Mapping clinicopathological entities within colorectal mucinous adenocarcinomas: a hierarchical clustering approach.

The aim of this study was to interrogate the heterogeneity of colorectal mucinous adenocarcinomas. This study is based on hierarchical clustering approach combining clinicopathological and molecular patterns known to be relevant to oncogenesis and therapeutic management of patients with colorectal carcinoma, ie, microsatellite instability, O6-methylguanine-DNA methyltransferase (MGMT) status, KRAS, and BRAF mutations and wnt signaling pathway activation. Comparison of the study group of 60 mucinous adenocarcinomas defined according to World Health Organization classification with control group of 136 colorectal adenocarcinomas successively removed shows higher frequency of BRAF and KRAS mutations and microsatellite instability-high status and lower frequency of wnt signaling pathway activation in mucinous adenocarcinomas. Hierarchical clustering isolated three relevant clusters: (i) cluster of microsatellite stable mucinous adenocarcinomas (54%) with KRAS mutation, and frequent MGMT changes, more frequently located in the left colon, often associated with contiguous precursor adenoma; (ii) cluster of BRAF-mutated mucinous adenocarcinomas (28%) with either microsatellite instability-high or microsatellite stable status, occurring in elderly female patients, nearly all located in the right colon, having the signature of serrated pathway of carcinomas; and (iii) a heterogeneous cluster of microsatellite instability-high mucinous carcinomas (18%), including inherited colorectal carcinomas, displaying a high-grade histological pattern. Age, TNM stage, and BRAF mutation had prognostic value. Hierarchical clustering analysis led to the identification of several clinicopathological entities of colorectal mucinous adenocarcinomas with epidemiologic, prognostic, and therapy relevance. Both KRAS and BRAF mutations appear as drivers in the alternate oncogenetic pathways governing the development of sporadic colorectal mucinous adenocarcinomas.

Heterogeneity of subordination of the IL-18/IFN-γ axis to caspase-1 among patients with Crohn's disease.

In Crohn's disease (CD), hierarchical architecture of the inflammatory network, including subordination of IL-18, an IFN-γ-inducing cytokine, to the inflammasome, have remained undeciphered. Heterogeneity among patients of such a subordination cannot be evaluated by animal models, monofactorial in their etiology and homogenous in disease progression. To address these issues, we set up an ex vivo model of inflamed mucosa explant cultures from patients with active long-standing CD. Th1 cytokine production, especially IFN-γ and IL-18, was assessed in relation with inflammation intensity. Subordination of the Th1 response to caspase-1, effector of the inflammasome, was determined in explant cultures subjected to pharmacological inhibition of caspase-1 by YVAD. We showed a correlation between secreted IFN-γ/IL-18 levels, and caspase-1 activation, with inflammation intensity of intestinal CD mucosa explants. Inhibition of caspase-1 activation using the specific inhibitor YVAD identified a homogenous non responder group featuring a caspase-1-independent IL-18/IFN-γ response, and a heterogenous responder group, in which both IL-18 and IFN-γ responses were caspase-1-dependent, with a 40-70% range of inhibition by YVAD. These findings bring out the concept of heterogeneity of subordination of the Th1 response to inflammasome activation among CD patients. This ex vivo model should have therapeutic relevance in allowing to determine eligibility of CD patients for new targeted therapies.

HLA-E/ß2 microglobulin overexpression in colorectal cancer is associated with recruitment of inhibitory immune cells and tumor progression.

