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The Netrin-1 receptor UNC5B is an axon guidance regulator that is also expressed in endothelial cells (ECs), where it finely controls developmental and tumor angiogenesis. In the absence of Netrin-1, UNC5B induces apoptosis that is blocked upon Netrin-1 binding. Here, we identify an UNC5B splicing isoform (called UNC5B-Δ8) expressed exclusively by ECs and generated through exon skipping by NOVA2, an alternative splicing factor regulating vascular development. We show that UNC5B-Δ8 is a constitutively pro-apoptotic splicing isoform insensitive to Netrin-1 and required for specific blood vessel development in an apoptosis-dependent manner. Like NOVA2, UNC5B-Δ8 is aberrantly expressed in colon cancer vasculature where its expression correlates with tumor angiogenesis and poor patient outcome. Collectively, our data identify a mechanism controlling UNC5B’s necessary apoptotic function in ECs and suggest that the NOVA2/UNC5B circuit represents a post-transcriptional pathway regulating angiogenesis., Nature Communications, 12, ISSN:2041-1723

ObjectiveDiagnostic tests, such as Immunoscore, predict prognosis in patients with colon cancer. However, additional prognostic markers could be detected on pathological slides using artificial intelligence tools.DesignWe have developed a software to detect colon tumour, healthy mucosa, stroma and immune cells on CD3 and CD8 stained slides. The lymphocyte density and surface area were quantified automatically in the tumour core (TC) and invasive margin (IM). Using a LASSO algorithm, DGMate (DiGital tuMor pArameTErs), we detected digital parameters within the tumour cells related to patient outcomes.ResultsWithin the dataset of 1018 patients, we observed that a poorer relapse-free survival (RFS) was associated with high IM stromal area (HR 5.65; 95% CI 2.34 to 13.67; pConclusionThese findings suggest that artificial intelligence can potentially improve patient care by assisting pathologists in better defining stage III colon cancer patients’ prognosis.

International audience; The anti-diabetic biguanide metformin may exert health-promoting effects via metabolic regulation of the epigenome. Here we show that metformin promotes global DNA methylation in non-cancerous, cancer-prone and metastatic cancer cells by decreasing S-adenosylhomocysteine (SAH), a strong feedback inhibitor of S-adenosylmethionine (SAM)-dependent DNA methyltransferases, while promoting the accumulation of SAM, the universal methyl donor for cellular methylation. Using metformin and a mitochondria/complex I (mCI)-targeted analog of metformin (norMitoMet) in experimental pairs of wild-type and AMP-activated protein kinase (AMPK)-, serine hydroxymethyltransferase 2 (SHMT2)- and mCI-null cells, we provide evidence that metformin increases the SAM:SAH ratio-related methylation capacity by targeting the coupling between serine mitochondrial one-carbon flux and CI activity. By increasing the contribution of one-carbon units to the SAM from folate stores while decreasing SAH in response to AMPK-sensed energetic crisis, metformin can operate as a metabolo-epigenetic regulator capable of reprogramming one of the key conduits linking cellular metabolism to the DNA methylation machinery.

Colon carcinogenesis is ranked second globally among human diseases after cardiovascular failures. Bee venom (BV) has been shown to possess in vitro anticancer effects against several types of cancer cells. The two main biopeptides of Apis mellifera BV, namely, melittin (MEL) and phospholipase A2 (PLA2), are suspected to be the biomolecules responsible for the anticancer activity. The present work aims to evaluate the cytotoxic effect of the A. mellifera venom on human colon carcinoma cells (HCT116), and to assess the synergistic effect of MEL and PLA2 on these cells. After analyzing, through high-pressure liquid chromatography, the proportions of MEL and PLA2 on BV, we have established a cell viability assay to evaluate the effect of BV, MEL, PLA2, and a mixture of MEL and PLA2 on the HCT116 cells. Results obtained showed a strong cytotoxicity effect induced by the A. mellifera venom and to a lower extent MEL or PLA2 alone. Remarkably, when MEL and PLA2 were added together, their cytotoxic effect was greatly improved, suggesting a synergistic activity on HCT116 cells. These findings confirm the cytotoxic effect of the A. mellifera venom and highlight the presence of synergistic potential activities between MEL and PLA2, possibly inducing membrane disruption of HCT116 cancer cells. Altogether, these results could serve as a basis for the development of new anticancer treatments.

International audience; Aberrant activation of the SRC family kinase hematopoietic cell kinase (HCK) triggers hematological malignancies as a tumor cell-intrinsic oncogene. Here we find that high HCK levels correlate with reduced survival of colorectal cancer patients. Likewise, increased Hck activity in mice promotes the growth of endogenous colonic malignancies and of human colorectal cancer cell xenografts. Furthermore, tumor-associated macrophages of the corresponding tumors show a pronounced alternatively activated endotype, which occurs independently of mature lymphocytes or of Stat6-dependent Th2 cytokine signaling. Accordingly, pharmacological inhibition or genetic reduction of Hck activity suppresses alternative activation of tumor-associated macrophages and the growth of colon cancer xenografts. Thus, Hck may serve as a promising therapeutic target for solid malignancies.

International audience

Mammalian cells express two closely related MEK isoforms, MEK1 and MEK2, upstream of the ERK1/ERK2 MAPK module. Although genetic studies have suggested that MEK1 and MEK2 do not have overlapping functions in vivo, little is known about their specific contribution to the activation of ERKs and to tumor cell proliferation. We used Tet-inducible shRNA to investigate the independent role of MEK1 and MEK2 for the oncogenic and the serum-induced activation of ERK1 and ERK2 in LS174T colon carcinoma cells. We show that MEK1 is the main activator of both ERK1 and ERK2. MEK2 removal has no impact by itself but it can cooperate with MEK1 ablation for the inhibition of ERK1/2 activity. In addition, we show that MEK1 is the critical isoform regulating tumor cell proliferation in vitro and in vivo.

Recent studies suggest that secreted phospholipases A2 (sPLA2s) represent attractive potential tumour biomarkers and therapeutic targets for various cancers. As a first step to address this issue in human colorectal cancer, we examined the expression of the full set of sPLA2s in sporadic adenocarcinomas and normal matched mucosa from 21 patients by quantitative PCR and immunohistochemistry. In normal colon, PLA2G2A and PLA2G12A were expressed at high levels, PLA2G2D, PLA2G5, PLA2G10 and PLA2G12B at moderate levels, and PLA2G1B, PLA2G2F and PLA2G3 at low levels. In adenocarcinomas from left and right colon, the expression of PLA2G3 was increased by up to 40-fold, while that of PLA2G2D and PLA2G5 was decreased by up to 23- and 14-fold. The variations of expression for sPLA2-IID, sPLA2-III and sPLA2-V were confirmed at the protein level. The expression pattern of these sPLA2s appeared to be linked respectively to the overexpression of interleukin-8, defensin alpha6, survivin and matrilysin, and downregulation of SFRP-1 and RLPA-1, all these genes being associated to colon cancer. This original sPLA2 profile observed in adenocarcinomas highlights the potential role of certain sPLA2s in colon cancer and suggests that sPLA2-III might be a good candidate as a novel biomarker for both left and right colon cancers.

