The role of extracellular proteases in stromal-epithelial interactions in gastric cancer

Cancers of the upper gastrointestinal tract present at an advanced stage and carry a poor prognosis. Oesophageal and gastric tumours have a rich stroma composed of vascular cells, immune cells, and myofibroblasts, which promotes tumour growth, invasion and metastasis. In addition, mesenchymal stromal cells (MSCs) are recruited from the bone marrow to the tumour stroma; the mechanisms underpinning this have not yet been defined. Extracellular proteases play a role in cell migration, invasion, and cell signalling and are known to influence cancer growth in conflicting ways. Myofibroblasts present in normal tissue differ from those found in cancer. Cancer-associated myofibroblasts (CAMs) are known to modulate extracellular protease activity by secreting plasminogen activator inhibitor-1 (PAI-1), an inhibitor of the serine protease urokinase plasminogen activator, and matrix metalloproteinases (MMPs). This investigation studies the role of PAI-1 in gastric cancer and assesses the contribution of myofibroblast-derived MMPs to tumour growth. Finally, the role of chemerin in recruiting MSCs has been investigated. The expression of PAI-1 in myofibroblasts was found to be higher than in gastric cancer cells. Overexpression of PAI-1 in gastric cancer cells resulted in decreased cell adhesion and decreased tumour growth in an in vivo subcutaneous xenograft model of gastric tumour growth. The addition of gastric CAMs potentiated the growth of gastric cancer subcutaneous xenografts. This was not accounted for by differences in cell proliferation rate, apoptosis or final stromal content. Xenografts containing CAMs suppress the growth of a contralateral xenograft without CAMs, demonstrating that a long-range signal can be generated as a result of stromal-epithelial interactions. MMP and cathepsin activity was compared between xenografts containing myofibroblasts to those without. MMP activity is increased in xenografts injected with CAMs, compared to those injected with myofibroblasts taken from normal stomach or those with gastric cancer cells alone. In an organotypic co-culture system, MMP inhibition resulted in a decrease in gastric cancer cell invasion. The injection of fluorescently labelled MSCs injected resulted in homing of these cells to subcutaneous oesophageal tumours containing CAMs. Antagonism at the ChemR23 receptor inhibited this MSC homing to oesophageal xenografts containing CAMs. This work emphasises the importance of assessing the contribution of specific proteases and their inhibitors in gastric cancer. The stroma is an important contributor to extracellular protease activity and myofibroblasts contribute both proteases and their inhibitors to the tumour microenvironment, resulting in the modulation of tumour growth and cell adhesion. MSCs are recruited to oesophageal tumours via a novel signalling pathway

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

The role of chemerin and ChemR23 in stimulating the invasion of squamous oesophageal cancer cells

BACKGROUND: Stromal cells, including cancer-associated myofibroblasts (CAMs), are recognised to be determinants of cancer progression, but the mechanisms remain uncertain. The chemokine-like protein, chemerin, is upregulated in oesophageal squamous cancer (OSC) CAMs compared with adjacent tissue myofibroblasts (ATMs). In this study, we hypothesised that chemerin stimulates OSC cell invasion. METHODS: Expression of the chemerin receptor, ChemR23, in OSC was examined by immunohistochemistry. The invasion of OSC cells was studied using Boyden chambers and organotypic assays, and the role of chemerin was explored using siRNA, immunoneutralisation and a ChemR23 receptor antagonist. Matrix metalloproteinases (MMPs) were detected by western blot, enzyme assays or immunohistochemistry. RESULTS: Immunohistochemistry indicated expression of the putative chemerin receptor ChemR23 in OSC. It was also expressed in the OSC cell line, OE21. Chemerin stimulated OE21 cell migration and invasion in Boyden chambers. Conditioned medium (CM) from OSC CAMs also stimulated OE21 cell invasion and this was inhibited by chemerin immunoneutralisation, the ChemR23 antagonist CCX832, and by pretreatment of CAMs with chemerin siRNA. In organotypic cultures of OE21 cells on Matrigel seeded with either CAMs or ATMs, there was increased OE21 cell invasion by CAMs that was again inhibited by CCX832. Chemerin increased MMP-1, MMP-2 and MMP-3 abundance, and activity in OE21 cell media, and this was decreased by inhibiting protein kinase C and p44/42 MAPK kinase but not PI-3 kinase. CONCLUSIONS: The data indicate that OSC myofibroblasts release chemerin that stimulates OSC cell invasion. Treatments directed at inhibiting chemerin-ChemR23 interactions might be therapeutically useful in delaying progression in OSC

Increased Expression of Chemerin in Squamous Esophageal Cancer Myofibroblasts and Role in Recruitment of Mesenchymal Stromal Cells