The host immune response plays a major role in colorectal carcinoma (CRC) progression. A mechanism of tumor immune escape might involve expression of the human leucocyte antigen (HLA)-E/ß2m on tumor cells. The inhibitory effect of HLA-E/ß2m on CD8+ cytotoxic T lymphocytes and natural killer (NK) cells is mediated by the main HLA-E receptor CD94/NKG2A. As the pathophysiological relevance of this mechanism in CRC remains unknown, this prompted us to examine, in situ, in a series of 80 CRC (i) the HLA-E and ß2m coexpression by tumor cells, (ii) the density of CD8+, cytotoxic, CD244+ and NKP46+ intraepithelial tumor-infiltrating lymphocyte (IEL-TIL) and (iii) the expression of CD94/NKG2 receptor on IEL-TIL. These data were then correlated to patient survival. We provided (i) the in situ demonstration of HLA-E/ß2m overexpression by tumor cells in 21% of CRC characterized by an overrepresentation of signet ring cell carcinomas, mucinous carcinomas and medullary carcinomas, (ii) the significant association between HLA-E/ß2m overexpression by tumor cells and increased density of CD8+ cytotoxic, CD244+ and CD94+ IEL-TIL and (iii) finally, the unfavorable prognosis associated with HLA-E/ß2m overexpression by tumor cells. Our findings show that HLA-E/ß2m overexpression is a surrogate marker of poor prognosis and point to a novel mechanism of tumor immune escape in CRC in restraining inhibitory IEL-TIL.

ADAM15 to α5ß1 integrin switch in colon carcinoma cells: a late event in cancer progression associated with tumor dedifferentiation and poor prognosis.

ADAM15, a member of the A Disintegrin And Metalloproteinase (ADAM) family, is a membrane protein containing an adhesion domain that binds to α5ß1 integrin through a unique RGD domain. ADAM15, expressed by human normal colonocytes, is involved in epithelial wound healing and tissue remodeling in inflammatory bowel disease. The aims of our study were (i) to analyze ADAM15 expression in a series of colon carcinomas and paired normal mucosa and (ii) to integrate the spatial relationship of ADAM15 with its binding partners α5ß1 integrin, a mesenchymal marker, as well as with other adhesion molecules, α3ß1 integrin and E-cadherin. A series of 94 colon carcinomas of the non other specified category were graded according to the World Health Organization classification. Immunohistochemistry was performed on frozen tissue sections using antibodies directed to ADAM15, α5ß1 and α3ß1 integrins, and E-cadherin. ADAM15 was quantified at the mRNA level. Finally, promoter methylation of ADAM15 was examined as well as the microsatellite instability status (MSS/MSI). Thirty-six percent of colorectal carcinomas displayed a reduced expression of ADAM15 in cancer cells, confirmed at the mRNA level in most cases, without promoter methylation. ADAM15 down-regulation was associated with histologically poorly differentiated carcinomas. In addition, it was associated with the acquisition of α5ß1 by cancer cells and down-regulation of α3ß1 integrin and E-cadherin. Finally this profile that includes characteristic of epithelial to mesenchymal transition is a late progression event of colon cancer with a poor prognosis.

Loss of interleukin-10 or transforming growth factor ß signaling in the human colon initiates a T-helper 1 response via distinct pathways.

BACKGROUND & AIMS: Signaling via interleukin (IL)-10 or transforming growth factor (TGF)-ß is disrupted in subpopulations of patients with inflammatory bowel disease, but it is not clear how a T-helper (Th) 1 cell response is induced. We studied conversion of human mucosal innate immune cells into inflammatory cells and the initiation of a Th1 cell response following loss of IL-10 or TGF-ß signaling. METHODS: We depleted IL-10 or TGF-ß from explant cultures of human normal colonic mucosa using immunoneutralization. Pharmacologic inhibitors and antibodies were used to determine the factors involved in the initiation of an interferon (IFN)-γ response following loss of TGF-ß or IL-10 signaling. Cytokines produced by mucosal cells were assessed by enzyme-linked immunosorbent assay and quantitative reverse-transcriptase polymerase chain reaction. The subsets of cells involved in cytokine production were determined by in situ immunofluorescence analysis and flow cytometry after digestion of the explants with collagenase. RESULTS: Depletion of IL-10 from human normal colonic mucosa resulted in an IFN-γ response, characterized by early-stage secretion of mature IL-18 and production of the active form of caspase-1 by macrophages and some epithelial cells. A caspase-1 inhibitor or the IL-18 antagonist IL-18-binding protein blocked this response. By contrast, depletion of TGF-ß resulted in an IFN-γ response that was preceded by and required secretion of IL-12 from macrophages, dendritic cells, and epithelial cells. CONCLUSIONS: Innate immune cells (macrophages and epithelial cells) activate a Th1 cell response in explant cultures of human normal colonic mucosa depleted in IL-10 or TGF-ß via distinct, nonredundant pathways. These pathways might contribute to the pathogenesis of inflammatory bowel disease.