International audience; Helix pomatia agglutinin (HPA) is a lectin that has been used extensively in histopathology, since its binding to tissue sections from breast and colon cancers is correlated with the worst prognosis for the patients. The lectin recognizes alpha-d-N-acetylgalactosamine (alphaGalNAc) containing epitopes which are only present in cancer cell lines having a high likelihood to undergo metastasis, such as the HT29 cancer colon cell line. Several breast cancer cell lines have also been shown to be labeled, although IGROV1, an ovarian cancer cell line, is not. Inhibition studies, using GalNAc monosaccharides, are reported here, showing that the labeling is dependent upon the presence of carbohydrate epitopes. The crystal structures of the lectin complexed with two GalNAc containing epitopes associated with cancer, the Tn (alphaGalNAc-Ser) and Forssman (alphaGalNAc1-3GalNAc) antigens, show the lectin's specificity for GalNAc is due to a particular network of hydrogen bonds. A histidine residue makes hydrophobic contact with the aglycon, rationalizing the preference for GalNAc bearing an additional sugar or amino acid in the alpha position. These structures provide the molecular basis for the use of HPA in metastasis research.

International audience; The BRCA1 (breast cancer 1) breast cancer susceptibility gene is recognized as responsible for most familial breast and ovarian cancers and is suggested to be a tissue-specific tumor suppressor gene. In this report, we investigated the tissue specificity of tumor inhibitory activities induced by a recombinant adenovirus coding for wild-type BRCA1 (wtAdBRCA1). We demonstrated a pronounced in vitro antiproliferative effect on H1299 lung and HT29 colon cells upon infection with AdBRCA1. We describe a prolonged G1 cell cycle arrest associated with a decrease in the hyperphosphorylated form of Rb, suggesting that the Rb/E2F pathway is implicated in BRCA1-induced cell growth arrest. We also observed a significant antitumor effect in these pre-established subcutaneous tumors after in situ delivery of AdBRCA1, although these two tumors do not express wt p53, and also estrogen alpha and beta, progesterone and androgen receptors. Moreover, BRCA1 can induce a strong prolonged cell cycle arrest and apoptotic cell death but no significant antiangiogenic effect in H1299 tumors. Finally, our data indicate that intratumor administration of wtAdBRCA1 significantly inhibits growth of lung and colon steroid hormone-independent tumors.

International audience; The in vivo disposition and antitumor efficacy of a newly developed phosphinic matrix metalloproteinase inhibitor (RXP03) were examined. RXP03 potently inhibits MMP-11, MMP-8 and MMP-13, but not MMP-1 and MMP-7. Twenty-four hours after i.p. injection into mice, most of the RXP03 was recovered intact in plasma, feces (biliary excretion) and tumor tissue. Pharmacokinetic parameters indicated that, after an i.p. dose of 100 microg/day, the plasma concentration of RXP03 over 24 hr remained higher than the Ki values determined for MMP-11, MMP-8 and MMP-13. Efficacy of RXP03 on the growth of primary tumors induced by s.c. injection of C(26) colon carcinoma cells in mice was observed to depend both on RXP03 doses and treatment schedules. Tumor volumes in mice treated for 18 days with 50, 100 and 150 microg/day of RXP03 were decreased compared with control tumor volumes, 100 microg/day being the most effective dose. Treatment at higher dose (600 microg/day) did not significantly reduce the tumor size as compared to control. Short treatments with RXP03 100 microg/day, 3 to 7 days after C(26) inoculation, were more effective on tumor growth than continuous treatment over 18 days. Strikingly, RXP03 treatment started 6 days after the C(26) injection and continued until day 18 led to stimulation of tumor growth, as compared to control. These paradoxical effects, depending on the RXP03 treatment schedule, underline the need to define carefully the spatiotemporal function of each MMP at various stages of tumor growth to achieve optimal therapeutic effects by MMP inhibitor treatment.

Scope Resveratrol may function as a chemopreventive agent. A recent clinical study demonstrates a reduction in tumor cell proliferation in colorectal patients receiving repeated oral ingestion of resveratrol. However, gaps remain in our knowledge of the molecular mechanisms by which resveratrol exerts its chemopreventive effect. We have previously demonstrated that resveratrol induces apoptosis in colon cancer cells and that resveratrol can sensitize chemoresistant colon cancer cells to various drugs. Based on its ability to activate peroxisome proliferator-activated receptor gamma (PPARγ) in colon cancer cells, we sought to determine the implication of this nuclear transcription factor in resveratrol-induced apoptosis. Methods and results Transient transfection of cancer cells with a dominant-negative PPARγ mutant or treatment with a PPARγ antagonist (GW9662) reversed the inhibitory effect of resveratrol. Moreover, GW9662 prevented disruption of the cell cycle induced by resveratrol and consequently abrogated resveratrol-induced apoptosis. Tumor cell death was potentiated by combining resveratrol with rosiglitazone, a PPARγ agonist. Conclusion The results show that PPARγ plays a role in resveratrol-induced apoptosis of colon carcinoma cells. The combination of resveratrol with a PPARγ agonist could be a promising pharmacological approach for treatment of colorectal cancer.

International audience; Dysfunctional telomeres suppress tumour progression by activating cell-intrinsic programs that lead to growth arrest. Increased levels of TRF2, a key factor in telomere protection, are observed in various human malignancies and contribute to oncogenesis. We demonstrate here that a high level of TRF2 in tumour cells decreased their ability to recruit and activate natural killer (NK) cells. Conversely, a reduced dose of TRF2 enabled tumour cells to be more easily eliminated by NK cells. Consistent with these results, a progressive upregulation of TRF2 correlated with decreased NK cell density during the early development of human colon cancer. By screening for TRF2-bound genes, we found that HS3ST4--a gene encoding for the heparan sulphate (glucosamine) 3-O-sulphotransferase 4--was regulated by TRF2 and inhibited the recruitment of NK cells in an epistatic relationship with TRF2. Overall, these results reveal a TRF2-dependent pathway that is tumour-cell extrinsic and regulates NK cell immunity.

International audience; Nitrite-preserved meats (e.g., hot dogs) may help cause colon cancer because they contain N-nitroso compounds. We tested whether purified hot-dog-derived total apparent N-nitroso compounds (ANC) could induce colonic aberrant crypts, which are putative precursors of colon cancer. We purified ANC precursors in hot dogs and nitrosated them to produce ANC. In preliminary tests, CF1 mice received 1 or 3 i.p. injections of 5 mg azoxymethane (AOM)/kg. In Experiments 1 and 2, female A/J mice received ANC in diet. In Experiment 1, ANC dose initially dropped sharply because the ANC precursors had mostly decomposed but, later in Experiment 1 and throughout Experiment 2, ANC remained at 85 nmol/g diet. Mice were killed after 8 (AOM tests) or 17-34 (ANC tests) wk. Median numbers of aberrant crypts in the distal 2 cm of the colon for 1 and 3 AOM injections, CF1 controls, ANC (Experiment 1), ANC (Experiment 2),and untreated A/J mice were 31, 74, 12, 20, 12, and 5-6, with P < 0.01 for both ANC tests. Experiment 2 showed somewhat increased numbers of colonic mucin-depleted foci in the ANC-treated group. We conclude that hot-dog-derived ANC induced significant numbers of aberrant crypts in the mouse colon.

International audience; The effect of meat consumption on cancer risk is a controversial issue. However, recent meta-analyses show that high consumers of cured meats and red meat are at increased risk of colorectal cancer. This increase is significant but modest (20-30%). Current WCRF-AICR recommendations are to eat no more than 500 g per week of red meat, and to avoid processed meat. Moreover, our studies show that beef meat and cured pork meat promote colon carcinogenesis in rats. The major promoter in meat is heme iron, via N-nitrosation or fat peroxidation. Dietary additives can suppress the toxic effects of heme iron. For instance, promotion of colon carcinogenesis in rats by cooked, nitrite-treated and oxidized high-heme cured meat was suppressed by dietary calcium and by α-tocopherol, and a study in volunteers supported these protective effects in humans. These additives, and others still under study, could provide an acceptable way to prevent colorectal cancer.

