation. By these mechanisms, it might prevent CAC.

Figure 1. Inhibition of Notch1 signaling results in increased susceptibility to CAC

CAC was induced in WT and MMP-9^−/− mice as described in the Methods section. Notch1 signaling was inhibited by 5 consecutive i.p. injections of DAPT or vehicle during each DSS cycle. Mice were weighed once a week and sacrificed after 56 days. A) graphical presentation of change in body weight of mice treated with Notch1 inhibitor and placebo. B) left panel shows representative colonoscopy images and right panel shows representative gross anatomy of the colon of mice treated with Notch1 inhibitor and vehicle. C) polyp count and D) polyp size. E) H&E staining of Swiss rolls showing dysplasia. F) histological score of mice treated with Notch1 inhibitor and vehicle. Each bar represents mean ± S.E. Data is representative of three experiments (n=20/group). *p< 0.05.

Figure 2. Inhibition of Notch1 signaling alters mRNA levels of cytokines

As described in the Methods section, Notch1 signaling was inhibited by 5 consecutive i.p. injections of DAPT or vehicle alone at each DSS cycle during CAC induction and mice were sacrificed after 56 days. RNA was extracted from colonic mucosal stripping and mRNA levels were quantified using real time PCR A) TNF-α mRNA, B) IF-γ mRNA, C) IL-1β mRNA and D) IL-6 mRNA. Each bar represents mean ± S.E., n=6, ^ap<0.05 vs water, ^bp<0.05 vs vehicle.

Figure 3. Inhibition of Notch1 is associated with altered levels of p53, Bax-1 and p21^WAF1/Cip1

As described in the Methods section, Notch1 signaling was inhibited by 5 consecutive i.p. injections of DAPT or vehicle alone at each DSS cycle during CAC induction and mice were sacrificed after 56 days. Western blots of proteins (30μg/lane) from the mucosal stripping of the colons were probed with A) anti-MMP9, B) anti-NICD, C) anti-p53, D) anti-Bax-1 and E) anti-p21^WAF1/Cip1. Western blots are quantified by scanning densitometry. Values are representative of three experiments, each bar represents mean ± S.E., *p<0.05.

Figure 4. Inhibition of Notch signaling is associated with decreased apoptosis during CAC

CAC was induced in WT mice as described in the Methods section. Notch1 signaling was inhibited by 5 consecutive i.p. injections of DAPT or vehicle alone during each DSS cycle. A) colons of mice were processed for immunofluorescence using TUNEL and DAPI staining (X20 magnification). B) bar graph presentation of the percentage of apoptotic cells/crypt (12 crypts/mice) among mice treated with Notch1 inhibitor and placebo. C) Western blot of proteins (30μg/lane) from the mucosal stripping of the colons probed with anti-caspase-3. Western blot was quantified by scanning densitometry. Values are representative of three experiments, each bar represents mean ± S.E., *p<0.05.

Figure 5

A) MEFs exposed to γ-radiation induce DNA damage and indicate that MMP9 activates p53 expression. MEFs obtained from WT and MMP9^−/− mice embryos were harvested on six well plates. Cultured cells were γ-irradiated (12Gy) and cell lysates were collected after 24 hrs. Western blots of proteins (15μg/lane) probed with i) anti-p53, ii) anti-NICD, iii) anti-Bax-1 and iv) anti-p21^WAF1/Cip1. Western blots were quantified by scanning densitometry. Values are representative of three experiments, each bar represents mean ± S.E., *p<0.05. B) Inhibition of Notch1 signaling among MEFs exposed to γ-radiation exhibited decreased NICD, p53 and p21^WAF1/Cip1 expression. MEFs obtained from WT mice embryos were harvested on six well plates. Cultured cells were γ-irradiated (12Gy) and then treated with DAPT (Notch1 inhibitor) or vehicle and cell lysates were collected after 24 hrs. Western blot of proteins (15μg/lane) probed with i) anti-NICD, ii) anti-p53 and iii) anti-p21^WAF1/Cip1. Western blots are quantified by scanning densitometry. Values are representative of three experiments, each bar represents mean ± S.E., *p<0.05.

Figure 6. MMP9 overexpression results in altered expression of NICD, p53, Bax-1 and p21^WAF1/Cip1

Stably transfected HCT116 cells overexpressing MMP9 were harvested from six well plates. Cultured cells were γ-irradiated (12Gy) and collected after 24 hrs. Western blot of proteins (15μg/lane) probed with A) anti-MMP9, B) anti-NICD, C) anti-p53 D) anti-Bax-1 and E) p21^WAF1/Cip1. Western blots were quantified by scanning densitometry. Values are representative of three experiments, each bar represents mean ± S.E., *p<0.05.

Figure 7. Schematic representation of mechanistic pathway showing that MMP9 acts as a tumor suppressor in CAC

During inflammation, MMP9 expression is elevated and we have shown previously that MMP9 activates Notch1. Active Notch1 (NICD) then translocates to nucleus and activates p53. Activation of p53 activates p21^WAF1/Cip1 which promotes cell cycle arrest and thereby promoting the protection from CAC and/or activates caspase cascade leading to an increase in apoptosis and thereby resulting in protection from CAC. Thus MMP-9 acts as a tumor suppressor during CAC, likely through p53 activation via activation of Notch1.