Mucosal IL-10 and TGF-beta play crucial roles in preventing LPS-driven, IFN-gamma-mediated epithelial damage in human colon explants.

IL-10 is an immunomodulatory cytokine that plays an obligate role in preventing spontaneous enterocolitis in mice. However, little is known about IL-10 function in the human intestinal mucosa. We showed here that IL-10 was constitutively expressed and secreted by the human normal colonic mucosa, including epithelial cells. Depletion of IL-10 in mucosal explants induced both downregulation of the IL-10-inducible, immunosuppressive gene BCL3 and upregulation of IFN-gamma, TNF-alpha, and IL-17. Interestingly, TGF-beta blockade also strongly induced IFN-gamma production. In addition, the high levels of IFN-gamma produced upon IL-10 depletion were responsible for surface epithelium damage and crypt loss, mainly by apoptosis. Polymyxin B, used as a scavenger of endogenous LPS, abolished both IFN-gamma production and epithelial barrier disruption. Finally, adding a commensal bacteria strain to mucosa explant cultures depleted of both IL-10 and LPS reproduced the ability of endogenous LPS to induce IFN-gamma secretion. These findings demonstrate that IL-10 ablation leads to an endogenous IFN-gamma-mediated inflammatory response via LPS from commensal bacteria in the human colonic mucosa. We also found that both IL-10 and TGF-beta play crucial roles in maintaining human colonic mucosa homeostasis.

KLF4-dependent, PPARgamma-induced expression of GPA33 in colon cancer cell lines.

The glycoprotein A33 (GPA33) is a colon cancer antigen. Phase I trials with 131I and 125I monoclonal antibody A33 in colon carcinoma patients showed excellent localization to colorectal cancer and some evidence of tumor response. Using DNA microarrays, we have identified the GPA33 gene as a target of PPARgamma in HT29-Cl.16E colon cancer cells. Treatment of HT29-Cl.16E, Caco2, SW1116 and LS174T colon cancer cells with the PPARgamma agonist GW7845 induced a 2- to 6-fold increase in GPA33 mRNA as determined by real-time PCR. This induction was also found in HT29-Cl.16E cells treated with rosiglitazone and ciglitazone and was prevented by cotreatment with the PPARgamma antagonist GW9662, indicating that this regulation was PPARgamma dependent. No canonical PPAR responsive element was found in the GPA33 promoter. We therefore analyzed the expression of transcription factors involved in GPA33 expression. CDXl, CDX2 and KLF5 expression was not modified by PPARgamma activation. By contrast, a significant increase in KLF4 was seen, both at mRNA and protein levels. Furthermore, chromatin immunoprecipitation studies demonstrated that an increased amount of KLF4 protein was bound to the GPA33 promoter in cells treated with rosiglitazone. Finally, downregulation of KLF4 expression by siRNA reduced rosiglitazone-induced GPA33 expression. This indicates that PPARgamma activation induces KLF4 expression, which in turn increases GPA33 expression. We also demonstrate that PPARgamma activation leads to increased (p21WAF1/Cip1 and keratin 19) or decreased (cyclin D1) expression of known KLF4 targets, suggesting that KLF4 is a nodal player in a network of PPARgamma-regulated genes.

N-cadherin serves as diagnostic biomarker in intrahepatic and perihilar cholangiocarcinomas.