International audience; BACKGROUND & AIMS: Fas belongs to the family of tumor necrosis factor receptors which induce apoptosis. Many cancer cells express Fas but do not undergo Fas-mediated apoptosis. Nitric oxide reverses this resistance by increasing levels of Fas at the plasma membrane. We studied the mechanisms by which NO affects Fas function. METHODS: Colon and mammary cancer cell lines were incubated with the NO donor glyceryl trinitrate or lipid A; S-nitrosylation of Fas was monitored using the biotin switch assay. Fas constructs that contained mutations at cysteine residues that prevent S-nitrosylation were used to investigate the involvement of S-nitrosylation in Fas-mediated cell death. Apoptosis was monitored according to morphologic criteria. RESULTS: NO induced S-nitrosylation of cysteine residues 199 and 304 in the cytoplasmic part of Fas. In cancer cells that overexpressed wild-type Fas, S-nitrosylation induced Fas recruitment to lipid rafts and sensitized the cells to Fas ligand. In cells that expressed a mutant form of Fas in which cysteine 304 was replaced by valine residue, NO-mediated translocation of Fas to lipid rafts was affected and the death-inducing signal complex and synergistic effect of glyceryl trinitrate-Fas ligand were inhibited significantly. These effects were not observed in cells that expressed Fas with a mutation at cysteine 199. CONCLUSIONS: We identified post-translational modifications (S-nitrosylation of cysteine residues 199 and 304) in the cytoplasmic domain of Fas. S-nitrosylation at cysteine 304 promotes redistribution of Fas to lipid rafts, formation of the death-inducing signal complex, and induction of cell death.

A monoclonal anti-CD160 antibody inhibits the growth of new vessels in pathological ocular and tumor neoangiogenesis but not in healthy tissues., Angiogenesis plays an essential role in several diseases of the eye and in the growth of solid tumors, but existing antiangiogenic therapies have limited benefits in several cases. We report the antiangiogenic effects of a monoclonal antibody, CL1-R2, in several animal models of neovascularization. CL1-R2 recognizes human CD160, a membrane receptor which is conserved in various mammal species. We show that CD160 is expressed on the endothelial cells of newly formed blood vessels in human colon carcinoma and mouse B16 melanoma but not in vessels of healthy tissues. CL1-R2 reduced fibroblast growth factor 2–induced neovascularization in the rabbit cornea, in a mouse model of oxygen-induced retinopathy, and in a mouse Matrigel plug assay. Treatment of B16 melanoma-bearing mice with CL1-R2 combined with cyclophosphamide chemotherapy caused regression of the tumor vasculature and normalization of the remaining vessels as shown by Doppler ultrasonography, intravital microscopy, and histology. These studies validate CD160 as a potential new target in cases of human pathological ocular and tumor neoangiogenesis that do not respond or become resistant to existing antiangiogenic drugs.

Background NSAIDs exhibit protective properties towards some cancers, especially colon cancer. Yet, it is not clear how they play their protective role. PGE2 is generally shown as the only target of the NSAIDs anticancerous activity. However, PGE2 known targets become more and more manifold, considering both the molecular pathways involved and the target cells in the tumour. The role of PGE2 in tumour progression thus appears complex and multipurpose. Methods To gain understanding into the role of PGE2 in colon cancer, we focused on the activity of PGE2 in apoptosis in colon cancer cell lines. Results We observed that an increase in intracellular PGE2 induced an apoptotic cell death, which was dependent on the expression of the proapoptotic protein Bax. This increase was induced by increasing PGE2 intracellular concentration, either by PGE2 microinjection or by the pharmacological inhibition of PGE2 exportation and enzymatic degradation. Conclusions We present here a new sight onto PGE2 in colon cancer cells opening the way to a new prospective therapeutic strategy in cancer, alternative to NSAIDs.

Despite recent advances in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. To better elucidate the molecular mechanisms involved in resistance to irinotecan (and its active metabolite SN38), we established SN38-resistant clones derived from HCT-116 and SW48 cell lines. These clones show various levels (6- to 60-fold) of resistance to SN-38 and display enhanced levels of activated MAPK p38 as compared with the corresponding parental cells. Because four different isoforms of p38 have been described, we then studied the effect of p38 overexpression or downregulation of each isoform on cell sensivity to SN38 and found that both α and β isoforms are involved in the development of resistance to SN38. In this line, we show that cell treatment with SB202190, which inhibits p38α and p38β, enhanced the cytotoxic activity of SN38. Moreover, p38 inhibition sensitized tumor cells derived from both SN38-sensitive and -resistant HCT116 cells to irinotecan treatment in xenograft models. Finally, we detected less phosphorylated p38 in primary colon cancer of patients sensitive to irinotecan-based treatment, compared with nonresponder patients. This indicates that enhanced level of phosphorylated p38 could predict the absence of clinical response to irinotecan. Altogether, our results show that the p38 MAPK pathway is involved in irinotecan sensitivity and suggest that phosphorylated p38 expression level could be used as a marker of clinical resistance to irinotecan. They further suggest that targeting the p38 pathway may be a potential strategy to overcome resistance to irinotecan-based chemotherapies in colorectal cancer. Cancer Res; 71(3); 1041–9. ©2010 AACR.

International audience; In human colon cancer cells, cisplatin-induced apoptosis involves the Fas death receptor pathway independent of Fas ligand. The present study explores the role of ezrin and actin cytoskeleton in relation with Fas receptor in this cell death pathway. In response to cisplatin treatment, a rapid and transient actin reorganisation is observed at the cell membrane by fluorescence microscopy after Phalloidin-FITC staining. This event is dependent on the membrane fluidification studied by electron paramagnetic resonance and necessary for apoptosis induction. Moreover, early after the onset of cisplatin treatment, ezrin co-localised with Fas at the cell membrane was visualised by membrane microscopy and was redistributed with Fas, FADD and procaspase-8 into membrane lipid rafts as shown on Western blots. In fact, cisplatin exposure results in an early small GTPase RhoA activation demonstrated by RhoA-GTP pull down, Rho kinase (ROCK)-dependent ezrin phosphorylation and actin microfilaments remodelling. Pretreatment with latrunculin A, an inhibitor of actin polymerisation, or specific extinction of ezrin or ROCK by RNA interference prevents both cisplatin-induced actin reorganisation and apoptosis. Interestingly, specific extinction of Fas receptor by RNA interference abrogates cisplatin-induced ROCK-dependent ezrin phosphorylation, actin reorganisation and apoptosis suggesting that Fas is a key regulator of cisplatin-induced actin remodelling and is indispensable for apoptosis. Thus, these findings show for the first time that phosphorylation of ezrin by ROCK via Fas receptor is involved in the early steps of cisplatin-induced apoptosis.

