Andrea Varro

Bone Marrow-Derived Myofibroblasts Contribute to the Mesenchymal Stem Cell Niche and Promote Tumor Growth

Carcinoma-associated fibroblasts (CAFs) that express α-smooth muscle actin (αSMA) contribute to cancer progression, but their precise origin and role are unclear. Using mouse models of inflammation-induced gastric cancer, we show that at least 20% of CAFs originate from bone marrow (BM) and derive from mesenchymal stem cells (MSCs). αSMA+ myofibroblasts (MFs) are niche cells normally present in BM and increase markedly during cancer progression. MSC-derived CAFs that are recruited to the dysplastic stomach express IL-6, Wnt5α and BMP4, show DNA hypomethylation, and promote tumor growth. Moreover, CAFs are generated from MSCs and are recruited to the tumor in a TGF-β- and SDF-1α-dependent manner. Therefore, carcinogenesis involves expansion and relocation of BM-niche cells to the tumor to create a niche to sustain cancer progression.

Synergistic interaction between hypergastrinemia and Helicobacter infection in a mouse model of gastric cancer.

BACKGROUND & AIMS Hypergastrinemia occurs frequently in association with acid suppression and Helicobacter infection, but its role in the progression to gastric atrophy and gastric cancer has not been well defined. METHODS The effects of hypergastrinemia, and possible synergy with Helicobacter felis infection, were investigated in insulin-gastrin (INS-GAS) transgenic mice. RESULTS INS-GAS mice initially showed mild hypergastrinemia, increased maximal gastric acid secretion, and increased parietal cell number but later progressed to decreased parietal cell number and hypochlorhydria. Development of gastric atrophy was associated with increased expression of growth factors, heparin-binding epidermal growth factor and transforming growth factor alpha. At 20 months of age, INS-GAS mice showed no evidence of increased enterochromaffin-like cell number, but instead exhibited gastric metaplasia, dysplasia, carcinoma in situ, and gastric cancer with vascular invasion. Invasive gastric carcinoma was observed in 6 of 8 INS-GAS mice that were >20 months old. Helicobacter felis infection of INS-GAS mice led to accelerated (< or = 8 mo) development of intramucosal carcinoma (85%), with submucosal invasion (54%) and intravascular invasion (46%; P < or = 0.05). CONCLUSIONS These findings support the unexpected conclusion that chronic hypergastrinemia in mice can synergize with Helicobacter infection and contribute to eventual parietal cell loss and progression to gastric cancer.

Processing and proliferative effects of human progastrin in transgenic mice.

Incompletely processed gastrins have been postulated to play a role in growth of the gastrointestinal tract, but few studies have examined the effects of progastrin on mucosal proliferation in vivo. Human gastrin gene expression and progastrin processing were therefore studied in transgenic mice containing a human gastrin (hGAS) minigene, and compared to processing in mice bearing an insulin gastrin (INS-GAS) transgene that overexpresses amidated gastrin. Progastrin processing was studied using region-specific antisera and radioimmunoassays, biosynthetic labeling, immunoprecipitation, and HPLC. Proliferative effects due to overexpression of processed and unprocessed gastrin in INS-GAS and hGAS mice, respectively, were determined using routine histology and BrdU incorporation. The pancreatic islets of INS-GAS mice were able to produce carboxyamidated G-17, resulting in a twofold elevation of serum amidated gastrin, marked thickening of the oxyntic mucosa, and an increased BrdU labeling index (LI) of the gastric body. In contrast, livers of adult hGAS mice expressed abundant human gastrin mRNA and human progastrin but were unable to process this peptide to the mature amidated form, resulting in markedly elevated serum progastrin levels and normal amidated gastrin levels. Nevertheless, there was a marked increase in the BrdU labeling index of the colon in hGAS mice (LI 7.46+/-1.90%), as well as in INS-GAS mice (LI 6.16+/-1.17%), compared to age-matched, wild type control mice (LI 4.01+/-0.98%, P < 0.05). These studies suggest that incompletely processed gastrin precursors may contribute to colonic mucosal proliferation in vivo.

Expression of cannabinoid CB1 receptors by vagal afferent neurons is inhibited by cholecystokinin

Both inhibitory (satiety) and stimulatory (orexigenic) factors from the gastrointestinal tract regulate food intake. In the case of the satiety hormone cholecystokinin (CCK), these effects are mediated via vagal afferent neurons. We now report that vagal afferent neurons expressing the CCK-1 receptor also express cannabinoid CB1 receptors. Retrograde tracing established that these neurons project to the stomach and duodenum. The expression of CB1 receptors determined by RT-PCR, immunohistochemistry and in situ hybridization in rat nodose ganglia was increased by withdrawal of food for ≥12 hr. After refeeding of fasted rats there was a rapid loss of CB1 receptor expression identified by immunohistochemistry and in situ hybridization. These effects were blocked by administration of the CCK-1 receptor antagonist lorglumide and mimicked by administration of CCK to fasted rats. Because CCK is a satiety factor that acts via the vagus nerve and CB1 agonists stimulate food intake, the data suggest a new mechanism modulating the effect on food intake of satiety signals from the gastrointestinal tract.

