Cell cycle dependent expression of the CCK2 receptor by gastrointestinal myofibroblasts: putative role in determining cell migration.

The well-known action of the gastric hormone gastrin in stimulating gastric acid secretion is mediated by activation of cholecystokinin-2 receptors (CCK2R). The latter are expressed by a variety of cell types suggesting that gastrin is implicated in multiple functions. During wound healing in the stomach CCK2R may be expressed by myofibroblasts. We have now characterized CCK2R expression in cultured myofibroblasts. Immunocytochemistry showed that a relatively small proportion (1-6%) of myofibroblasts expressed the receptor regardless of the region of the gut from which they were derived, or whether from cancer or control tissue. Activation of CCK2R by human heptadecapeptide gastrin (hG17) increased intracellular calcium concentrations in a small subset of myofibroblasts indicating the presence of a functional receptor. Unexpectedly, we found over 80% of cells expressing CCK2R were also labeled with 5-ethynyl-2'-deoxyuridine (EdU) which is incorporated into DNA during S-phase of the cell cycle. hG17 did not stimulate EdU incorporation but increased migration of both EdU-labeled and unlabelled myofibroblasts; the migratory response was inhibited by a CCK2R antagonist and by an inhibitor of IGF receptor tyrosine kinase; hG17 also increased IGF-2 transcript abundance. The data suggest myofibroblasts express CCK2R in a restricted period of the cell cycle during S-phase, and that gastrin accelerates migration of these cells; it also stimulates migration of adjacent cells probably through paracrine release of IGF. Together with previous findings, the results raise the prospect that gastrin controls the position of dividing myofibroblasts which may be relevant in wound healing and cancer progression in the gastrointestinal tract

Glycine-extended gastrin promotes the growth of lung cancer

The less processed forms of gastrin have recently been shown to act as trophic factors for both normal and malignant colonic cells. Although incompletely processed forms of gastrin such as glycine-extended gastrin and progastrin are also expressed in human lung cancers, the clinical significance of this expression has not been addressed. Consequently, we investigated the effects of overexpression of glycine-extended gastrin in a mouse strain that is prone to developing lung cancer and also examined the expression of incompletely processed gastrins in primary human lung cancers. We found that transgenic overexpression of glycine-extended gastrin in FVB/N mice resulted in a significant increase in the prevalence and growth of bronchoalveolar carcinoma. In addition, a substantial subset of human lung cancers was found to express progastrin and/or glycine-extended gastrin. Overexpression of glycine-extended gastrin by human lung cancers was associated with a significantly decreased survival. Taken together, these results suggest that glycine-extended gastrin may play a role in the growth and progression of some human lung cancers

Mapping proteolytic processing in the secretome of gastric cancer-associated myofibroblasts reveals activation of MMP-1, MMP-2, and MMP-3

Cancer progression involves changes in extracellular proteolysis, but the contribution of stromal cell secretomes to the cancer degradome remains uncertain. We have now defined the secretome of a. specific stromal cell type, the rnyofibroblast, in gastric cancer and its modification by proteolysis. SILAC labeling and COFRADIC isolation of methionine containing peptides allowed us to quantify differences in gastric cancer-derived myofibroblasts compared with myofibroblasts from adjacent tissue, revealing increased abundance of several proteases in cancer myofibroblasts including matrix metalloproteinases (MMP)-1 and -3. Moreover, N-terminal COFRADIC analysis identified cancer-restricted proteolytic cleavages, including liberation of the active forms of MMP-1, -2, and -3 from their inactive precursors. In vivo imaging confirmed increased MMP activity when gastric cancer cells were xenografted in mice together with gastric cancer myofibroblasts. Western blot and enzyme activity assays confirmed increased MMP-1, -2, and -3 activity in cancer myofibroblasts, and cancer cell migration assays indicated stimulation by MMP-1, -2, and -3 in cancer-associated rnyofibroblast media. Thus, cancer-derived myofibroblasts differ from their normal counterparts by increased production and activation of MMP-1, -2, and -3, and this may contribute to the remodelling of the cancer cell microenvironment

Stable isotope dynamic labeling of secretomes (SIDLS) identifies authentic secretory proteins released by cancer and stromal cells