International audience; One of the major angiogenic factor released by tumor cells is VEGF. Its high expression is correlated with poor prognosis in colorectal tumors. In colon cancer, gastrin gene expression is also upregulated. In these tumors, gastrin precursors are mainly produced and act as growth factors. Recently, a study has also shown that the gastrin precursor, G-gly induced in vitro tubules formation by vascular endothelial cells suggesting a potential proangiogenic role. Here, we demonstrate that stimulation of human colorectal cancer cell lines with G-gly increases the expression of the proangiogenic factor VEGF at the mRNA and protein levels. In addition, blocking the progastrin autocrine loop leads to a downregulation of VEGF. Although HIF-1 is a major transcriptional activator for VEGF our results suggest an alternative mechanism for VEGF regulation in normoxic conditions, independent of HIF-1 that involves the PI3K/AKT pathway. Indeed we show that G-gly does not lead to HIF-1 accumulation in colon cancer cells. Moreover, we found that G-gly activates the PI3K/AKT pathway and inhibition of this pathway reverses the effects of G-gly observed on VEGF mRNA and protein levels. In correlation with these results, we observed in vivo, on colon tissue sections from transgenic mice overexpressing G-gly, an increase in VEGF expression in absence of HIF-1 accumulation. In conclusion, our study demonstrates that gastrin precursors, known to promote colon epithelial cells proliferation and survival can also contribute to the angiogenesis process by stimulating the expression of the proangiogenic factor VEGF via the PI3K pathway and independently of hypoxia conditions.

International audience; The nonreceptor tyrosine kinases of the Src family (SFK) are frequently deregulated in human colorectal cancer (CRC), and they have been implicated in tumour growth and metastasis. How SFK are activated in this cancer has not been clearly established. Here, we show that the SFK-dependent invasion is induced by inactivation of the negative regulator C-terminal Src kinase, Csk. While the level of Csk was inconsistent with SFK activity in colon cancer cells, its membrane translocation, needed for efficient regulation of membrane-localized SFK activity, was impaired. Accordingly, Csk downregulation did not affect SFK oncogenic activity in these cells, whereas expression of a membrane-localized form of this kinase affected their invasive activity. Downregulation of the transmembrane and rafts-localized Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomain (PAG), was instrumental for the cytoplasmic accumulation of Csk. Re-expression of PAG in cells from late-stage CRC inhibited SFK invasive activity in a Csk-dependent manner. Conversely, inactivation of its residual expression in early-stage CRC cells promoted SFK invasive activity. Finally, this mechanism was specific to CRC as Csk coupling to SFK was readily detected in breast cancer cells. Therefore, Csk mis-localization defines a novel mechanism for SFK oncogenic activation in CRC cells.

International audience; 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.

International audience; Prominin 1/CD133 is a marker of transplantable cancer stem cells. We have generated anti-peptide antibodies against a N-terminal epitope of CD133 belonging to a ganglioside-binding domain. The labelling of colon cancer cells with these antibodies was inhibited by various gangliosides including GM1 and GD3, but not GT1b. CD133 immunolabelling progressively decreased to undetectable levels in post-confluent cultures, possibly through ganglioside-mediated epitope masking since the staining was partially recovered after chemical disruption of lipid rafts. We suggest that selected gangliosides could modulate the accessibility of CD133 and regulate cell-cell contacts involving CD133(+) stem cells at the earliest steps of tumour development.

Background Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard technique for mRNA quantification, but appropriate normalization is required to obtain reliable data. Normalization to accurately quantitated RNA has been proposed as the most reliable method for in vivo biopsies. However, this approach does not correct differences in RNA integrity. Results In this study, we evaluated the effect of RNA degradation on the quantification of the relative expression of nine genes (18S, ACTB, ATUB, B2M, GAPDH, HPRT, POLR2L, PSMB6 and RPLP0) that cover a wide expression spectrum. Our results show that RNA degradation could introduce up to 100% error in gene expression measurements when RT-qPCR data were normalized to total RNA. To achieve greater resolution of small differences in transcript levels in degraded samples, we improved this normalization method by developing a corrective algorithm that compensates for the loss of RNA integrity. This approach allowed us to achieve higher accuracy, since the average error for quantitative measurements was reduced to 8%. Finally, we applied our normalization strategy to the quantification of EGFR, HER2 and HER3 in 104 rectal cancer biopsies. Taken together, our data show that normalization of gene expression measurements by taking into account also RNA degradation allows much more reliable sample comparison. Conclusion We developed a new normalization method of RT-qPCR data that compensates for loss of RNA integrity and therefore allows accurate gene expression quantification in human biopsies.

Acidosis of the tumor microenvironment is typical of a malignant phenotype, particularly in hypoxic tumors. All cells express multiple isoforms of carbonic anhydrase (CA), enzymes catalyzing the reversible hydration of carbon dioxide into bicarbonate and protons. Tumor cells express membrane-bound CAIX and CAXII that are controlled via the hypoxia-inducible factor (HIF). Despite the recognition that tumor expression of HIF-1α and CAIX correlates with poor patient survival, the role of CAIX and CAXII in tumor growth is not fully resolved. To understand the advantage that tumor cells derive from expression of both CAIX and CAXII, we set up experiments to either force or invalidate the expression of these enzymes. In hypoxic LS174Tr tumor cells expressing either one or both CA isoforms, we show that (a) in response to a “CO2 load,” both CAs contribute to extracellular acidification and (b) both contribute to maintain a more alkaline resting intracellular pH (pHi), an action that preserves ATP levels and cell survival in a range of acidic outside pH (6.0–6.8) and low bicarbonate medium. In vivo experiments show that ca9 silencing alone leads to a 40% reduction in xenograft tumor volume with up-regulation of ca12 mRNA levels, whereas invalidation of both CAIX and CAXII gives an impressive 85% reduction. Thus, hypoxia-induced CAIX and CAXII are major tumor prosurvival pHi-regulating enzymes, and their combined targeting shows that they hold potential as anticancer targets. [Cancer Res 2009;69(1):358–68]

International audience; Tumors that progress do so via their ability to escape the antitumor immune response through several mechanisms, including developing ways to induce the differentiation and/or recruitment of CD4(+)CD25(+) Tregs. The Tregs, in turn, inhibit the cytotoxic function of T cells and NK cells, but whether they have an effect on the cytotoxic function of tumor-infiltrating DCs (TIDCs) has not been determined. Here we have shown, in 2 rodent models of colon cancer, that CD4(+)CD25(+) Tregs inhibit the ability of CD11b(+) TIDCs to mediate TNF-related apoptosis-inducing ligand-induced (TRAIL-induced) tumor cell death. In both models of cancer, combination treatment with Mycobacterium bovis Bacillus Calmette-Guérin (BCG), which activates the innate immune system via TLR2, TLR4, and TLR9, and cyclophosphamide (CTX), which depletes Tregs, eradicated the tumors. Further analysis revealed that the treatment led to a marked increase in the number of CD11b(+) TIDCs that killed the tumor cells via a TRAIL-dependent mechanism. Furthermore, acquisition of TRAIL expression by the CD11b(+) TIDCs was induced by BCG and dependent on signaling through TLR2, TLR4, and TLR9. In vivo transfer of Tregs abrogated the ability of BCG to induce CD11b(+) TIDCs to express TRAIL and thereby nullified the efficacy of the CTX-BCG treatment. Our data have therefore delineated what we believe to be a novel mechanism by which Tregs inhibit the antitumor immune response.

