Mesenchymal-epithelial signalling in tumour microenvironment: role of high-mobility group Box 1.

Glucose deprivation, hypoxia and acidosis are characteristic features of the central core of most solid tumours. Myofibroblasts are stromal cells present in many such solid tumours, including those of the colon, and are known to be involved in all stages of tumour progression. HMGB1 is a nuclear protein with an important role in nucleosome stabilisation and gene transcription; it is also released from immune cells and is involved in the inflammatory process. We report that the microenvironmental condition of glucose deprivation is responsible for the active release of HMGB1 from various types of cancer cell lines (HT-29, MCF-7 and A549) under normoxic conditions. Recombinant HMGB1 (10 ng/ml) triggered proliferation in myofibroblast cells via activation of PI3K and MEK1/2. Conditioned medium collected from glucose-deprived HT-29 colon cancer cells stimulated the migration and invasion of colonic myofibroblasts, and these processes were significantly inhibited by immunoneutralising antibodies to HMGB1, RAGE and TLR4, together with specific inhibitors of PI3K and MEK1/2. Our data suggest that HMGB1 released from cancer cells under glucose deprivation is involved in stimulating colonic myofibroblast migration and invasion and that this occurs through the activation of RAGE and TLR4, resulting in the activation of the MAPK and PI3K signalling pathways. Thus, HMGB1 might be released by cancer cells in areas of low glucose in solid tumours with the resulting activation of myofibroblasts and is a potential therapeutic target to inhibit solid tumour growth

Gene expression profile and genomic alterations in colonic tumours induced by 1,2-dimethylhydrazine (DMH) in rats

<p>Abstract</p> <p>Background</p> <p>Azoxymethane (AOM) or 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats shares many phenotypical similarities with human sporadic colon cancer and is a reliable model for identifying chemopreventive agents. Genetic mutations relevant to human colon cancer have been described in this model, but comprehensive gene expression and genomic analysis have not been reported so far. Therefore, we applied genome-wide technologies to study variations in gene expression and genomic alterations in DMH-induced colon cancer in F344 rats.</p> <p>Methods</p> <p>For gene expression analysis, 9 tumours (TUM) and their paired normal mucosa (NM) were hybridized on 4 × 44K Whole rat arrays (Agilent) and selected genes were validated by semi-quantitative RT-PCR. Functional analysis on microarray data was performed by GenMAPP/MappFinder analysis. Array-comparative genomic hybridization (a-CGH) was performed on 10 paired TUM-NM samples hybridized on Rat genome arrays 2 × 105K (Agilent) and the results were analyzed by CGH Analytics (Agilent).</p> <p>Results</p> <p>Microarray gene expression analysis showed that <it>Defcr4</it>, <it>Igfbp5</it>, <it>Mmp7, Nos2, S100A8 </it>and <it>S100A9 </it>were among the most up-regulated genes in tumours (Fold Change (FC) compared with NM: 183, 48, 39, 38, 36 and 32, respectively), while <it>Slc26a3</it>, <it>Mptx</it>, <it>Retlna </it>and <it>Muc2 </it>were strongly down-regulated (FC: -500; -376, -167, -79, respectively). Functional analysis showed that pathways controlling cell cycle, protein synthesis, matrix metalloproteinases, TNFα/NFkB, and inflammatory responses were up-regulated in tumours, while Krebs cycle, the electron transport chain, and fatty acid beta oxidation were down-regulated. a-CGH analysis showed that four TUM out of ten had one or two chromosomal aberrations. Importantly, one sample showed a deletion on chromosome 18 including <it>Apc</it>.</p> <p>Conclusion</p> <p>The results showed complex gene expression alterations in adenocarcinomas encompassing many altered pathways. While a-CGH analysis showed a low degree of genomic imbalance, it is interesting to note that one of the alterations concerned <it>Apc</it>, a key gene in colorectal carcinogenesis. The fact that many of the molecular alterations described in this study are documented in human colon tumours confirms the relevance of DMH-induced cancers as a powerful tool for the study of colon carcinogenesis and chemoprevention.</p

Gene Expression Profiles of Beta-Cell Enriched Tissue Obtained by Laser Capture Microdissection from Subjects with Type 2 Diabetes