As a definite immunoprofile of this tumor is missing, the histopathologic diagnosis of intrahepatic cholangiocarcinoma is difficult. The aim of this study was to explore E- and N-cadherin expressions in intrahepatic bile duct tumors, and to determine their potential interest in differential diagnosis. Normal liver tissue, 5 cirrhosis with ductular reaction, 5 focal nodular hyperplasia, 5 bile duct hamartomas, 5 bile duct adenomas, and 45 intrahepatic cholangiocarcinomas from Caucasian patients were studied. Tissue-microarrays including 20 esophageal, 86 gastric, 8 small bowel, 64 colonic, 18 pancreatic, 6 gallbladder, and 7 extrahepatic biliary tract adenocarcinomas, 22 hepatocellular carcinomas, and normal tissues were constructed. Immunohistochemistry was performed using E-cadherin, N-cadherin, NCAM, Hep Par1, and cytokeratins 7, 19 and 20. Immunoblot analysis of frozen liver tissues was performed to control the specificity of E- and N-cadherin antibodies used. In normal liver, epithelial cells of intrahepatic bile ducts, whatever their caliber, as well as hepatocytes, coexpressed E- and N-cadherins at their plasma membranes. In cirrhosis, ductular reactions completely expressed E- and N-cadherins. All the benign lesions and 30 of the 45 intrahepatic cholangiocarcinomas (23/29 peripheral and 7/16 hilar) also expressed N-cadherin. E-cadherin was detected in all the lesions. The expression of N-cadherin at the plasma membrane of tumor cells was significantly more frequent in peripheral than in hilar intrahepatic cholangiocarcinomas (P=0.003). Among noncholangiocarcinomas, only 1% gastric and 66% gallbladder adenocarcinomas and all the hepatocellular carcinomas expressed N-cadherin at the membrane of tumor cells. Finally, for the diagnosis of intrahepatic cholangiocarcinomas, the specificity value of membranous expression of N-cadherin was 88%, whereas that of the combination cytokeratin 7/membranous N-cadherin was 98%. In the gastrointestinal and liver tract, membranous N-cadherin is restricted to the hepatocytes and intrahepatic biliary cells. In combination with cytokeratin 7 and Hep Par1, N-cadherin is a reliable tool for the histopathological diagnosis of primary hepatic tumors.

ADAM-15: a metalloprotease that mediates inflammation.

Cell-cell and cell-matrix interactions are of utmost importance in the pathogenesis of inflammatory diseases. For example, cell-cell and cell-matrix interactions are crucial for leukocyte homing and recruitment to inflammatory sites. The discovery of the disintegrin and metalloprotease (ADAM) proteins, which have both adhesive and proteolytic activities, raised the question of their involvement in inflammatory processes. More interestingly, the presence of the RGD integrin-binding sequence in the disintegrin domain of ADAM-15 (MDC-15; metargidin) highlighted ADAM-15 as a protein particularly involved in cell-cell interactions. These findings therefore prompted authors to investigate the roles of ADAM-15 in inflammatory diseases. Because of the early description of ADAM-15 expression in endothelial cells, work first focused on the roles of ADAM-15 in vascular diseases, and ADAM-15 was found to be associated with atherosclerosis. Other studies also pointed at ADAM-15 as a mediator of rheumatoid arthritis and intestinal inflammation as well as inherent angiogenesis. The roles of ADAM-15 in these diseases appear to involve mechanisms as different as cell-cell interactions, cell-extracellular matrix (ECM) interactions, and shedding activity. Here we review and discuss these recent discoveries pointing to ADAM-15 as a mediator of mechanisms underlying inflammation and as a possible therapeutic target for prevention of inflammatory diseases.

TACE inhibition amplifies TNF-alpha-mediated colonic epithelial barrier disruption.