International audience; PURPOSE: Tumor necrosis factor-alpha (TNF-alpha) enhances radiotherapy (RT) killing of tumor cells in vitro and in vivo. To overcome systemic side effects, we used a bispecific antibody (BsAb) directed against carcinoembryonic antigen (CEA) and TNF-alpha to target this cytokine in a CEA-expressing colon carcinoma. We report the evaluation of this strategy in immunocompetent CEA-transgenic mice. METHODS AND MATERIALS: The murine CEA-transfected colon carcinoma MC-38 was used for all experiments. In vitro, clonogenic assays were performed after RT alone, TNF-alpha alone, and RT plus TNF-alpha. In vivo, the mice were randomly assigned to treatment groups: control, TNF-alpha, BsAb, BsAb plus TNF-alpha, RT, RT plus TNF-alpha, and RT plus BsAb plus TNF-alpha. Measurements of endogenous TNF-alpha mRNA levels and evaluation of necrosis (histologic evaluation) were assessed per treatment group. RESULTS: In vitro, combined RT plus TNF-alpha resulted in a significant decrease in the survival fraction at 2 Gy compared with RT alone (p < 0.00001). In vivo, we observed a complete response in 5 (50%) of 10, 2 (20%) of 10, 2 (18.2%) of 11, and 0 (0%) of 12 treated mice in the RT plus BsAb plus TNF-alpha, RT plus TNF-alpha, RT alone, and control groups, respectively. This difference was statistically significant when TNF-alpha was targeted with the BsAb (p = 0.03). The addition of exogenous TNF-alpha to RT significantly increased the endogenous TNF-alpha mRNA level, particularly when TNF-alpha was targeted with BsAb (p < 0.01). The percentages of necrotic area were significantly augmented in the RT plus BsAb plus TNF-alpha group. CONCLUSION: These results suggest that targeting TNF-alpha with the BsAb provokes RT curability in a CEA-expressing digestive tumor syngenic model and could be considered as a solid rationale for clinical trials.

Chronic alcohol consumption is associated with increased risk of gastrointestinal cancer. High concentrations of ethanol trigger mucosal hyperregeneration, disrupt cell adhesion, and increase the sensitivity to carcinogens. Most of these effects are thought to be mediated by acetaldehyde, a genotoxic metabolite produced from ethanol by alcohol dehydrogenases. Here, we studied the role of low ethanol concentrations, more likely to mimic those found in the intestine in vivo, and used intestinal cells lacking alcohol dehydrogenase to identify the acetaldehyde-independent biological effects of ethanol. Under these conditions, ethanol did not stimulate the proliferation of nonconfluent cells, but significantly increased maximal cell density. Incorporation of phosphatidylethanol, produced from ethanol by phospholipase D, was instrumental to this effect. Phosphatidylethanol accumulation induced claudin-1 endocytosis and disrupted the claudin-1/ZO-1 association. The resulting nuclear translocation of ZONAB was shown to mediate the cell density increase in ethanol-treated cells. In vivo, incorporation of phosphatidylethanol and nuclear translocation of ZONAB correlated with increased proliferation in the colonic epithelium of ethanol-fed mice and in adenomas of chronic alcoholics. Our results show that phosphatidylethanol accumulation after chronic ethanol exposure disrupts signals that normally restrict proliferation in highly confluent intestinal cells, thus facilitating abnormal intestinal cell proliferation. (Mol Cancer Res 2007;5(11):1147–57)

Depending on its cellular localization, p120 catenin (p120ctn) can participate in various processes, such as cadherin-dependent cell-cell adhesion, actin cytoskeleton remodeling, and intracellular trafficking. Recent studies also indicate that p120ctn could regulate cell proliferation and contact inhibition. This report describes a new function of p120ctn in the regulation of cell cycle progression. Overexpression of the p120ctn isoform 3A in human colon adenocarcinoma cells (HT-29) results in cytoplasmic accumulation of the protein, as observed in many tumors. This cytoplasmic increase is correlated with a reduction in proliferation and inhibition of DNA synthesis. Under these conditions, experiments on synchronized cells revealed a prolonged S phase associated with cyclin E stabilization. Both confocal microscopy and biochemical analysis showed that cyclin E and cyclin-dependent kinase 2 colocalized with p120ctn in centrosomes during mitosis. These proteins are associated in a functional complex evidenced by coimmunoprecipitation experiments and the emergence of Thr199-phosphorylated nucleophosmin/B23. Such post-translational modification of this centrosomal target has been shown to trigger the initiation of centrosome duplication. Therefore, p120ctn-mediated accumulation of cyclin E in centrosomes may participate in abnormal amplification of centrosomes and the inhibition of DNA replication, thus leading to aberrant mitosis and polyploidy. Because these modifications are often observed in cancer, p120ctn may represent a new therapeutic target for future therapy. [Cancer Res 2007;67(20):9781–90]

International audience; The abnormal expression of gastric M1/MUC5AC mucin in precancerous lesions and colon cancer evidenced by immunohistochemistry led us to check for its presence in the mucus obtained directly from patients undergoing surgery for cancerous (adenocarcinoma) or inflammatory (diverticulitis or ulcerative colitis) diseases. In parallel, the authors quantified aberrant crypt foci (ACF) and their immunolabelling by M1/MUC5AC in mucosae of cancer and diverticulitis patients. Immuno-Radio-Metric Assay of M1/MUC5AC mucin developed by the authors was used to detect M1/MUC5AC mucin in the colonic mucus scraped from surgical specimens. M1/MUC5AC mucin was detected in the mucus of 51/69 (74%) patients with colon adenocarcinoma, versus 7/27 (26%) patients with diverticulitis (threshold: 30 units of M1 mucin per mg protein, area under ROC curve: 0.80). M1/MUC5AC was present in significantly (p < 0.001) larger amounts in the mucus of cancer versus diverticulitis patients. All (10/10) patients with ulcerative colitis tested showed levels above the threshold and their mucosae were strongly labelled with the anti-M1/MUC5AC antibody by immunohistochemistry. Patients with cancer exhibited 3 fold more ACF than those with diverticulitis, but no significant difference was observed in the mean size and M1/MUC5AC expression pattern of ACF between these two groups. The expression of M1/MUC5AC was in correlation with their size. In macroscopically normal mucosa, ACF were the most important source of M1/MUC5AC mucin. Testing of M1/MUC5AC can enhance the detection of precancerous lesions and colon cancer.

We have previously shown that cisplatin triggers an early acid sphingomyelinase (aSMase)-dependent ceramide generation concomitantly with an increase in membrane fluidity and induces apoptosis in HT29 cells. The present study further explores the role and origin of membrane fluidification in cisplatin-induced apoptosis. The rapid increase in membrane fluidity following cisplatin treatment was inhibited by membrane-stabilizing agents such as cholesterol or monosialoganglioside-1. In HT29 cells, these compounds prevented the early aggregation of Fas death receptor and of membrane lipid rafts on cell surface and significantly inhibited cisplatin-induced apoptosis without altering drug intracellular uptake or cisplatin DNA adducts formation. Early after cisplatin treatment, Na+/H+ membrane exchanger-1 (NHE1) was inhibited leading to intracellular acidification, aSMase was activated, and ceramide was detected at the cell membrane. Treatment of HT29 cells with Staphylococcus aureus sphingomyelinase increased membrane fluidity. Moreover, pretreatment with cariporide, a specific inhibitor of NHE1, inhibited cisplatin-induced intracellular acidification, aSMase activation, ceramide membrane generation, membrane fluidification, and apoptosis. Finally, NHE1-expressing PS120 cells were more sensitive to cisplatin than NHE1-deficient PS120 cells. Altogether, these findings suggest that the apoptotic pathway triggered by cisplatin involves a very early NHE1-dependent intracellular acidification leading to aSMase activation and increase in membrane fluidity. These events are independent of cisplatin-induced DNA adducts formation. The membrane exchanger NHE1 may be another potential target of cisplatin, increasing cell sensitivity to this compound. [Cancer Res 2007;67(16):7865–74]

Shiga-toxin (Stx) is the cardinal virulence factor of enterohemorrhagic Escherichia coli (EHEC). The genes encoding Stx are carried by a lambdoid phage integrated in the bacterial genome and are fully expressed after a bacterial SOS response induced by DNA-damaging agents. Because nitric oxide (NO) is an essential mediator of the innate immune response of infected colonic mucosa, we aimed to determine its role in Stx production by EHEC. Here we demonstrate that chemical or cellular sources of NO inhibit spontaneous and mitomycin C-induced stx mRNA expression and Stx synthesis, without altering EHEC viability. The synthesis of stx phage is also reduced by NO. This inhibitory effect apparently occurs through the NO-mediated sensitization of EHEC because mutation of the NO sensor nitrite-sensitive repressor results in loss of NO inhibiting activity on stx expression. Thus our findings identify NO as an inhibitor of stx expressing-phage propagation and Stx release and thus as a potential protective factor limiting the development of hemolytic syndromes.