Supported by a grant from North West Cancer Research.Analysis of secretomes critically underpins the capacity to understand the mechanisms determining interactions between cells and between cells and their environment. In the context of cancer cell micro-environments, the relevant interactions are recognised to be an important determinant of tumor progression. Global proteomic analyses of secretomes are often performed at a single time point and frequently identify both classical secreted proteins (possessing an N-terminal signal sequence), as well as many intracellular proteins, the release of which is of uncertain biological significance. Here, we describe a mass spectrometry-based method for stable isotope dynamic labeling of secretomes (SIDLS) that, by dynamic SILAC, discriminates the secretion kinetics of classical secretory proteins and intracellular proteins released from cancer and stromal cells in culture. SIDLS is a robust classifier of the different cellular origins of proteins within the secretome and should be broadly applicable to non-proliferating cells and cells grown in short term culture.PostprintPeer reviewe

Cell cycle dependent expression of the CCK2 receptor by gastrointestinal myofibroblasts: putative role in determining cell migration

The well-known action of the gastric hormone gastrin in stimulating gastric acid secretion is mediated by activation of cholecystokinin-2 receptors (CCK2R). The latter are expressed by a variety of cell types suggesting that gastrin is implicated in multiple functions. During wound healing in the stomach CCK2R may be expressed by myofibroblasts. We have now characterized CCK2R expression in cultured myofibroblasts. Immunocytochemistry showed that a relatively small proportion (1-6%) of myofibroblasts expressed the receptor regardless of the region of the gut from which they were derived, or whether from cancer or control tissue. Activation of CCK2R by human heptadecapeptide gastrin (hG17) increased intracellular calcium concentrations in a small subset of myofibroblasts indicating the presence of a functional receptor. Unexpectedly, we found over 80% of cells expressing CCK2R were also labeled with 5-ethynyl-2'-deoxyuridine (EdU) which is incorporated into DNA during S-phase of the cell cycle. hG17 did not stimulate EdU incorporation but increased migration of both EdU-labeled and unlabelled myofibroblasts; the migratory response was inhibited by a CCK2R antagonist and by an inhibitor of IGF receptor tyrosine kinase; hG17 also increased IGF-2 transcript abundance. The data suggest myofibroblasts express CCK2R in a restricted period of the cell cycle during S-phase, and that gastrin accelerates migration of these cells; it also stimulates migration of adjacent cells probably through paracrine release of IGF. Together with previous findings, the results raise the prospect that gastrin controls the position of dividing myofibroblasts which may be relevant in wound healing and cancer progression in the gastrointestinal tract

Chemerin acts via CMKLR1 and GPR1 to stimulate migration and invasion of gastric cancer cells: putative role of decreased TIMP-1 and TIMP-2

The chemokine-like peptide, chemerin, stimulates chemotaxis in several cell types. In this study we examined the expression of putative chemerin receptors in gastric cancer and the action of chemerin on cancer cell migration and invasion. Immunohistochemical studies of gastric tumors identified expression of two putative receptors, chemokine-like receptor-1 (CMKLR1) and G-protein coupled receptor 1(GPR1), in cancer cells; there was also some expression in stromal myofibroblasts although generally at a lower intensity. The expression of both receptors was detected in a gastric cancer cell line, AGS; chemerin itself was expressed in cultured gastric cancer myofibroblasts but not AGS cells. Chemerin stimulated (a) morphological transformation of AGS cells characterized by extension of processes and cell scattering, (b) migration in scratch wound assays and (c) both migration and invasion in Boyden chamber chemotaxis assays. These responses were inhibited by two putative receptor antagonists CCX832 and α-NETA. Inhibition of receptor expression by siRNA selectively reduced CMKLR1 or GPR1 and inhibited the action of chemerin indicating that both receptors contributed to the functional response. Using a proteomic approach employing stable isotope dynamic labeling of secretomes (SIDLS) to selectively label secreted proteins, we identified down regulation of tissue inhibitors of metalloproteinease (TIMP)1 and TIMP2 in media in response to chemerin. When cells were treated with chemerin and TIMP1 or TIMP2 the migration response to chemerin was reduced. The data suggest a role for chemerin in promoting the invasion of gastric cancer cells via CMKLR1 and GPR1at least partly by reducing TIMP1 and TIMP2 expression. Chemerin receptor antagonists have potential in inhibiting gastric cancer progression

Distinct miRNA profiles in normal and gastric cancer myofibroblasts and significance in Wnt signaling