Inflammatory bowel diseases (IBD) are characterized by tumor necrosis factor alpha (TNF-alpha)-mediated epithelial barrier disruption. TNF-alpha production and the bioavailability of its receptors on the cell surface are regulated by TACE (TNF-alpha converting enzyme), a pleiotropic metalloprotease also known as ADAM17, and its specific inhibitor TIMP3. We therefore examined ADAM17 and TIMP3 expression in human intestinal epithelial cells (IEC) using immunohistochemistry on tissue microarrays and real-time PCR on preparations of IEC isolated from human normal and IBD colon. The effects of TACE inhibition by TIMP3 or a pharmacological inhibitor were assessed in inflammatory conditions on a TIMP3-deficient colonic cell line HT29-Cl.16E. Both TACE and TIMP3 were found to be constitutively expressed by intestinal epithelial cells in the normal and inflammatory human intestinal barrier. In the TIMP3-deficient cell line, the addition of recombinant human TIMP3 or of Tapi-2, a pharmacological ADAM17 inhibitor, i) sensitized the cells to TNF-alpha-mediated hyperpermeability, ii) down-regulated tight junction-associated protein expression and iii) inhibited TNFRI shedding. In conclusion, our data showed that TACE and TIMP3 were co-expressed in the human intestinal barrier and that TACE inhibition, either physiologically or pharmacologically, amplified TNF-alpha-mediated hyperpermeability. TIMP3 could thus play a major role in inflammatory conditions by creating an autocrine effect leading to amplified epithelial barrier hyperpermeability.

The double stranded RNA analog poly-IC elicits both robust IFN-λ production and oncolytic activity in human gastrointestinal cancer cells.

PURPOSE: Type III IFN (IFN-λ) is the dominant frontline response over type I IFN in human normal intestinal epithelial cells upon viral infection, this response being mimicked by the dsRNA analog poly-IC. Poly-IC also induces cell death in murine intestinal crypts ex vivo. Here we examined whether these innate defense functions of normal intestinal epithelial cells are recapitulated in gastrointestinal carcinoma cells so that they could be harnessed to exert both immunoadjuvant and oncolytic functions, an unknown issue yet. EXPERIMENTAL DESIGN: Four human gastrointestinal carcinoma cell lines versus the Jurkat lymphoma cell line were used to assess the effects of intracellular poly-IC on i) IFN-λ secretion and cell proliferation and ii) role of NFκB signaling using the NFκB inhibitory peptide SN50 as a screening probe and a siRNA approach. RESULTS: Poly-IC induced in all cell lines except Jurkat both a robust IFN-λ secretion and a cytoreductive effect on adherent cells, restricted to proliferating cells and associated with cellular shedding and reduced clonogenicity of the shed cells. Collectively these findings demonstrate the oncolytic activity of poly-IC. Inhibiting NFκB in T84 cells using a siRNA approach decreased IFN-λ production without protecting the cells from the poly-IC oncolytic effects. In line with these findings IFN-λ, that upregulated the anti-viral protein MxA, was unable per se to alter T84 cell proliferation. CONCLUSION: Our demonstration that poly-IC-induced concomitant recapitulation of two innate functions of normal intestine, i.e. IFN-λ production and cell death, by human gastrointestinal cancer cells opens new perspectives in gastrointestinal cancer treatment.

PAR-2 activation increases human intestinal mucin secretion through EGFR transactivation.

PAR-2 (protease-activated receptors-2) are G protein-coupled receptors whose action on mucin secretion by intestinal epithelial cells is still unknown. The aim of this study was to examine the effect of PAR-2 activation on mucin secretion in the human colonic goblet cell line HT29-Cl.16E and the intracellular pathways involved. We found that PAR-2 mRNA was constitutively expressed by HT29-Cl.16E cells as well as by isolated human normal colonocytes. The PAR-2-activating peptide SLIGKV-NH(2) elicited rapid mucin secretion in HT29-Cl.16E, which was partially inhibited by calcium chelator BAPTA. Inhibitors of MAPK activation (PD98059) and EGFR tyrosine kinase activity (AG1478) abrogated PAR-2-induced ERK1/2 and EGFR tyrosine phosphorylation, respectively, and subsequent mucin secretion. Finally, PAR-2-induced EGFR transactivation was involved upstream of ERK1/2 activation. Our results show that the activation of PAR-2 expressed by human intestinal epithelial cells enhances mucin secretion, a component of the intestinal innate defence, via a pathway involving EGFR transactivation.