Small-intestinal dysfunction accompanies the complex endocrinopathy of human proprotein convertase 1 deficiency

We have previously described the only reported case of human proprotein convertase 1 (PC1) deficiency, in a female (Subject A) with obesity, hypogonadism, hypoadrenalism, and reactive hypoglycemia. We now report the second case of human PC1 deficiency (Subject B), also due to compound heterozygosity for novel missense and nonsense mutations. While both subjects shared the phenotypes of obesity, hypoadrenalism, reactive hypoglycemia, and elevated circulating levels of certain prohormones, the clinical presentation of Subject B was dominated by severe refractory neonatal diarrhea, malabsorptive in type. Subsequent investigation of Subject A revealed marked small-intestinal absorptive dysfunction, which was not previously clinically suspected. We postulate that PC1, presumably in the enteroendocrine cells, is essential for the normal absorptive function of the human small intestine. The differences in the nature and severity of presentation between the two cases cannot readily be explained on the basis of allelic heterogeneity, as the nonsense and missense mutations from both subjects had comparably severe effects on the catalytic activity of PC1. Despite Subject As negligible PC1 activity, some mature ACTH and glucagon-like peptide 17-36(amide) were detectable in her plasma, suggesting that the production of these hormones, at least in humans, does not have an absolute dependence on PC1. The presence of severe obesity and the absence of growth retardation in both subjects contrast markedly with the phenotype of mice lacking PC1 and suggest that the precise physiological repertoire of this enzyme may vary between mammalian species.

Regulation by gastric acid of the processing of progastrin‐derived peptides in rat antral mucosa

1 Inhibition of gastric acid secretion by proton pump inhibitors like omeprazole increases the synthesis and secretion of the pyloric antral hormone gastrin. We report here how omeprazole influences the conversion of the gastrin precursor to its final products, and the abundance of mRNAs encoding proteins associated with progastrin processing in rat antral mucosa. 2 Progastrin processing was studied using a pulse–chase protocol in antral mucosa, incubated in vitro, from rats treated with omeprazole for up to 5 days. Labelled peptides were detected by on–line scintillation counting after immunoprecipitation and HPLC. The mRNAs encoding prohormone‐processing enzymes were identified by Northern blot, polymerase chain reaction or ribonuclease protection assay, and their cellular origins identified by immunocytochemistry. 3 Cleavage of [3H]‐ and [35S]‐labelled progastrins at Arg‐94–95 or Arg‐57–58, and amidation at Phe‐92 were not influenced by pretreatment with omeprazole. In contrast, cleavage of G34 (the thirty‐four amino acid amidated gastrin) at Lys‐74–75 to give G17 (the seventeen amino acid amidated gastrin), and of G34–Gly to Gl7–Gly (G34 and G17 with COOH‐terminal glycine), was increased 3‐fold after treatment with omeprazole for either 1 or 5 days. 4 Approximately 20% of newly synthesized amidated and Gly‐extended gastrins were secreted within 240 min of the labelling period in omeprazole‐treated samples, but secretion of labelled gastrins from control tissue was undetectable over a comparable period. 5 The amidating enzyme, peptidylglycine α‐amidating mono‐oxygenase (PAM), the prohormone convertases PC1/3, PC2, PC5 and the PC2 chaperone 7B2 were localized to rat antral gastrin cells by immunocytochemistry. The relative abundance of mRNA species encoding 7B2, PC5 and PAM were unchanged after treatment with omeprazole for 5 days, whereas gastrin, PC1/3 and PC2 mRNAs are known to increase at this time. 6 The main consequence of increased cleavage at Lys‐74–75 is the production of G17 and G17–Gly at the expense of G34 and G34–Gly, respectively. The latter have longer plasma half‐lives, and so their increased cleavage may serve to limit the rise in plasma gastrin concentration after inhibition of acid secretion. Changes in the abundance of mRNAs encoding prohormone‐processing enzymes cannot account for the rapidity of the changes in cleavage of progastrin at Lys residues after omeprazole.

Gastrin is a target of the β-catenin/TCF-4 growth-signaling pathway in a model of intestinal polyposis

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene occur in most colorectal cancers and lead to activation of beta-catenin. Whereas several downstream targets of beta-catenin have been identified (c-myc, cyclin D1, PPARdelta), the precise functional significance of many of these targets has not been examined directly using genetic approaches. Previous studies have shown that the gene encoding the hormone gastrin is activated during colon cancer progression and the less-processed forms of gastrin are important colonic trophic factors. We show here that the gastrin gene is a downstream target of the beta-catenin/TCF-4 signaling pathway and that cotransfection of a constitutively active beta-catenin expression construct causes a threefold increase in gastrin promoter activity. APC(min-/+) mice overexpressing one of the alternatively processed forms of gastrin, glycine-extended gastrin, show a significant increase in polyp number. Gastrin-deficient APC(min-/+) mice, conversely, showed a marked decrease in polyp number and a significantly decreased polyp proliferation rate. Activation of gastrin by beta-catenin may therefore represent an early event in colorectal tumorigenesis and may contribute significantly toward neoplastic progression. The identification of gastrin as a functionally relevant downstream target of the beta-catenin signaling pathway provides a new target for therapeutic modalities in the treatment of colorectal cancer.

