Marc G. Denis

Up‐regulated expression of ADAM17 in human colon carcinoma: co‐expression with EGFR in neoplastic and endothelial cells

The ADAM17 metalloproteinase (a disintegrin and metalloprotease 17) controls epidermal growth factor receptor (EGFR) activation through regulated shedding of EGFR ligands. With the advent of new therapeutic options targeting EGFR signalling in colon carcinoma, it was decided to determine ADAM17 status in relation to clinico‐pathological parameters and EGFR status. To this end, a series of 39 colon carcinomas were analysed.

Zoledronic acid potentiates mTOR inhibition and abolishes the resistance of osteosarcoma cells to RAD001 (Everolimus): pivotal role of the prenylation process.

Despite recent improvements in therapeutic management of osteosarcoma, ongoing challenges in improving the response to chemotherapy warrants new strategies still needed to improve overall patient survival. In this study, we investigated in vivo the effects of RAD001 (Everolimus), a new orally available mTOR inhibitor, on the growth of human and mouse osteosarcoma cells either alone or in combination with zoledronate (ZOL), an anti-osteoporotic drug used to treat bone metastases. RAD001 inhibited osteosarcoma cell proliferation in a dose- and time-dependent manner with no modification of cell-cycle distribution. Combination with ZOL augmented this inhibition of cell proliferation, decreasing PI3K/mTOR signaling compared with single treatments. Notably, in contrast to RAD001, ZOL downregulated isoprenylated membrane-bound Ras concomitantly with an increase of nonisoprenylated cytosolic Ras in sensitive and resistant osteosarcoma cell lines to both drugs. Moreover, ZOL and RAD001 synergized to decrease Ras isoprenylation and GTP-bound Ras levels. Further, the drug combination reduced tumor development in two murine models of osteoblastic or osteolytic osteosarcoma. We found that ZOL could reverse RAD001 resistance in osteosarcoma, limiting osteosarcoma cell growth in combination with RAD001. Our findings rationalize further study of the applications of mTOR and mevalonate pathway inhibitors that can limit protein prenylation pathways.

Over‐expression of a novel member of the immunoglobulin superfamily in Min mouse intestinal adenomas

Germline mutations of the APC (adenomatous polyposis coli) gene lead to multiple intestinal tumors in familial adenomatous polyposis patients and in Min (multiple intestinal neoplasia) mice. Consequently, these mice provide an excellent model for familial colon cancer. We have identified an Mr approx. 66 kDa glycoprotein which is preferentially expressed at the cell surface of cell lines established from chemically induced rat colon carcinomas. Cloning of the corresponding Tage4 cDNA has revealed that this protein contains the conserved amino acids characteristic of members of the immunoglobulin gene superfamily. Here, we analyze expression of the mouse Tage4gene in Min mouse intestinal adenomas. RT‐PCR analysis allowed us to detect expression of this gene in all the mouse adenomas tested. In contrast, lower levels of Tage4 mRNA were found in the intestinal tract and barely detectable levels in other tissues of normal mice. Furthermore, Tage4 mRNA was detected in a series of mouse intestinal adenomas by in situ hybridization. A strong signal was seen in the samples analyzed.

Identification of secreted CD155 isoforms.

The CD155 gene is a member of the immunoglobulin superfamily. We first demonstrate the existence of soluble CD155 (sCD155) isoforms in culture medium conditioned by CD155-expressing cells, in human serum and in cerebrospinal fluid. sCD155 concentration was measured in human serum and cerebrospinal fluid using a specific ELISA. Analysis of conditioned media indicated that sCD155 release does not require protease activity. In order to determine which tissues are responsible for sCD155 expression, we have quantified CD155 mRNAs in human normal tissues. The highest expression was observed in liver. The CD155alpha transcript is the most abundant and the proportion of the CD155beta and CD155gamma variants was similar between the tissues. Finally, serum purified sCD155 reduces poliovirus entry mediated by membrane-bound CD155. The high level of CD155 synthesis in many tissues and the presence of sCD155 in biological fluids suggest the existence of an important role for the protein in cellular function.

