The neuroendocrine phenotype of gastric myofibroblasts and its loss with cancer progression.

Stromal cells influence cancer progression. Myofibroblasts are an important stromal cell type, which influence the tumour microenvironment by release of extracellular matrix (ECM) proteins, proteases, cytokines and chemokines. The mechanisms of secretion are poorly understood. Here, we describe the secretion of marker proteins in gastric cancer and control myofibroblasts in response to insulin-like growth factor (IGF) stimulation and, using functional genomic approaches, we identify proteins influencing the secretory response. IGF rapidly increased myofibroblast secretion of an ECM protein, TGFßig-h3. The secretory response was not blocked by inhibition of protein synthesis and was partially mediated by increased intracellular calcium (Ca(2+)). The capacity for evoked secretion was associated with the presence of dense-core secretory vesicles and was lost in cells from patients with advanced gastric cancer. In cells responding to IGF-II, the expression of neuroendocrine marker proteins, including secretogranin-II and proenkephalin, was identified by gene array and LC-MS/MS respectively, and verified experimentally. The expression of proenkephalin was decreased in cancers from patients with advanced disease. Inhibition of secretogranin-II expression decreased the secretory response to IGF, and its over-expression recovered the secretory response consistent with a role in secretory vesicle biogenesis. We conclude that normal and some gastric cancer myofibroblasts have a neuroendocrine-like phenotype characterized by Ca(2+)-dependent regulated secretion, dense-core secretory vesicles and expression of neuroendocrine marker proteins; loss of the phenotype is associated with advanced cancer. A failure to regulate myofibroblast protein secretion may contribute to cancer progression.

NHE1 activity contributes to migration and is necessary for proliferation of human gastric myofibroblasts.

Myofibroblasts play central roles in wound healing, deposition of the extracellular matrix and epithelial function. Their functions depend on migration and proliferation within the subepithelial matrix, which results in accelerated cellular metabolism. Upregulated metabolic pathways generate protons which need to be excreted to maintain intracellular pH (pH(i)). We isolated human gastric myofibroblasts (HGMs) from surgical specimens of five patients. Then we characterized, for the first time, the expression and functional activities of the Na(+)/H(+) exchanger (NHE) isoforms 1, 2 and 3, and the functional activities of the Na(+)/HCO(3)(-) cotransporter (NBC) and the anion exchanger (AE) in cultured HGMs using microfluorimetry, immunocytochemistry, reverse transcription polymerase chain reaction and immunoblot analysis. We showed that NHE1-3, NBC and AE activities are present in HGMs and that NHE1 is the most active of the NHEs. In scratch wound assays we also demonstrated (using the selective NHE inhibitor HOE-642) that carbachol and insulin like growth factor II (IGF-II) partly stimulate migration of HGMs in a NHE1-dependent manner. EdU incorporation assays revealed that IGF-II induces proliferation of HGMs which is inhibited by HOE-642. The results indicate that NHE1 is necessary for IGF-II-induced proliferation response of HGMs. Overall, we have characterized the pH(i) regulatory mechanisms of HGMs. In addition, we demonstrated that NHE1 activity contributes to both IGF-II- and carbachol-stimulated migration and that it is obligatory for IGF-II-induced proliferation of HGMs.

Loss of parietal cell expression of Sonic hedgehog induces hypergastrinemia and hyperproliferation of surface mucous cells.

BACKGROUND & AIMS: Sonic Hedgehog (Shh) is expressed in the adult stomach, but its role as a gastric morphogen is unclear. We sought to identify mechanisms by which Shh might regulate gastric epithelial cell function and differentiation. METHODS: Mice with a parietal cell-specific deletion of Shh (HKCre/Shh(KO)) were created. Gastric morphology and function were studied in control and HKCre/Shh(KO) mice between 1 and 8 months of age. RESULTS: In contrast to control mice, HKCre/Shh(KO) mice developed gastric hypochlorhydria, hypergastrinemia, and a phenotype that resembled foveolar hyperplasia. The fundic mucosa of HKCre/Shh(KO) mice had an expanded surface pit cell lineage that was documented by increased incorporation of bromodeoxyuridine and was attributed to the hypergastrinemia. Compared with controls, numbers of total mucous neck and zymogen cells were significantly decreased in stomachs of HKCre/Shh(KO) mice. In addition, zymogen and neck cell markers were coexpressed in the same cell populations, indicating disrupted differentiation of the zymogen cell lineage from the mucous neck cells in the stomachs of HKCre/Shh(KO) mice. Laser capture microdissection of the surface epithelium, followed by quantitative reverse-transcription polymerase chain reaction, revealed a significant increase in expression of Indian Hedgehog, glioma-associated oncogene homolog 1, Wnt, and cyclin D1. Laser capture microdissection analysis also showed a significant increase in Snail with a concomitant decrease in E-cadherin. CONCLUSIONS: In the stomachs of adult mice, loss of Shh from parietal cells results in hypochlorhydria and hypergastrinemia. Hypergastrinemia might subsequently induce increased Hedgehog and Wnt signaling in the surface pit epithelium, resulting in hyperproliferation.