Over-expression of neurotensin high-affinity receptor 1 (NTS1) in relation with its ligand neurotensin (NT) and nuclear beta-catenin in inflammatory bowel disease-related oncogenesis.

We investigated the expression of the neurotensin high-affinity receptor 1 (NTS1) during inflammatory bowel disease (IBD)-related colorectal oncogenesis, in colonic samples from 30 patients with IBD-related adenocarcinomas, dysplasias, and inflammatory mucosa (IM). The percentage of NTS1-positive epithelial cells progressively increased from the inflammatory condition to adenocarcinoma and was significantly higher in adenocarcinomas than in IM (p=0.0169). In parallel, the percentage of neurotensin (NT)-positive epithelial cells increased during the IBD-related oncogenesis. Finally, as NTS1 is a ss-catenin inducible gene, we found that a number of preneoplastic lesions and adenocarcinomas co-expressed NTS1 and beta-catenin without NT expression. Therefore, this study suggests two pathways of NTS1 overexpression during IBD-related oncogenesis: one triggered by NT overexpression, and a second associated with an activation of the APC/beta-catenin pathway, these two pathways being not mutually exclusive.

Involvement of the serrated neoplasia pathway in inflammatory bowel disease-related colorectal oncogenesis.

The purpose of this study is to identify colorectal serrated lesions in the inflammatory mucosa of inflammatory bowel disease (IBD), to characterize their molecular status based on BRAF and KRAS mutations, mismatch-repair (MMR) deficiency and microsatellite instability (MSI), and to verify that these molecular alterations are specific to the 'serrated neoplasia pathway' in IBD. Neoplastic lesions from 36 patients with IBD were reviewed retrospectively, including 13 adenocarcinomas (1 mucinous and 12 conventional), 28 dysplasias [1 traditional serrated adenoma (TSA) and 27 conventional adenomas] and 1 hyperplastic polyp (HP). Both the HP and TSA exhibited the V600E BRAF mutation without MSI or MMR deficiency. The mucinous adenocarcinoma, close to the TSA, exhibited the BRAF mutation and MSI with loss of hMLH1. No KRAS mutations were found in these 3 lesions, and no BRAF mutations were found in the conventional ones. Serrated lesions exist in the inflammatory mucosa of IBD and are associated with a characteristic molecular profile, i.e. the appearance of the BRAF mutation as early as the hyperplastic polyp stage followed by MSI at the carcinoma stage. We therefore identified the serrated neoplasia pathway in IBD-related colorectal oncogenesis.

Low expression of ORF4, a dominant negative variant of peroxisome proliferator-activated receptor gamma, in colorectal adenocarcinoma.

Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists have been demonstrated to exert an inhibitory effect on cell growth, and to induce the cell differentiation and apoptosis of colorectal cancer cells. PPARgamma was therefore proposed as a therapeutic target. Recently, a variant of PPARgamma which functions as a dominant negative (ORF4) was described. Expression of this protein may prevent PPARgamma ligand efficiency in colon cancer treatment. In an effort to evaluate the importance of this variant, we determined the expression level of PPARgamma and that of the splicing variant ORF4 in a series of 28 human colon adenocarcinomas relative to paired normal mucosa by real-time PCR. PPARgamma expression was found to be heterogeneous among tumors. ORF4 was also expressed, but represented <10% of the PPARgamma transcripts. This low level was also found in several human colon cancer cell lines treated or not with a specific PPARgamma ligand in preparations of isolated human colonic epithelial cells and in mouse colon. We conclude that ORF4 expression is a general phenomenon, and that its low level should not affect the efficiency of selective PPARgamma modulators in colon cancer treatment.