International audience; Patients with cancer produce specific autoantibodies against protein antigens present in limited amount among a large background of immunoglobulins (Igs), nonrelevant as biomarkers, including natural antibodies. Multiple affinity protein profiling (MAPPing) that combines 2-D immunoaffinity chromatography, enzymatic digestion of the isolated proteins, and identification by MS/MS, may facilitate the identification of these so far unknown patient antibodies. The first immunoaffinity chromatography is crucial, as it is used for selectively removing proteins (autoantigens) recognized by natural antibodies. Application of this depletion step to colon cancer cell proteins is specifically described along with the identification of the natural autoantigens, as well as the coupling of this depletion step with the next steps. By enabling to separate antibody-binding proteins recognized by either natural autoantibodies or patient-specific antibodies this approach may contribute significantly towards the definition of autoantibody signatures.

International audience; Aberrant crypt foci (ACF) are microscopic lesions which have been postulated to precede the development of adenomas, precursors of colon cancer. The gastric M1/MUC5AC mucin has also been described as an early marker of colon carcinogenesis in the human and in the rat. To study changes in mucin expression associated with the genesis of tumors, Wistar rats were treated by intrarectal instillations of MNNG, twice a week for 2 weeks, and were sacrificed 10 (n = 20), 14 (n = 20), 22 (n = 20), 30 (n = 10) and 66 (n = 16) weeks after the beginning of the treatment. In the treated rats, the MUC5AC mucin was mainly expressed in ACF compared with the histologically normal mucosae, which showed few isolated MUC5AC-positive normal crypts. During carcinogenesis, the percentage of large ACF [> or =10 aberrant crypts] increased and the number of MUC5AC-positive (NCs) decreased. At Week 30, small tumors were observed arising from large ACF, both types of lesions expressing MUC5AC. At Week 66, large tumors showed remnants of MUC5AC-positive ACF in their adjacent mucosae. This observation suggests that the expression of MUC5AC is associated with the ACF/adenoma sequence and supports the notion of large ACF as precursors of adenomas/adenocarcinomas. Moreover, the expression of MUC5AC in the transitional mucosa adjacent to both rat and human colon tumors suggests that some human tumors could arise from large ACF, and reinforces the concept of the premalignant potential of these lesions.

Tumor protein 53-induced nuclear protein 1 (TP53INP1) is an antiproliferative and proapoptotic protein involved in cell stress response. To address its physiological roles in colorectal cancer and colitis, we generated and tested the susceptibility of Trp53inp1-deficient mice to the development of colorectal tumors induced by injection of the carcinogen azoxymethane followed by dextran sulfate sodium (DSS)-induced chronic colitis. Trp53inp1-deficient mice showed an increased incidence and multiplicity of tumors compared to those of wild-type (WT) mice. Furthermore, acute colitis induced by DSS treatment was more severe in Trp53inp1-deficient mice than in WT mice. Treatment with the antioxidant N-acetylcysteine prevented colitis and colitis-associated tumorigenesis more efficiently in WT mice than in Trp53inp1-deficient mice, suggesting a higher oxidative load in the latter. Consistently, we demonstrated by electron spin resonance and spin trapping that colons derived from deficient mice produced more free radicals than those of the WT during colitis and that the basal blood level of the antioxidant ascorbate was decreased in Trp53inp1-deficient mice. Collectively, these results indicate that the oxidative load is higher in Trp53inp1-deficient mice than in WT mice, generating a more-severe DSS-induced colitis, which favors development of colorectal tumors in Trp53inp1-deficient mice. Therefore, TP53INP1 is a potential target for the prevention of colorectal cancer in patients with inflammatory bowel disease.

Conventional cancer treatments rely on radiotherapy and chemotherapy. Such treatments supposedly mediate their effects via the direct elimination of tumor cells. Here we show that the success of some protocols for anticancer therapy depends on innate and adaptive antitumor immune responses. We describe in both mice and humans a previously unrecognized pathway for the activation of tumor antigen-specific T-cell immunity that involves secretion of the high-mobility-group box 1 (HMGB1) alarmin protein by dying tumor cells and the action of HMGB1 on Toll-like receptor 4 (TLR4) expressed by dendritic cells (DCs). During chemotherapy or radiotherapy, DCs require signaling through TLR4 and its adaptor MyD88 for efficient processing and cross-presentation of antigen from dying tumor cells. Patients with breast cancer who carry a TLR4 loss-of-function allele relapse more quickly after radiotherapy and chemotherapy than those carrying the normal TLR4 allele. These results delineate a clinically relevant immunoadjuvant pathway triggered by tumor cell death.

Tumor necrosis factor-α–related apoptosis-inducing ligand (TRAIL) is a potential anticancer agent that induces apoptosis in cancer cells but not in most normal cells. How tumor physiology, particularly acidic extracellular pH (pHe), would modify sensitivity of cancer cells to TRAIL-induced cell death is not known. We have previously shown that cancer cells, resistant to TRAIL-induced apoptosis at physiologic pHe (7.4), could be sensitized to TRAIL at acidic pHe (6.5). However, at this acidic pHe, cell death was necrotic. We show here that, in spite of a necrosis-like cell death morphology, caspases are activated and are necessary for TRAIL-induced cell death at acidic pHe in HT29 human colon cancer cells. Furthermore, we observed that, whereas receptor-interacting protein (RIP) was cleaved following TRAIL treatment at physiologic pHe (7.4), it was not cleaved following TRAIL treatment at acidic pHe (6.5). Moreover, RIP degradation by geldanamycin or decrease expression of RIP by small RNA interference transfection inhibited TRAIL-induced necrosis at acidic pHe, showing that RIP was necessary for this necrotic cell death pathway. We also show that RIP kinase activity was essential for this cell death pathway. Altogether, we show that, under acidic pHe conditions, TRAIL induces a necrosis-like cell death pathway that depends both on caspases and RIP kinase activity. Thus, our data suggest for the first time that RIP-dependent necrosis might be a major death pathway in TRAIL-based therapy in solid tumors with acidic pHe. [Cancer Res 2007;67(1):218–26]

International audience; Anthracyclin-treated tumor cells are particularly effective in eliciting an anticancer immune response, whereas other DNA-damaging agents such as etoposide and mitomycin C do not induce immunogenic cell death. Here we show that anthracyclins induce the rapid, preapoptotic translocation of calreticulin (CRT) to the cell surface. Blockade or knockdown of CRT suppressed the phagocytosis of anthracyclin-treated tumor cells by dendritic cells and abolished their immunogenicity in mice. The anthracyclin-induced CRT translocation was mimicked by inhibition of the protein phosphatase 1/GADD34 complex. Administration of recombinant CRT or inhibitors of protein phosphatase 1/GADD34 restored the immunogenicity of cell death elicited by etoposide and mitomycin C, and enhanced their antitumor effects in vivo. These data identify CRT as a key feature determining anticancer immune responses and delineate a possible strategy for immunogenic chemotherapy.