ADAM15 upregulation and interaction with multiple binding partners in inflammatory bowel disease

A disintegrin and metalloproteinase (ADAM)15 is upregulated in some tissues undergoing remodeling. This glycoprotein is characterized by adhesive function through its interaction with members of the integrin family and protease properties. The goal of this work was to describe the tissue distribution of ADAM15 and its spatial relationship with its known binding partners in inflammatory bowel disease. ADAM15 expression was examined using frozen tissues from eight patients with ulcerative colitis or Crohns disease and four normal colons by immunohistochemistry, immunoblotting and quantitative reverse transcription-polymerase chain reaction. In addition expression of α5β1- and αvβ3-integrins, VE-cadherin, α-smooth mucle actin (α-SMA) and collagen IV was examined using immunohistochemistry and confocal microscopy. In the normal colon, ADAM15 was expressed by all epithelial cells throughout the crypt and by pericryptic myofibroblasts coexpressing α-SMA and collagen IV. ADAM15 was also expressed by endothelial cells and vascular myocytes in all layers of the intestinal wall as well as by nonvascular myocytes of the muscularis mucosae and muscularis propria. In inflammatory bowel diseases, ADAM15 was strongly upregulated at the mRNA level and expressed only as an active form as shown by immunoblotting analysis. Parallel to its upregulation, ADAM15 expression was found both at the plasma membrane and in the cytoplasm of epithelial cells in acute attacks of the disease. In the crypt abcesses, ADAM15-positive epithelial cells were in close contact with α5β1-integrin-positive leukocytes localized between these cells and in the crypt lumen. In the regenerative areas, ADAM15-positive epithelial cells were in close contact with α5β1- and αvβ3-positive pericryptic myofibroblasts. In endothelial cells, VE-cadherin was decreased. In contrast, ADAM15 was strongly expressed by endothelial cells and was in close contact with α5β1-positive leukocytes. There is a differential expression of ADAM15 in epithelial cells during inflammatory bowel disease compared with the normal colon. In addition, the spatial relationships with its binding partners suggest a role for ADAM15 in the differentiation of regenerative colonic mucosa as well as in leukocyte transmigration across epithelial and endothelial barriers.

Organization of the rat Tage4 gene and herpesvirus entry activity of the encoded protein

The Tage4 gene (Tumor-Associated Glycoprotein E4) is a member of the immunoglobulin superfamily overexpressed in rat colon tumors and Min mouse intestinal adenomas. The Tage4 cDNA presents approximately 60% identity with the human CD155, a member of the immunoglobulin superfamily coding for a transmembrane protein capable of serving as an entry receptor for poliovirus, porcine pseudorabies virus and bovine herpesvirus 1. We determined the structure of the Tage4 gene. This gene covers approximately 15 kb and is composed of eight exons and seven introns. We also isolated approximately 2 kb of the 5 flanking region of the Tage4 gene and demonstrated the existence of closely clustered transcription start sites. No splicing variant was identified by RT-PCR indicating that the Tage4 gene is transcribed as a unique mRNA. Finally, the protein encoded by the Tage4 gene was tested for ability to mediate entry of several viruses. These structural and functional features of the rat Tage4 gene were compared to those of the human CD155 gene. The results indicated that the Tage4 gene is probably orthologous to the gene for CD155.

Human colonic myocytes are involved in postischemic inflammation through ADAM17-dependent TNFα production

The aim of this study was to identify human colonic resident cells able to initiate an inflammatory response in postischemic injury. Postischemic colonic injury, a condition relevant to various clinical settings, involves an inflammatory cascade in intestinal tissues through the recruitment of circulating inflammatory cells. However, there is no information on the nature of resident cells of the different intestinal layers able to initiate a postischemic inflammatory response. It is however an important issue in the context of a pharmacological approach of the early phase of intestinal ischemia. We reasoned that maintaining the different colonic layers as explant cultures in an oxygenated medium immediately after colonic resection, that is, after an ischemic period, would allow one to identify the resident cells able to initiate an inflammatory cascade, without interference of recruited inflammatory/immune cells. To this end, we designed an explant culture system that operationally defines three compartments in surgical specimens of the human colon, based on the microdissected layers, that is, mucosa, submucosa (containing muscularis mucosae) and muscularis propria. To validate the results obtained in explant cultures in the clinical setting of ischemic colitis, eight cases of sigmoid volvulus were examined. Only the myocytes‐containing explants produced tumor necrosis factor alpha (TNFα), via an ADAM17 (a disintegrin and metalloproteinase‐17)‐dependent pathway, as shown by the abrogation of TNFα production by the inhibitor Tapi‐2. Immunofluorescence studies identified nonvascular and vascular myocytes as resident cells coexpressing TNFα and ADAM17, both in our postischemic explant system and in surgical specimens from ischemic colitis patients. Finally, time‐course experiments on explanted tissues showed that TNFα production by myocytes was an early event triggered by a postischemic oxidative stress involving nuclear factor kappa B (NF‐κB). In conclusion, this study identifies human intestinal myocytes as resident cells able to initiate an inflammatory reaction through TNFα production in postischemic conditions, and delineates two points of control in TNFα production, NF‐κB and ADAM17, which can be targeted by pharmacological manipulation.