Interferon-Alpha Promotes Th1 Response and Epithelial Apoptosis via Inflammasome Activation in Human Intestinal Mucosa.

BACKGOUND & AIMS: Several lines of investigation suggest that interferon (IFN) alpha can alter human intestinal mucosa homeostasis. These include the endogenous production of IFN alpha in celiac disease or inflammatory bowel diseases, as well as the occurrence of intestinal side effects of exogenous IFN alpha used as a therapeutic tool. Here, we present an ex vivo translational approach to investigate the effects of IFN alpha on the human normal intestinal mucosa, as well as its underlying mechanisms. METHODS: Human normal colonic mucosa explants were cultured in the presence or absence of IFN alpha 2a. Epithelial homeostasis was assessed using the immunohistochemical marker of apoptosis M30. The Wnt inhibitor Dickkopf-Homolog-1 (DKK1) was assayed in the supernatants by enzyme-linked immunosorbent assay. Activation of the inflammasome (caspase-1/interleukin [IL]18) and of a Th1 response was determined by in situ detection of active caspase-1, as well as by measurement of mature IL18 production and the prototype Th1 cytokine IFN gamma by enzyme-linked immunosorbent assay. In addition, mechanistic studies were performed using the specific caspase-1 inhibitor Tyr-Val-Ala-Asp(OMe)-fluoromethylketone (YVAD-FMK), IL18-binding protein, neutralizing anti-IFN gamma, and anti-DKK1 antibodies. RESULTS: IFN alpha 2a elicited a rapid (24 hours) disruption of surface and crypt colonic epithelial cells via apoptosis that was variable in intensity among the 20 individuals studied. This apoptotic effect was dependent on the initiation of an IFN gamma response elicited by resident T box expressed in T cells-positive lamina propria cells. Both apoptosis and Th1 response were subordinated to active caspase-1 and IL18 production. Finally, neutralization of IFN gamma-induced DKK1 partially protected against IFN alpha-induced epithelial apoptosis. CONCLUSIONS: By using an ex vivo model, we show an interindividual heterogeneity of IFN alpha effects. We show that IFN alpha is able to disrupt both epithelial and immune homeostasis in the human intestine, by activation of an innate immunity platform, the inflammasome, which drives a Th1 response and leads to epithelial barrier disruption.

Human colonic myocytes are involved in postischemic inflammation through ADAM17-dependent TNFalpha 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 (TNFalpha), via an ADAM17 (a disintegrin and metalloproteinase-17)-dependent pathway, as shown by the abrogation of TNFalpha production by the inhibitor Tapi-2. Immunofluorescence studies identified nonvascular and vascular myocytes as resident cells coexpressing TNFalpha 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 TNFalpha production by myocytes was an early event triggered by a postischemic oxidative stress involving nuclear factor kappa B (NF-kappaB). In conclusion, this study identifies human intestinal myocytes as resident cells able to initiate an inflammatory reaction through TNFalpha production in postischemic conditions, and delineates two points of control in TNFalpha production, NF-kappaB and ADAM17, which can be targeted by pharmacological manipulation.

Growth phase-dependent expression of ICAD-L/DFF45 modulates the pattern of apoptosis in human colonic cancer cells.