Stromal cells influence epithelial function in both health and disease. Myofibroblasts are abundant stromal cells that influence the cellular microenvironment by release of extracellular matrix (ECM) proteins, growth factors, proteases, cytokines, and chemokines. Cancer-associated myofibroblasts (CAMs) differ from adjacent tissue (ATMs) and normal tissue myofibroblasts (NTMs), but the basis of this is incompletely understood. We report now the differential expression of miRNAs in gastric cancer CAMs. MicroRNA arrays identified differences in the miRNA profile in gastric and esophageal NTMs and in CAMs from stomach compared with NTMs. miR-181d was upregulated in gastric CAMs. Analysis of differentially regulated miRNAs indicated an involvement in Wnt signaling. Examination of a microarray data set then identified Wnt5a as the only consistently upregulated Wnt ligand in gastric CAMs. Wnt5a stimulated miR-181d expression, and knockdown of miR-181d inhibited Wnt5a stimulation of CAM proliferation and migration. Analysis of miR-181d targets suggested a role in chemotaxis. Conditioned medium from CAMs stimulated gastric cancer cell (AGS) migration more than that from ATMs, and miR-181d knockdown reduced the effect of CAM-CM on AGS cell migration but had no effect on AGS cell responses to ATM conditioned media. The data suggest that dysregulation of miRNA expression in gastric CAMs, secondary to Wnt5a signaling, accounts at least in part for the effect of CAMs in promoting cancer cell migration. stromal cells have emerged in recent years as important determinants of epithelial cell function in the gastrointestinal mucosa in health and disease (7, 23, 25). There are multiple stromal cell types, including inflammatory and immune cells, fibroblasts, pericytes, and myofibroblasts. The latter are sparse in many tissues, but in the gut there is normally a sheath of myofibroblasts that surrounds intestinal crypts and gastric glands. They may arise from activation of fibroblasts, for example, by TGFβ, by transdifferentiation of mesenchymal stem cells (26), or by epithelial-mesenchymal transition (20). Physiologically, they play a role in wound healing. They may also influence tumor progression (26). Myofibroblasts are often operationally defined as expressing α-smooth muscle actin (SMA), vimentin, and fibroblast activation protein and are negative for cytokeratin and usually desmin (7). An emerging body of evidence from multiple experimental platforms supports the idea that there are marked differences between different myofibroblast populations in both health and disease. For example, microarray studies reveal differences between myofibroblasts from different regions of the normal gastrointestinal tract (12). Moreover, there are marked differences in cancer at the levels of transcripts, proteins, and functions. Previously, we showed that myofibroblasts from gastric or esophageal cancer differ from their counterparts in adjacent tissue with evidence that myofibroblasts from advanced gastric tumors promote more aggressive phenotypes in cancer cells (3, 13, 14, 17). We also showed that esophageal cancer-associated myofibroblasts (CAMs) exhibit increased secretion of the chemokine-like peptide chemerin, which plays a role in mesenchymal stem cell recruitment (17). MicroRNAs (miRNAs) are short RNAs of ∼22 nucleotides that act posttranscriptionally to determine mRNA stability and translation (1). They regulate an impressive diversity of biological processes and importantly may contribute to cancer initiation and progression. In stomach and esophagus, previous studies have identified differentially expressed miRNAs (8, 11, 19). However, it is not known whether miRNAs contribute to the differences in function of different myofibroblast populations. In view of differences in the secretomes and proteomes of gastric or esophageal cancer-derived myofibroblasts compared with their respective adjacent tissue myofibroblasts (ATMs), in the present study we sought to determine whether there might also be differences in their miRNA expression profiles compared both with each other and with normal tissue myofibroblasts (NTMs). We now report that gastric and esophageal NTM miRNA profiles are readily distinguishable, that gastric CAMs differ from their respective NTMs in their miRNA profiles, and that Wnt5a (which is upregulated in gastric CAMs) may act in part via miR-181d to influence mesenchymal-epithelial signaling

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

Supported by grants from North West Cancer Research (Grant number: CR945), The Wellcome Trust (Grant number: 074287/Z/03/Z) and a research studentship (HG) from the Libyan Government.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.Publisher PDFPeer reviewe

Gut feelings about appetite

The obesity epidemic is now widely recognized as a major threat to health in many different countries. Some surgical therapies for obesity are efficacious and indicate that signals from the gastrointestinal tract are capable of exerting beneficial long-term effects on food intake and body weight. The development of non-surgical therapies targeting this system depends on understanding how food, and the absence of food, in the gastrointestinal tract signals to those parts of the brain that regulate feeding behaviour. What can be called gastrointestinal surveillance systems include both nervous pathways linking the gut and brain, and some gut hormones. Nutrient sensing mechanisms within the gastrointestinal tract determine the release of satiety hormones such as cholecystokinin (CCK), or appetite stimulating hormones such as ghrelin, and offer potential therapeutic targets. It seems that CCK and ghrelin both act on the vagus nerve that links the gut to the brain. Examples of interactions between different factors regulating this pathway are discussed. It is argued that sufficient is now known to indicate that this signalling system can provide new targets for the treatment of obesity.