Overexpression of glycine-extended gastrin in transgenic mice results in increased colonic proliferation

Gastrin is a peptide hormone involved in the growth of both normal and malignant gastrointestinal tissue. Recent studies suggest that the glycine-extended biosynthetic intermediates mediate many of these trophic effects, but the in vivo relevance of glycine-extended gastrin (G-Gly) has not been tested. We have generated mice (MTI/G-GLY) that overexpress progastrin truncated at glycine-72 to evaluate the trophic effects of G-Gly in an in vivo model. MTI/G-GLY mice have elevated serum and colonic mucosal levels of G-Gly compared with wild-type mice. MTI/G-GLY mice had a 43% increase in colonic mucosal thickness and a 41% increase in the percentage of goblet cells per crypt. MTI/G-GLY mice exhibited increased colonic proliferation compared with wild-type controls, with an expansion of the proliferative zone into the upper third of the colonic crypts. Continuous infusion of G-Gly into gastrin-deficient mice for two weeks also resulted in elevated G-Gly levels, a 10% increase in colonic mucosal thickness, and an 81% increase in colonic proliferation when compared with gastrin-deficient mice that received saline alone. To our knowledge, these studies demonstrate for the first time that G-Glys contribute to colonic mucosal proliferation in vivo.

Lack of Commensal Flora in Helicobacter pylori–Infected INS-GAS Mice Reduces Gastritis and Delays Intraepithelial Neoplasia

BACKGROUND & AIMS Transgenic FVB/N insulin-gastrin (INS-GAS) mice have high circulating gastrin levels, and develop spontaneous atrophic gastritis and gastrointestinal intraepithelial neoplasia (GIN) with 80% prevalence 6 months after Helicobacter pylori infection. GIN is associated with gastric atrophy and achlorhydria, predisposing mice to nonhelicobacter microbiota overgrowth. We determined if germfree INS-GAS mice spontaneously develop GIN and if H pylori accelerates GIN in gnotobiotic INS-GAS mice. METHODS We compared gastric lesions, levels of messenger RNA, serum inflammatory mediators, antibodies, and gastrin among germfree and H pylori-monoinfected INS-GAS mice. Microbiota composition of specific pathogen-free (SPF) INS-GAS mice was quantified by pyrosequencing. RESULTS Germfree INS-GAS mice had mild hypergastrinemia but did not develop significant gastric lesions until 9 months old and did not develop GIN through 13 months. H pylori monoassociation caused progressive gastritis, epithelial defects, oxyntic atrophy, marked foveolar hyperplasia, dysplasia, and robust serum and tissue proinflammatory immune responses (particularly males) between 5 and 11 months postinfection (P<0.05, compared with germfree controls). Only 2 of 26 female, whereas 8 of 18 male, H pylori-infected INS-GAS mice developed low to high-grade GIN by 11 months postinfection. Stomachs of H pylori-infected SPF male mice had significant reductions in Bacteroidetes and significant increases in Firmicutes. CONCLUSIONS Gastric lesions take 13 months longer to develop in germfree INS-GAS mice than male SPF INS-GAS mice. H pylori monoassociation accelerated gastritis and GIN but caused less severe gastric lesions and delayed onset of GIN compared with H pylori-infected INS-GAS mice with complex gastric microbiota. Changes in gastric microbiota composition might promote GIN in achlorhydric stomachs of SPF mice.

Identification of progastrin derived peptides in colorectal carcinoma extracts.

The possible production of gastrin by colorectal carcinomas has been studied. Extracts of 44 tumours and adjacent macroscopically normal tissue were examined in radioimmunoassay using the following antibodies: (i) L289 raised to a C-terminal fragment of progastrin which shows specificity for intact progastrin, but not the extreme C-terminal tryptic peptide; (ii) LW60 raised to a C-terminal fragment of progastrin which reacts with progastrin and its C-terminal tryptic peptide; (iii) 109-21 which was raised to, and reacts with, Gly-extended forms of heptadecapeptide gastrin--that is, biosynthetic intermediates on the pathway producing active gastrin; and (iv) L2 which reacts with amidated, biologically active gastrins. All samples contained detectable material in assays using LW60; in general, concentrations measured with this antibody were higher than with the other antibodies, and in particular there were higher concentrations in tumour compared with normal tissue extracts. Tumour extracts also contained higher concentrations of immunoreactivity compared with normal tissue, in assays using antibodies L289 and 109-21. In contrast, amidated gastrins were found in similar concentrations in tumour and normal tissue, and concentrations were the lowest of those recorded in the four assays. Separation on Sephadex G50 revealed peaks compatible with progastrin and its C-terminal flanking peptide, and two other peaks that are so far unidentified. In conclusion most colorectal carcinomas contain peptides derived from the gastrin precursor, progastrin, but for the most part these tumours do not convert progastrin into biologically active products.