The inhibitor of caspase-3-activated DNase (ICAD) is a caspase-3 substrate that controls nuclear apoptosis. ICAD has two isoforms: a functional isoform of M(r) 45,000, ICAD-L/DNA fragmentation factor (DFF) 45; and a M(r) 35,000 isoform, ICAD-S/DFF35. ICAD-deficient murine cells display resistance to apoptotic stimuli and absence of typical nuclear changes of apoptosis. Our aim was to: (a) characterize the ICAD expression in several human colonic cancer cell lines compared with human normal colonocytes; and (b) correlate the phenotypic features of apoptosis to the level of ICAD expression. ICAD expression was assessed by immunoblot analysis. Early markers of apoptosis of cultured cells included lactate dehydrogenase retention in dying cells, cytokeratin 18 cleavage, and caspase-3 activation. Nuclear markers of apoptosis were assessed by Hoechst staining of nuclei, electron microscopy, and DNA electrophoresis. Inhibition of caspases was performed using a broad-spectrum caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone. ICAD expression was restricted to the functional ICAD-L/DFF45 isoform in colonic cancer cells as well as in human normal colonocytes. In a clonal derivative of HT29 cells (HT29-Cl.16E cells), ICAD expression was found to be down-regulated during the exponential phase of growth, and the cell death triggered by IFN-gamma, anti-Fas antibody plus Adriamycin was characterized by the expression of early markers of apoptosis, whereas the key nuclear features of apoptosis were absent. In contrast, exposure of confluent cells to this treatment led to a typical apoptotic nuclear fragmentation. Both forms of apoptosis, in exponentially growing and confluent cells, were sensitive to the broad spectrum inhibitor of caspases, z-Val-Ala-Asp-fluoromethyl ketone. Our findings support the concept that the expression of ICAD is essential to the execution of full-blown apoptosis in colonic cancer cells. Altogether, our results point to ICAD as a potential target for restoring a normal apoptotic signal transduction pathway in colonic cancer cells.

Position in cell cycle controls the sensitivity of colon cancer cells to nitric oxide-dependent programmed cell death.

Mounting evidence suggests that the position in the cell cycle of cells exposed to an oxidative stress could determine their survival or apoptotic cell death. This study aimed at determining whether nitric oxide (NO)-induced cell death in colon cancer cells might depend on their position in the cell cycle, based on a clone of the cancer cell line HT29 exposed to an NO donor, in combination with the manipulation of the cell entry into the cell cycle. We show that PAPA NONOate (pNO), from 10(-4) m to 10(-3) m, exerted early and reversible cytostatic effects through ribonucleotide reductase inhibition, followed by late resumption of cell growth at 5 x 10(-4) m pNO. In contrast, 10(-3) m pNO led to late programmed cell death that was accounted for by the progression of cells into the cell cycle as shown by (a) the accumulation of apoptotic cells in the G(2)-M phase at 10(-3) m pNO treatment; and (b) the prevention of cell death by inhibiting the entry of cells into the cell cycle. The entry of pNO-treated cells into the G(2)-M phase was associated with actin depolymerization and its S-glutathionylation in the same way as in control cells. However, the pNO treatment interfered with the build-up of a high reducing power, associated in control cells with a dramatic increase in reduced glutathione biosynthesis in the G(2)-M phase. This oxidative stress prevented the exit from the G(2)-M phase, which requires a high reducing power for actin deglutathionylation and its repolymerization. Finally, our demonstration that programmed cell death occurred through a caspase-independent pathway is in line with the context of a nitrosative/oxidative stress. In conclusion, this work, which deciphers the connection between the position of colonic cancer cells in the cell cycle and their sensitivity to NO-induced stress and their programmed cell death, could help optimize anticancer protocols based on NO-donating compounds.

The PPAR(gamma) K422Q mutation does not contribute to troglitazone inefficiency in colon cancer treatment.

Peroxisome proliferator-activated receptor gamma (PPAR(gamma)) ligands inhibit cell growth of colorectal cancer cells in most experimental models, but no significant effect could be observed in patients with colorectal cancer. We therefore, screened human colorectal tumors to determine the prevalence of the PPAR(gamma) K422Q loss-of-function mutation, recently identified in 50% of colonic cancer cell lines. A sensitive allele-specific real-time amplification assay was developed and 170 colorectal primary tumors and 12 liver metastasis were analyzed. We did not find the K422Q mutation in any of these samples. We can therefore exclude this alteration as a mechanism of resistance to PPAR(gamma) ligands in patients with colon cancer.