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.

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.

Expanding the clinical spectrum of hereditary fibrosing poikiloderma with tendon contractures, myopathy and pulmonary fibrosis due to FAM111B mutations.

BACKGROUND: Hereditary Fibrosing Poikiloderma (HFP) with tendon contractures, myopathy and pulmonary fibrosis (POIKTMP [MIM 615704]) is a very recently described entity of syndromic inherited poikiloderma. Previously by using whole exome sequencing in five families, we identified the causative gene, FAM111B (NM_198947.3), the function of which is still unknown. Our objective in this study was to better define the specific features of POIKTMP through a larger series of patients. METHODS: Clinical and molecular data of two families and eight independent sporadic cases, including six new cases, were collected. RESULTS: Key features consist of: (i) early-onset poikiloderma, hypotrichosis and hypohidrosis; (ii) multiple contractures, in particular triceps surae muscle contractures; (iii) diffuse progressive muscular weakness; (iv) pulmonary fibrosis in adulthood and (v) other features including exocrine pancreatic insufficiency, liver impairment and growth retardation. Muscle magnetic resonance imaging was informative and showed muscle atrophy and fatty infiltration. Histological examination of skeletal muscle revealed extensive fibroadipose tissue infiltration. Microscopy of the skin showed a scleroderma-like aspect with fibrosis and alterations of the elastic network. FAM111B gene analysis identified five different missense variants (two recurrent mutations were found respectively in three and four independent families). All the mutations were predicted to localize in the trypsin-like cysteine/serine peptidase domain of the protein. We suggest gain-of-function or dominant-negative mutations resulting in FAM111B enzymatic activity changes. CONCLUSIONS: HFP with tendon contractures, myopathy and pulmonary fibrosis, is a multisystemic disorder due to autosomal dominant FAM111B mutations. Future functional studies will help in understanding the specific pathological process of this fibrosing disorder.

Acute cytotoxicity of MIRA-1/NSC19630, a mutant p53-reactivating small molecule, against human normal and cancer cells via a caspase-9-dependent apoptosis.

Although numerous studies have focused on the mechanisms of action of the candidate chemotherapeutic drug MIRA-1/NSC19630, initially described as a mutant p53-reactivating small molecule, the issue of its toxicological evaluation remains open. Here, we devised a strategy to examine the effects of MIRA-1 on a variety of human normal cells and cancer cell lines. First, we demonstrated a massive and rapid (within 2 hours) MIRA-1 apoptotic effect on human normal primary epithelial cells as shown using an intestinal mucosa explant assay. MIRA-1 was also cytotoxic to primary and subcultured human mesenchymal cells. Interestingly these effects were restricted to actively proliferating cells. Second, MIRA-1 acute toxicity was independent of p53, since it occurred in human normal cells with increased or silenced p53 expression level, in cancer cells derived from solid or liquid tumors, with either mutated or wt TP53, and in cancer cells devoid of p53. Third, combined pharmacological and genetic approaches showed that MIRA-1 acute cytotoxicity was mediated by a caspase-9-dependent apoptosis. In conclusion, our strategy unveils the limitations of the targeted action of a small molecule designed to reactivate mutant p53.

Association of HER1 amplification with poor prognosis in well differentiated gastric carcinomas.

AIMS: The pattern of E-cadherin expression and the HER1/HER2 status were studied in European patients with gastric carcinomas in relation with their differentiation and prognosis. METHODS: 82 gastric carcinomas (five papillary, 52 tubular, 19 poorly cohesive and six mixed according to WHO classification) were investigated for E-cadherin distribution (normal: restricted to the membrane; abnormal: absent or cytoplasmic expression), HER1 and HER2 expression using HercepTest and amplification using fluorescent in situ hybridisation. Statistical analysis assessed the association between the markers and their correlation with clinicopathological parameters and follow-up information. RESULTS: Abnormal E-cadherin distribution was found in 34 of the 82 gastric carcinomas (41%) (18/25 poorly cohesive or mixed (72%); 16/57 papillary or tubular type (28%)). HER1 overexpression (3+) and equivocal expression (2+) were found in five carcinomas (6%; four tubular and one poorly cohesive) and eight carcinomas (10%; six tubular and two poorly cohesive), respectively. HER2 overexpression (3+) and equivocal expression (2+) were found in seven carcinomas (8%; five papillary and two tubular) and three carcinomas (4%; three tubular), respectively. Amplification of HER1 or HER2 was detected in 14 gastric carcinomas (five papillary and nine tubular). All of them showed a normal E-cadherin distribution. In the univariate analysis, only HER1 amplification had a prognostic impact, while HER2 amplification and E-cadherin expression/distribution were not per se prognostically relevant. CONCLUSIONS: E-cadherin immunostaining and HER1 in situ hybridisation define a group of well differentiated gastric carcinomas with poor prognosis eligible for an aggressive therapeutic approach.

Mutations in FAM111B cause hereditary fibrosing poikiloderma with tendon contracture, myopathy, and pulmonary fibrosis.