Elevated serum gastrin is associated with a history of advanced neoplasia in Barrett's esophagus.

OBJECTIVES: Proton pump inhibitors (PPIs) are frequently prescribed to patients with Barrett's esophagus (BE), but in a subset, they can induce significant hypergastrinemia. Elevated levels of gastrin have been associated with tumorigenic effects in a number of gastrointestinal cancers. We decided to investigate the association between serum gastrin levels and dysplasia in BE. METHODS: We performed a cross-sectional study and enrolled patients with BE without dysplasia, low-grade dysplasia (LGD), high-grade dysplasia (HGD), or adenocarcinoma (AC), as well as gastroesophageal reflux disease controls, all chronically taking PPIs. Fasting serum gastrin was measured, and data were collected on patient characteristics, medication use, and the highest degree of BE neoplasia. RESULTS: A total of 95 patients were enrolled. The mean age was 64.7 (+/-10.0) years, and 70.5% were male. The median serum gastrin level was 40 pM. There was no significant difference in gastrin levels with increased degrees of BE neoplasia (overall P=0.68). In multivariable analysis, the highest quartile of gastrin was associated with significantly increased odds of advanced neoplasia (HGD or AC) (odds ratio (OR): 5.46, 95% confidence interval (CI): 1.20-24.8). CONCLUSIONS: In BE patients taking PPIs, an elevated serum gastrin is associated with a history of HGD or AC. Prospective studies are needed to determine whether patients with nondysplastic BE and elevated serum gastrin are at increased risk for neoplastic progression.

Gastrin is an essential cofactor for helicobacter-associated gastric corpus carcinogenesis in C57BL/6 mice.

We have previously described a synergistic interaction between hypergastrinemia and Helicobacter felis infection on gastric corpus carcinogenesis in FVB/N mice housed under specific-pathogen-free conditions. However, gastrin-deficient (GAS-KO) mice on a mixed C57BL/6/129Sv genetic background maintained in conventional housing were reported to develop spontaneous gastric antral tumors. Therefore, we investigated the role of gastrin in Helicobacter-associated gastric carcinogenesis in H. felis-infected mice on a uniform C57BL/6 background housed in specific-pathogen-free conditions. Hypergastrinemic transgenic (INS-GAS) mice, GAS-KO mice, and C57BL/6 wild-type mice were infected with H. felis for either 12 or 18 months. At 12 months postinfection, INS-GAS mice had mild corpus dysplasia, while B6 wild-type mice had either severe gastritis or metaplasia, and GAS-KO mice had only mild to moderate gastritis. At 18 months postinfection, both INS-GAS and B6 wild-type mice had both severe atrophic gastritis and corpus dysplasia, while GAS-KO mice had severe gastritis with mild gastric atrophy, but no corpus dysplasia. In contrast, both GAS-KO and B6 wild-type mice had mild to moderate antral dysplasia, while INS-GAS mice did not. H. felis antral colonization remained stable over time among the three groups of mice. These results point to a distinct effect of gastrin on carcinogenesis of both the gastric corpus and antrum, suggesting that gastrin is an essential cofactor for gastric corpus carcinogenesis in C57BL/6 mice.

Matrix metalloproteinase (MMP)-7 in Barrett's esophagus and esophageal adenocarcinoma: expression, metabolism, and functional significance.

Matrix metalloproteinase (MMP)-7, unlike many MMPs, is typically expressed in epithelial cells. It has been linked to epithelial responses to infection, injury, and tissue remodeling including the progression of a number of cancers. We have now examined how MMP-7 expression changes in the progression to esophageal adenocarcinoma (EAC), and have studied mechanisms regulating its expression and its functional significance. Immunohistochemistry revealed that MMP-7 was weakly expressed in normal squamous epithelium adjacent to EAC but was abundant in epithelial cells in both preneoplastic lesions of Barrett's esophagus and EAC particularly at the invasive front. In the stroma, putative myofibroblasts expressing MMP-7 were abundant at the invasive front but were scarce or absent in adjacent tissue. Western blot and ELISA revealed high constitutive secretion of proMMP-7 in an EAC cell line (OE33) that was inhibited by the phosphatidylinositol (PI) 3-kinase inhibitor LY294002 but not by inhibitors of protein kinase C, or MAP kinase activation. There was detectable proMMP-7 in cultured esophageal myofibroblasts but it was undetectable in media. Possible metabolism of MMP-7 by myofibroblasts studied by proteomic analysis indicated degradation via extensive endopeptidase, followed by amino- and carboxpeptidase, cleavages. Myofibroblasts exhibited increased migration and invasion in response to conditioned media from OE33 cells that was reduced by MMP-7 knockdown and immunoneutralization. Thus, MMP-7 expression increases at the invasive front in EAC which may be partly attributable to activation of PI 3-kinase. Secreted MMP-7 may modify the tumor microenvironment by stimulating stromal cell migration and invasion.