Neuropeptide Y (NPY) promotes inflammation-induced tumorigenesis by enhancing epithelial cell proliferation.

We have demonstrated that neuropeptide Y (NPY), abundantly produced by enteric neurons, is an important regulator of intestinal inflammation. However, the role of NPY in the progression of chronic inflammation to tumorigenesis is unknown. We investigated whether NPY could modulate epithelial cell proliferation and apoptosis, and thus regulate tumorigenesis. Repeated cycles of dextran sodium sulfate (DSS) were used to model inflammation-induced tumorigenesis in wild-type (WT) and NPY knockout (NPY-/-) mice. Intestinal epithelial cell lines (T84) were used to assess the effects of NPY (0.1 µM) on epithelial proliferation and apoptosis in vitro. DSS-WT mice exhibited enhanced intestinal inflammation, polyp size, and polyp number (7.5 ± 0.8) compared with DSS-NPY-/- mice (4 ± 0.5, P < 0.01). Accordingly, DSS-WT mice also showed increased colonic epithelial proliferation (PCNA, Ki67) and reduced apoptosis (TUNEL) compared with DSS-NPY-/- mice. The apoptosis regulating microRNA, miR-375, was significantly downregulated in the colon of DSS-WT (2-fold, P < 0.01) compared with DSS-NPY-/--mice. In vitro studies indicated that NPY promotes cell proliferation (increase in PCNA and ß-catenin, P < 0.05) via phosphatidyl-inositol-3-kinase (PI3-K)-ß-catenin signaling, suppressed miR-375 expression, and reduced apoptosis (increase in phospho-Bad). NPY-treated cells also displayed increased c-Myc and cyclin D1, and reduction in p21 (P < 0.05). Addition of miR-375 inhibitor to cells already treated with NPY did not further enhance the effects induced by NPY alone. Our findings demonstrate a novel regulation of inflammation-induced tumorigenesis by NPY-epithelial cross talk as mediated by activation of PI3-K signaling and downregulation of miR-375. NEW & NOTEWORTHY: Our work exemplifies a novel role of neuropeptide Y (NPY) in regulating inflammation-induced tumorigenesis via two modalities: first by enhanced proliferation (PI3-K/pAkt), and second by downregulation of microRNA-375 (miR-375)-dependent apoptosis in intestinal epithelial cells. Our data establish the existence of a microRNA-mediated cross talk between enteric neurons producing NPY and intestinal epithelial cells, and the potential of neuropeptide-regulated miRNAs as potential therapeutic molecules for the management of inflammation-associated tumors in the gut.

MicroRNA 375 mediates palmitate-induced enteric neuronal damage and high-fat diet-induced delayed intestinal transit in mice.

BACKGROUND & AIMS: A high-fat diet (HFD) can cause serious health problems, including alteration of gastrointestinal transit, the exact mechanism of which is not clear. Several microRNAs (miRNAs) are involved in energy homeostasis, lipid metabolism, and HFD-induced weight gain. We investigated the role of miRNAs in HFD-induced damage to the enteric nervous system. METHODS: Male mice were fed a HFD (60% calories from fat) or regular diets (18% calories from fat) for 11 weeks. Mice on regular diets and HFDs were given intraperitoneal injections of Mir375 inhibitor or a negative control. Body weights, food intake, stool indices, and gastrointestinal transit (following Evans blue gavage) were measured. An enteric neuronal cell line (immorto-fetal enteric neuronal) and primary enteric neurons were used for in vitro studies. RESULTS: HFD delayed intestinal transit, which was associated with increased apoptosis and loss of colonic myenteric neurons. Mice fed a low-palmitate HFD did not develop a similar phenotype. Palmitate caused apoptosis of enteric neuronal cells associated with mitochondrial dysfunction and endoplasmic reticulum stress. Palmitate significantly increased the expression of Mir375 in vitro; transfection of cells with a Mir375 inhibitor prevented the palmitate-induced enteric neuronal cell apoptosis. Mir375 expression was increased in myenteric ganglia of mice fed HFD and associated with decreased levels of Mir375 target messenger RNAs, including Pdk1. Systemic injection of a Mir375 inhibitor for 5 weeks prevented HFD-induced delay in intestinal transit and morphologic changes. CONCLUSIONS: HFDs delay colonic transit, partly by inducing apoptosis in enteric neuronal cells. This effect is mediated by Mir375 and is associated with reduced levels of Pdk1. Mir375 might be targeted to increase survival of enteric neurons and gastrointestinal motility.

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

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.

Tumor necrosis factor-neuropeptide Y cross talk regulates inflammation, epithelial barrier functions, and colonic motility.

BACKGROUND: Neuro-immune interactions play a significant role in regulating the severity of inflammation. Our previous work demonstrated that neuropeptide Y (NPY) is upregulated in the enteric nervous system during murine colitis and that NPY knockout mice exhibit reduced inflammation. Here, we investigated if NPY expression during inflammation is induced by tumor necrosis factor (TNF), the main proinflammatory cytokine. METHODS: Using primary enteric neurons and colon explant cultures from wild type and NPY knockout (NPY(-/-)) mice, we determined if NPY knockdown modulates TNF release and epithelial permeability. Further, we assessed if NPY expression is inducible by TNF in enteric neuronal cells and mouse model of experimental colitis, using the TNF inhibitors-etanercept (blocks transmembrane and soluble TNF) and XPro1595 (blocks soluble TNF only). RESULTS: We found that enteric neurons express TNF receptors (TNFR1 and R2). Primary enteric neurons from NPY(-/-) mice produced less TNF compared with wild type. Further, TNF activated NPY promoter in enteric neurons through phospho-c-Jun. NPY(-/-) mice had decreased intestinal permeability. In vitro, NPY increased epithelial permeability through phosphatidyl inositol-3-kinase (PI3-K)-induced pore-forming claudin-2. TNF inhibitors attenuated NPY expression in vitro and in vivo. TNF inhibitor-treated colitic mice exhibited reduced NPY expression and inflammation, reduced oxidative stress, enhanced neuronal survival, and improved colonic motility. XPro1595 had more protective effects on neuronal survival and motility compared with etanercept. CONCLUSIONS: We demonstrate a novel TNF-NPY cross talk that modulates inflammation, barrier functions, and colonic motility during inflammation. It is also suggested that selective blocking of soluble TNF may be a better therapeutic option than using anti-TNF antibodies.

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

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.