To investigate whether Src, JAK2 and phosphatidylinositol 3-kinase (PI3K) pathways are involved in the proliferation of human colonic tumour cells induced by glycine-extended gastrin (G-gly), the precursor of the mature amidated gastrin and to elucidate the molecular interaction between these three kinases in response to this peptide.Using the human colonic tumour cell line HCT116 as a model, we first measured the activation of PI3K, p60-Src and JAK2 in response to G-gly by in vitro kinase assays. Then we investigated the involvement of these kinases in G-gly-induced cell proliferation by MTT test.G-gly stimulation induced p60-Src, JAK2 and PI3K activation in HCT116. The different pathways were involved in proliferation of human colon cancer cells induced by G-gly. Furthermore, we found that both Src and JAK2 were necessary to PI3K regulation by this peptide. However, we did not find any cross-talk between the two tyrosine kinases.Our results suggest that the p60-Src/PI3K and JAK2/PI3K pathways act independently to mediate G-gly proliferative effect on human colonic tumour cells.

International audience; Evaluation of the role of clonal heterogeneity in colon tumour sensitivity/resistance to drugs and/or in conferring metastatic potential requires an adequate experimental model in which the tumour cells maintain the initial genetic alterations and intra-tumoral heterogeneity through maintenance of the genetic clones present in the initial tumour. Therefore, we xenografted subcutaneously into nude mice seven human colonic tumours (from stages B1 to D) that showed chromosome instability and transplanted them sequentially for up to 14 passages. Maintenance after xenografting of the genetic alterations present in the initial tumours was scored by allelotype studies targeting 45 loci localized on 18 chromosomes. We show that xenografting does not alter the genetic or the histological profiles of the tumours even after 14 passages. Screening of the entire genome of one tumour by comparative genome hybridization also showed overall stability of the alterations between the initial and the xenografted tumour. In addition, intra-tumoral heterogeneity was maintained over time, suggesting that no clonal selection occurred in the nude mice. The observation that some loci showed partial allelic imbalance in the initial tumour but loss of heterozygosity after the first passage in nude mice when all the normal cells were lost may allow identification of interesting genetic defects that could be involved in tumour expansion. Thus, sequential xenografts of colon tumours will provide a powerful model for further study of tumour clonality and for the identification of genetic profiles responsible for differential resistance to therapeutic treatments. Our data also suggest that tumour expansion can result from alterations in several distinct genetic pathways.

The initial invasive processes during cancer development remain largely unknown. Stromelysin-3/matrix metalloproteinase 11 (ST3/MMP11) is associated with tumor invasion and poor prognosis. We present novel evidence that adipocytes present at human breast tumor invasive front are induced by cancer cells to express ST3. Using mouse syngeneic model, light and electron microscopy showed that in ST3-deficient mice but not in wild-type mice, forced cancer cell-adipocyte interaction/crosstalk results in adipocyte membrane alteration, allowing cancer cell fat infiltration and death. Thus, adipocytes are involved in initial cancer cell survival into connective tissue, and this effect is ST3 mediated. This suggested that ST3 might play a role in adipocyte metabolism. Accordingly, ST3-deficient mice exhibited fat excess and increased mRNA levels of peroxisome proliferator-activated receptor γ (PPARγ) and adipocyte protein 2 (aP2) adipogenic markers, indicating that, in vivo, ST3 negatively regulates fat homeostasis. Moreover, ST3-deficient mouse embryonic fibroblasts exhibited a dramatic enhanced potential to differentiate into adipocytes associated with increased PPARγ and aP2 expression, and recombinant ST3 treatment reverted their differentiation. Thus, in vitro, ST3 reduces adipocyte differentiation in an autocrine manner. High fibroblasts/adipocytes ratio is a stroma feature, and peritumoral fibroblast origin remains debated. Our results support the concept that invading cancer cells aberrantly restore the negative ST3 function on adipogenesis into proximal adipocytes/preadipocytes, leading to the accumulation/maintenance of a particular peritumoral fibroblast subpopulation. Accordingly, in human breast tumors, we observed that ST3-expressing peritumoral fibroblasts are distinct from α-smooth muscle actin-expressing myofibroblasts. This constitutes the first report of implication of a MMP in cancer cell-adipocyte interaction/crosstalk during early steps of connective tissue invasion.

The Caudal-related homeodomain transcription factor Cdx2 plays a key role in intestinal cell fate determination. Reduction of Cdx2 expression is a feature of many human colon carcinomas and inactivation of one cdx2 allele facilitates the development of invasive adenocarcinoma in the murine colon. Here, we investigated the post-translational regulation of Cdx2. We showed that various forms of Cdx2 coexist in the intestine and colon cancer cell lines, some of them being phosphorylated forms. We found that cyclin-dependent kinase 2 phosphorylated Cdx2 in vitro and in vivo. Using site-specific mutagenesis, we identified serine 281 as a new key residue for Cdx2 phosphorylation. Intriguingly, serine 281 belongs to a conserved motif of four evenly spaced serines (the 4S motif) similar to the one controlling beta-catenin degradation by the proteasome pathway. A nonphosphorylated mutant Cdx2 lacking the 4S motif (4S>A) exhibited reduced polyubiquitination upon proteasome inhibition and increased stability compared to wild-type Cdx2. In addition, we found that this mutant was less efficient to suppress colony formation than wild-type Cdx2. Thus, our data highlight a novel post-translational mechanism controlling Cdx2 degradation via phosphorylation and polyubiquitination, which may be of importance for intestinal development and cancer.

International audience; Tumours in rodent and human colon share many histological and genetic features. To know if rodent models of colon carcinogenesis are good predictors of chemopreventive efficacy in humans, we conducted a meta-analysis of aspirin, beta-carotene, calcium, and wheat bran studies. Controlled intervention studies of adenoma recurrence in human volunteers were compared with chemoprevention studies of carcinogen-induced tumours in rats, and of polyps in Min (Apc(+/-)) mice: 6714 volunteers, 3911 rats and 458 mice were included in the meta-analyses. Difference between models was small since most global relative risks were between 0.76 and 1.00. A closer look showed that carcinogen-induced rat studies matched human trials for aspirin, calcium, carotene, and were compatible for wheat bran. Min mice results were compatible with human results for aspirin, but discordant for calcium and wheat bran (no carotene study). These few results suggest that rodent models roughly predict effect in humans, but the prediction is not accurate for all agents. Based on three cases only, the carcinogen-induced rat model seems better than the Min mouse model. However, rodent studies are useful to screen potential chemopreventive agents, and to study mechanisms of carcinogenesis and chemoprevention.