Congenital poikiloderma is characterized by a combination of mottled pigmentation, telangiectasia, and epidermal atrophy in the first few months of life. We have previously described a South African European-descent family affected by a rare autosomal-dominant form of hereditary fibrosing poikiloderma accompanied by tendon contracture, myopathy, and pulmonary fibrosis. Here, we report the identification of causative mutations in FAM111B by whole-exome sequencing. In total, three FAM111B missense mutations were identified in five kindreds of different ethnic backgrounds. The mutation segregated with the disease in one large pedigree, and mutations were de novo in two other pedigrees. All three mutations were absent from public databases and were not observed on Sanger sequencing of 388 ethnically matched control subjects. The three single-nucleotide mutations code for amino acid changes that are clustered within a putative trypsin-like cysteine/serine peptidase domain of FAM111B. These findings provide evidence of the involvement of FAM111B in congenital poikiloderma and multisystem fibrosis.

Differential roles of Hath1, MUC2 and P27Kip1 in relation with gamma-secretase inhibition in human colonic carcinomas: a translational study.

Hath1, a bHLH transcription factor negatively regulated by the γ-secretase-dependent Notch pathway, is required for intestinal secretory cell differentiation. Our aim was fourfold: 1) determine whether Hath1 is able to alter the phenotype of colon cancer cells that are committed to a differentiated phenotype, 2) determine whether the Hath1-dependent alteration of differentiation is coupled to a restriction of anchorage-dependent growth, 3) decipher the respective roles of three putative tumor suppressor genes Hath1, MUC2 and P27kip1 in this coupling and, 4) examine how our findings translate to primary tumors. Human colon carcinoma cell lines that differentiate along a mucin secreting (MUC2/MUC5AC) and/or enterocytic (DPPIV) lineages were maintained on inserts with or without a γ-secretase inhibitor (DBZ). Then the cells were detached and their ability to survive/proliferate in the absence of substratum was assessed. γ-secretase inhibition led to a Hath1-mediated preferential induction of MUC2 over MUC5AC, without DPPIV modification, in association with a decrease in anchorage-independent growth. While P27kip1 silencing relieved the cells from the Hath1-induced decrease of anchorage-independent growth, MUC2 silencing did not modify this parameter. Hath1 ectopic expression in the Hath1 negative enterocytic Caco2 cells led to a decreased anchorage-independent growth in a P27kip1-independent manner. In cultured primary human colon carcinomas, Hath1 was up-regulated in 7 out of 10 tumors upon DBZ treatment. Parallel MUC2 up-regulation occurred in 4 (4/7) and P27kip1 in only 2 (2/7) tumors. Interestingly, the response patterns of primary tumors to DBZ fitted with the hierarchical model of divergent signalling derived from our findings on cell lines.

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.

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.

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.

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 Crohn's disease and four normal colons by immunohistochemistry, immunoblotting and quantitative reverse transcription-polymerase chain reaction. In addition expression of alpha5beta1- and alphavbeta3-integrins, VE-cadherin, alpha-smooth muscle actin (alpha-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 alpha-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 alpha5beta1-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 alpha5beta1- and alphavbeta3-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 alpha5beta1-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.

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.

Metabolic control of resistance of human epithelial cells to H2O2 and NO stresses.

The carbon flux through the oxidative branch of the pentose phosphate pathway (PPP) can be viewed as an integrator of the antioxidant mechanisms via the generation of NADPH. It could therefore be used as a control point of the cellular response to an oxidative stress. Replacement of glucose by galactose sensitized the human epithelial cell line HGT-1 to H2O2 stress. Here we demonstrate that, due to the restricted galactose flux into the PPP, the H2O2 stress led to early cellular blebbing followed by cell necrosis, these changes being associated with a fall in the NADPH/NADP+ ratio and GSH depletion. H2O2 cytotoxicity was prevented by adding 2-deoxyglucose (2dGlc). This protection was associated with an increased flow of 2-deoxyglucose 6-phosphate into the oxidative branch of the PPP together with the prevention of the NADPH/NADP+ fall and the maintenance of intracellular GSH redox homoeostasis. Inhibitors of enzyme pathways connecting the PPP to GSH recycling abolished the 2dGlc protection. In carbohydrate-free culture conditions, 2dGlc dose-dependent protective effect was paralleled by a dose-dependent influx of 2dGlc into the PPP leading to the maintenance of the intracellular redox status. By contrast, in Glc-fed cells, the PPP was not a control point of the cellular resistance to H2O2 stress as they maintained a high NADPH/NADP+ ratio. Both 2dGlc and Glc inhibited, through the maintenance of GSH redox status, NO cytotoxicity on galactose-containing Dulbecco's modified Eagle's medium (Gal-DMEM)-fed cells. 2dGlc did not prevent the fall of ATP content in NO-treated Gal-DMEM-fed cells, indicating that NO cytotoxicity was essentially due to the disruption of GSH redox homoeostasis and not to the alteration of ATP production by the mitochondrial respiratory chain. The maintenance of ATP content in NO-treated glucose-fed cells was due to their ability to derive their energy from anaerobic glycolysis. In conclusion, Gal-DMEM and 2dGlc-supplemented Gal-DMEM provide a useful system to decipher and organize into a hierarchy the targets of several stresses at the level of intact barrier epithelial cells.

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.