Background: Changes in gene expression in pancreatic beta-cells from type 2 diabetes (T2D) should provide insights into their abnormal insulin secretion and turnover. Methodology/Principal Findings: Frozen sections were obtained from cadaver pancreases of 10 control and 10 T2D human subjects. Beta-cell enriched samples were obtained by laser capture microdissection (LCM). RNA was extracted, amplified and subjected to microarray analysis. Further analysis was performed with DNA-Chip Analyzer (dChip) and Gene Set Enrichment Analysis (GSEA) software. There were changes in expression of genes linked to glucotoxicity. Evidence of oxidative stress was provided by upregulation of several metallothionein genes. There were few changes in the major genes associated with cell cycle, apoptosis or endoplasmic reticulum stress. There was differential expression of genes associated with pancreatic regeneration, most notably upregulation of members of the regenerating islet gene (REG) family and metalloproteinase 7 (MMP7). Some of the genes found in GWAS studies to be related to T2D were also found to be differentially expressed. IGF2BP2, TSPAN8, and HNF1B (TCF2) were upregulated while JAZF1 and SLC30A8 were downregulated. Conclusions/Significance: This study made possible by LCM has identified many novel changes in gene expression tha

The role of High-Mobility Group Box 1 protein in mesenchymal-epithelial signalling in the tumour microenvironment.

Introduction Glucose depravation, hypoxia, and acidosis are characteristic features of the central core most solid tumours. Myofibroblasts are stromal cells present in many such solid tumours including those of the colon, and they are known to be involved in all stages of tumour progression. HMGB1 is a nuclear protein that plays an important role in nucleosome stabilisation and gene transcription. HMGB1 is also released from immune cells and is involved in the inflammatory process. This work reports a novel finding that the microenvironmental condition of glucose depravation is responsible for the active release of HMGB1 from different types of cancer cell lines (HT-29, MCF-7 and A549) under normoxic conditions. Materials and method Human colonic myofibroblasts cells CCD18COCo, and cancer cells, colon HT-29, lung A549 and breast MCF7 were obtained from the ATCC and bladder cancer cells EJ138 from the ECACC. The human recombinant HMGB1, anti-HMGB1 antibody, anti-RAGE primary antibody and anti-TLR4 antibody were purchased from R&D systems. MEK1/2 inhibitor (U0126) and PI3K inhibitor (LY294002) were purchased from Cell Signalling Technology (USA). Medium was collected from HT-29, MCF-7, EJ138 and A549 cells after 48h treatment with various conditioned or fresh medium and analysed by western blotting for the presence of HMGB1. The MTT assay was used measure proliferation of myofibroblasts after treatment with conditioned medium with or without an MEK1/2 inhibitor or a PI3K inhibitor. Migration of CCD18Co cells in response to medium previously conditioned on HT-29 cells for 20h was studied using 8µm pore Boyden chamber inserts in 24-well plate system (BD Bioscience, California, USA). In addition, invasion assays of myofibroblast cells were performed using 8µm pore matrigel matrix Biocoat® inserts according to the manufacturer’s instruction (Becton Dickinson). Results and discussion Recombinant HMGB1 (10ng/ml) was shown to trigger proliferation in myofibroblasts cells via activation of PI3K and MEK1/2. Conditioned medium collected from glucose deprived HT-29 colon cancer cells was shown to stimulate migration and invasion of colonic myofibroblasts, and these processes were significantly inhibited by immunoneutralising antibodies to HMGB1, RAGE and TLR4, along with specific inhibitors of PI3K and MEK1/2. Conclusion Together these data suggests that HMGB1 released from the cancer cells under glucose depravation is involved in stimulating colonic myofibroblast migration and invasion and that this was through activation of RAGE and TLR4, resulting in activation of the MAPK and PI3K signalling pathways. Thus, it is proposed that HMGB1 may be released by cancer cells in areas of low glucose in solid tumours with the resulting activation of myofibroblasts. Therefore, HMGB1 may be considered as potential therapeutic target to inhibit solid tumour growth

Insulin-like growth factor binding protein-5 is a target of matrix metalloproteinase-7: implications for epithelial-mesenchymal signaling

Matrix metalloproteinase-7 (MMP-7) is localized to epithelial cells and is up-regulated in many cancers and in inflammation. We now report that MMP-7 targets a key mesenchymal cell type, the myofibroblast. Recombinant MMP-7 stimulated the proliferation and migration of human colonic myofibroblasts. These responses were partly attributable to activation of other MMPs, notably MMP-3 and MMP-8, and to stimulation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways. Using a proteomic approach, we identified insulin-like growth factor binding protein-5 (IGFBP-5) as a previously unsuspected target of MMP-7 produced by colonic myofibroblasts. We present evidence that the MMP-7 cleavage of IGFBP-5 liberates IGF-II that functions as an autocrine myofibroblast growth factor. Thus, MMP-7 may act as a signal from epithelial cells for local recruitment of myofibroblasts and stimulation of their proliferation. Similar effects of MMP-7 produced in epithelial tumors might account for the expansion of stroma through activation of myofibroblasts