New chondrosarcoma cell lines and mouse models to study the link between chondrogenesis and chemoresistance

Chondrosarcomas are cartilage-forming, poorly vascularized tumors. They represent the second malignant primary bone tumor of adults after osteosarcoma, but in contrast to osteosarcoma they are resistant to chemotherapy and radiotherapy, surgical excision remaining the only therapeutic option. Few cell lines and animal models are available, and the mechanisms behind their chemoresistance remain largely unknown. Our goal was to establish new cell lines and animal cancer models from human chondrosarcoma biopsies to study their chemoresistance. Between 2007 and 2012, 10 chondrosarcoma biopsies were collected and used for cell culture and transplantation into nude mice. Only one transplanted biopsy and one injected cell line has engrafted successfully leading to conventional central high-grade chondrosarcoma similar to the original biopsies. In culture, two new stable cell lines were obtained, one from a dedifferentiated and one from a grade III conventional central chondrosarcoma biopsy. Their genetic characterization revealed triploid karyotypes, mutations in IDH1, IDH2, and TP53, deletion in CDKN2A and/or MDM2 amplification. These cell lines expressed mesenchymal membrane markers (CD44, 73, 90, 105) and were able to produce a hyaline cartilaginous matrix when cultured in chondrogenic three-dimensional (3D) pellets. Using a high-throughput quantitative RT-PCR approach, we observed that cell lines cultured in monolayer had lost expression of several genes implicated in cartilage development (COL2A1, COMP, ACAN) but restored their expression in 3D cultures. Chondrosarcoma cells in monolayer were sensitive to several conventional chemotherapeutic agents but became resistant to low doses of mafosfamide or doxorubicin when cultured in 3D pellets, in parallel with an altered nucleic accumulation of the drug. Our results indicate that the cartilaginous matrix produced by chondrosarcoma cells may impair diffusion of several drugs and thus contribute to chemoresistance. Therefore, 3D chondrogenic cell pellets constitute a more relevant model to study chondrosarcoma chemoresistance and may be a valuable alternative to animal experimentations.

Biological effects of glucocorticoid hormones on two rat colon adenocarcinoma cell lines

Glucocorticoid hormones are thought to play a role in carcinogenesis as they regulate cell differentiation and proliferation. We have investigated the effect of dexamethasone on two cell lines derived from a colon carcinoma, which differ by their tumorigenicity. Dexamethasone was found to inhibit growth of both the progressive (PROb) and the regressive clone (REGb). Upon glucocorticoid treatment, PROb cells were found to secrete an additional Mr approximately 40,000 protein. The synthesis and the release in the culture medium of this protein is stimulated specifically by glucocorticoid agonists, and not by other steroid hormones. The anti-glucocorticoid RU 38486 is inefficient and suppresses the induction of this protein by dexamethasone. Induction is sensitive to actinomycin D, suggesting that regulation may be related to an alteration of the rate of mRNA synthesis. The cellular effect of glucocorticoid hormones being mediated through a specific soluble receptor, we have characterized this protein. The PROb cells contained more specific glucocorticoid-binding sites (approximately 170,000 sites per cell) than the regressive ones (REGb cells; approximately 100,000 sites per cell). In both clones, the receptor was associated with the Mr approximately 90,000 heat shock protein to yield large complexes (Stokes radius Rs approximately 7.5 nm), which were dissociated to the same extent upon heat- and salt-treatment. The steroid- and DNA-binding unit of the receptor, characterized under denaturing conditions using an anti-receptor monoclonal antibody, was found to be more degraded in the PROb cell line.

Clinical significance of BRAF mutation status in circulating tumor DNA of metastatic melanoma patients at baseline

Circulating tumor DNA is a promising non‐invasive tool for cancer monitoring. The main objective of our work was to investigate the relationship between mutant BRAF DNA in plasma and clinical response. Thirty‐eight stage IV patients with a V600 mutated BRAF melanoma were included prior to any treatment. DNA was extracted from plasma and mutant DNA was detected using the amplification‐refractory mutation system method. Before the beginning of any treatment, the corresponding BRAF mutation was detected in 29 of the 38 tested plasma samples (76.3% positive per cent agreement). We observed a strong correlation between the presence of circulating mutated DNA and overall survival (OS; P=.02), and with the number of metastatic sites (P=.01). The presence of circulating mutated DNA was also strongly correlated with serum LDH activity (P<.01) and S100 protein concentration (P<.01). Finally, seven patients presented discordant BRAF status in different tumor sites. In all these patients, the test performed on ctDNA was positive, suggesting that ctDNA analysis might be less sensitive to tumor heterogeneity. Altogether, these results suggest that plasmatic mutant BRAF DNA is a prognostic factor of OS, correlated with tumor burden. In addition, it represents an interesting alternative source of DNA to detect BRAF mutations before treatment.