Stromal cells such as myofibroblasts influence tumor progression. The mechanisms 45 are unclear but may involve effects on both tumor cells and recruitment of bone 46 marrow-derived mesenchymal stromal cells (MSCs) which then colonize tumors. 47 Using iTRAQ and LC-MS/MS we identified the adipokine, chemerin, as 48 overexpressed in esophageal squamous cancer associated myofibroblasts (CAMs) 49 compared with adjacent tissue myofibroblasts (ATMs). The chemerin receptor, 50 ChemR23, is expressed by MSCs. Conditioned media (CM) from CAMs significantly 51 increased MSC cell migration compared to ATM-CM; the action of CAM-CM was 52 significantly reduced by chemerin-neutralising antibody, pretreatment of CAMs with 53 chemerin siRNA, pretreatment of MSCs with ChemR23 siRNA, and by a ChemR23 54 receptor antagonist, CCX832. Stimulation of MSCs by chemerin increased 55 phosphorylation of p42/44, p38 and JNK-II kinases and inhibitors of these kinases 56 and PKC reversed chemerin-stimulated MSC migration. Chemerin stimulation of 57 MSCs also induced expression and secretion of macrophage inhibitory factor (MIF) 58 that tended to restrict migratory responses to low concentrations of chemerin but not 59 higher concentrations. In a xenograft model consisting of OE21 esophageal cancer 60 cells and CAMs, homing of MSCs administered i.v. was inhibited by CCX832. Thus, 61 chemerin secreted from esophageal cancer myofibroblasts is a potential 62 chemoattractant for MSCs and its inhibition may delay tumor progression

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 myofibroblast, 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 myofibroblast 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

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 myofibroblast, 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 myofibroblast 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

Increased MSC homing to xenografts seeded with CAMs and inhibition of homing by the chemR23 receptor antagonist, CCX832.

<p><i>A</i>, Visualisation of PKH67-labelled MSCs in representative fields from xenografts established with OE21 cancer cells alone or co-injected with CAMs followed by treatment with vehicle (top) or CCX832 (bottom) and iv injection of PKH67-labelled MSCs. <i>B</i>, In xenografts with OE21 cancer cells and CAMs there was increased MSC homing expressed as labelled cells per unit area of xenograft compared with xenografts of OE21 cancer cell alone; treatment with CCX832 inhibited homing (OE21/vehicle, n = 3; OE21/CCX832, n = 4; OE21 and CAMs/vehicle, n = 6; OE21 and CAMs/CCX832, n = 6). Horizontal arrows, p<0.05, ANOVA.</p

ChemR23 mediates chemerin stimulation of MSC migration via PKC and MAP kinases.

<p><i>A</i>, Representative images from MSCs stained for vimentin (positive control) and chemR23 revealing knock-down (KD) after ChemR23 siRNA treatment (left). Knockdown of ChemR23, but not GPR1, inhibited MSC migration in response to chemerin (100 ng/ml)(center) and CAM-CM (right). <i>B</i>, Concentration-dependent inhibition of MSC migration in response to chemerin by the ChemR23 antagonist CCX832 (left) but not the control compound CCX826 (1 µM) (center). MSC migration in response to CAM-CM was inhibited similarly by chemerin neutralising antibody, and CCX832, but not CCX826 (1 µM)(right). <i>C</i>, Representative Western blot shows increased phosphorylation of p42/44, p38 and JNK-II kinases in MSCs treated with chemerin (100 ng/ml)(left). In Boyden chamber assays, chemerin-stimulated MSC migration was inhibited by the JNK-II inhibitor, SP600125 (50 µM), the p42/44 inhibitor, UO126 (10 µM), p38 inhibitor SB202190 (3 µM), and the PKC inhibitor Ro320432 (2 µM) but not by PIK3 inhibitor LY294002 (50 µM) (right). Horizontal arrows, p<0.05, ANOVA (n = 3 in each case).</p

Chemerin stimulates transendothelial migration of MSCs and requires MMP-2.

<p><i>A</i>, Representative fields from MSC transendothelial migration experiments showing migration of PKH67-labelled MSCs (left). CCX832 (1 µM) inhibited chemerin- (center) and CAM-CM stimulated MSC transendothelial migration but CCX826 (1 µM) had no effect (right). <i>B</i>, Chemerin, and IGF-II used as a positive control, promptly (30 min) stimulated proMMP2 abundance in media as detected by Western blot but had no effect on cellular proMMP2 abundance (left); chemerin significantly increased MMP-2 enzyme activity in MSC media detected by the selective substrate MCA-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH₂ (right). <i>C</i>, Human recombinant MMP-2 (80 ng/ml) stimulated transendothelial migration and there was dose-dependent inhibition by an MMP-2 selective inhibitor (MMP-2 inhibitor I) (left). The MMP-2 inhibitor (60 µM) significantly inhibited chemerin-stimulated MSC transendothelial migration (centre). Horizontal arrows, p<0.05, t- test (n = 3).</p