International audience; Red meat intake is associated with colon cancer risk. Puzzlingly, meat does not promote carcinogenesis in rat studies. However, we demonstrated previously that dietary heme promotes aberrant crypt foci (ACF) formation in rats given a low-calcium diet. Here, we tested the hypothesis that heme-rich meats promote colon carcinogenesis in rats treated with azoxymethane and fed low-calcium diets (0.8 g/kg). Three meat-based diets were formulated to contain varying concentrations of heme by the addition of raw chicken (low heme), beef (medium heme), or black pudding (blood sausage; high heme). The no-heme control diet was supplemented with ferric citrate and the heme control diet with hemoglobin to match iron and heme concentrations in the beef diet, respectively. After 100 d, colons were scored for ACF and mucin-depleted foci (MDF). Fecal water was assayed for lipoperoxides and cytotoxicity. Only diets with heme promoted the formation of MDF, but all meat diets promoted ACF formation. The number of MDF/colon was 0.55 +/- 0.68 in controls, but 1.2 +/- 0.6 (P = 0.13), 1.9 +/- 1.4 (P < 0.01), and 3.0 +/- 1.2 (P < 0.001) in chicken-, beef-, and black pudding-fed rats. MDF promotion by the high-heme black pudding diet was greater than that by the medium-heme beef diet. The number of ACF/colon was 72 +/- 16 in controls, but 91 +/- 18, 100 +/- 13, and 103 +/- 14 in chicken-, beef-, and black pudding-fed rats (all P < 0.001). ACF and MDF did not differ between rats fed the beef diet and those fed the heme control diet. MDF promotion was correlated with high fecal water lipoperoxides and cytotoxicity (r = 0.65, P < 0.01). This is the first study to show the promotion of experimental carcinogenesis by dietary meat and the association with heme intake.

International audience; TCF and SOX proteins belong to the high mobility group box transcription factor family. Whereas TCFs, the transcriptional effectors of the Wnt pathway, have been widely implicated in the development, homeostasis and disease of the intestine epithelium, little is known about the function of the SOX proteins in this tissue. Here, we identified SOX9 in a SOX expression screening in the mouse fetal intestine. We report that the SOX9 protein is expressed in the intestinal epithelium in a pattern characteristic of Wnt targets. We provide in vitro and in vivo evidence that a bipartite beta-catenin/TCF4 transcription factor, the effector of the Wnt signaling pathway, is required for SOX9 expression in epithelial cells. Finally, in colon epithelium-derived cells, SOX9 transcriptionally represses the CDX2 and MUC2 genes, normally expressed in the mature villus cells of the intestinal epithelium, and may therefore contribute to the Wnt-dependent maintenance of a progenitor cell phenotype.

Potential chemopreventive agents for colorectal cancer are assessed in rodents. We speculated that the magnitude of the effect is meaningful and ranked all published agents according to their potency. Data were gathered systematically from 137 articles with the aberrant crypt foci (ACF) end point and from 146 articles with the tumor end point. The potency of each agent to reduce the number of ACF is listed in one table and the potency of each agent to reduce the tumor incidence in another table. Both tables are shown in this review and on a website with sorting abilities (http://www.inra.fr/reseau-nacre/sci-memb/corpet/indexan.html). Potency was estimated as the ratio of the value in control rats to the value in treated rats. From each article, only the most potent agent was kept, except in articles reporting the effect of more than seven agents. Among the 186 agents in the ACF table, the median agent reduced the number of ACF by one-half. The most potent agents to reduce azoxymethane-induced ACF were Pluronic, polyethylene glycol, perilla oil with beta-carotene, and sulindac sulfide. Among the 160 agents in the tumor table, the median agent reduced the tumor incidence in rats by one-half. The most potent agents to reduce the incidence of azoxymethane-induced tumors were celecoxib, a protease inhibitor from soy, difluoromethylornithine with piroxicam, polyethylene glycol, and a thiosulfonate. For the 57 agents present in both tables, a significant correlation (r) was found between the potencies against ACF and tumors (r = 0.45, P < 0.001); without celecoxib, a major outlying point in the correlation, r = 0.68 (P < 0.001, n = 56). In conclusion, this review gathers most known chemopreventive agents, ranks the most promising agents against colon carcinogenesis in rats or mice, and further supports the use of ACF as a surrogate end point for tumors in rats.

International audience; Processed meat intake is associated with increased risk of colorectal cancer. This association may be explained by the endogenous formation of N-nitroso compounds (NOC). The hypothesis that meat intake can increase fecal NOC levels and colon carcinogenesis was tested in 175 Fischer 344 rats. Initiation was assessed by the number of aberrant crypt foci (ACF) in the colon of rats 45 days after the start of a high-fat bacon-based diet. Promotion was assessed by the multiplicity of ACF (crypts per ACF) in rats given experimental diets for 100 days starting 7 days after an azoxymethane injection. Three promotion studies were done, each in 5 groups of 10 rats, whose diets contained 7%, 14%, or 28% fat. Tested meats were bacon, pork, chicken, and beef. Fecal and dietary NOC were assayed by thermal energy analysis. Results show that feces from rats fed bacon-based diets contained 10-20 times more NOC than feces from control rats fed a casein-based diet (all p < 0.0001 in 4 studies). In bacon-fed rats, the amount of NOC input (diet) and output (feces) was similar. Rats fed a diet based on beef, pork, or chicken meat had less fecal NOC than controls (most p < 0.01). No ACF were detected in the colon of bacon-fed uninitiated rats. After azoxymethane injection, unprocessed but cooked meat-based diets did not change the number of ACF or the ACF multiplicity compared with control rats. In contrast, the bacon-based diet consistently reduced the number of large ACF per rat and the ACF multiplicity in the three promotion studies by 12%, 17%, and 20% (all p < 0.01). Results suggest that NOC from dietary bacon would not enhance colon carcinogenesis in rats.

Polyethylene-glycol (PEG) is a strong inhibitor of colon cancer in rats, and the most potent suppressor of aberrant crypt foci. 9 PEG-like block copolymers were tested in rodents, after an azoxymethane injection. Dietary pluronic F68 led to a 98.6% reduction in the number of aberrant crypt foci in a first rat study (P< 0.0001). Next 3 studies confirmed this pluronic efficacy in rats and mice. This non-toxic laxative seems roughly 5 times more potent than PEG for chemoprevention. © 2001 Cancer Research Campaign http://www.bjcancer.com

International audience; We speculated that a diet with a high glycemic index (GI) or a diet with a low nutrient density (nutrient-to-calorie ratio) would enhance colon carcinogenesis, presumably via increased insulin resistance. Forty-eight Sprague-Dawley (SD) rats received an azoxymethane injection (20 mg/kg) and were randomized into five groups given an AIN-76 diet containing 1) 65% starch by weight, 2) 65% glucose (GI = 100), 3) 65% fructose (GI = 23), 4) 82% starch, or 5) 39% oil and 39% sucrose. The nutrient density of Diets 4 and 5 was one-half that of Diets 1-3. Promotion was assessed by the multiplicity (number of crypts) of aberrant crypt foci (ACF), an early marker of colon carcinogenesis. Insulin resistance was estimated by the FIRI index (blood insulin x blood glucose), by plasma triglycerides, and by visceral fat. To confirm the results in another rat strain, the experiment was duplicated in 48 Fischer (F344) rats. Results show that 1) the ACF multiplicity was not different in glucose- and fructose-fed rats (p > 0.7): diets with contrasting GI had the same effect on ACF growth; 2) diets of low nutrient density increased visceral fat (p < 0.05) but reduced the ACF size in F344 rats (p < 0.001, no reduction in SD rats); and 3) indirect insulin resistance markers (FIRI index, blood triglycerides, and visceral fat) did not correlate with ACF multiplicity. These results do not support the hypothesis that diets with a high GI or low nutrient density or diets that increase some indirect insulin resistance markers can promote colon carcinogenesis in F344 or SD rats.