Intestinal tumorigenesis initiated by dedifferentiation and acquisition of stem-cell-like properties.

Cell-type plasticity within a tumor has recently been suggested to cause a bidirectional conversion between tumor-initiating stem cells and nonstem cells triggered by an inflammatory stroma. NF-κB represents a key transcription factor within the inflammatory tumor microenvironment. However, NF-κB's function in tumor-initiating cells has not been examined yet. Using a genetic model of intestinal epithelial cell (IEC)-restricted constitutive Wnt-activation, which comprises the most common event in the initiation of colon cancer, we demonstrate that NF-κB modulates Wnt signaling and show that IEC-specific ablation of RelA/p65 retards crypt stem cell expansion. In contrast, elevated NF-κB signaling enhances Wnt activation and induces dedifferentiation of nonstem cells that acquire tumor-initiating capacity. Thus, our data support the concept of bidirectional conversion and highlight the importance of inflammatory signaling for dedifferentiation and generation of tumor-initiating cells in vivo.

MEX3A regulates Lgr5+ stem cell maintenance in the developing intestinal epithelium.

Intestinal stem cells (ISCs) fuel the lifelong self-renewal of the intestinal tract and are paramount for epithelial repair. In this context, the Wnt pathway component LGR5 is the most consensual ISC marker to date. Still, the effort to better understand ISC identity and regulation remains a challenge. We have generated a Mex3a knockout mouse model and show that this RNA-binding protein is crucial for the maintenance of the Lgr5+ ISC pool, as its absence disrupts epithelial turnover during postnatal development and stereotypical organoid maturation ex vivo. Transcriptomic profiling of intestinal crypts reveals that Mex3a deletion induces the peroxisome proliferator-activated receptor (PPAR) pathway, along with a decrease in Wnt signalling and loss of the Lgr5+ stem cell signature. Furthermore, we identify PPARγ activity as a molecular intermediate of MEX3A-mediated regulation. We also show that high PPARγ signalling impairs Lgr5+ ISC function, thus uncovering a new layer of post-transcriptional regulation that critically contributes to intestinal homeostasis.

Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning.

During the suckling-to-weaning transition, the intestinal epithelium matures, allowing digestion of solid food. Transplantation experiments with rodent fetal epithelium into subcutaneous tissue of adult animals suggest that this transition is intrinsically programmed and occurs in the absence of dietary or hormonal signals. Here, we show that organoids derived from mouse primary fetal intestinal epithelial cells express markers of late fetal and neonatal development. In a stable culture medium, these fetal epithelium-derived organoids lose all markers of neonatal epithelium and start expressing hallmarks of adult epithelium in a time frame that mirrors epithelial maturation in vivoIn vitro postnatal development of the fetal-derived organoids accelerates by dexamethasone, a drug used to accelerate intestinal maturation in vivo Together, our data show that organoids derived from fetal epithelium undergo suckling-to-weaning transition, that the speed of maturation can be modulated, and that fetal organoids can be used to model the molecular mechanisms of postnatal epithelial maturation.

Anti-TNF therapy in IBD exerts its therapeutic effect through macrophage IL-10 signalling.

OBJECTIVE: Macrophage interleukin (IL)-10 signalling plays a critical role in the maintenance of a regulatory phenotype that prevents the development of IBD. We have previously found that anti-tumour necrosis factor (TNF) monoclonal antibodies act through Fcγ-receptor (FcγR) signalling to promote repolarisation of proinflammatory intestinal macrophages to a CD206+ regulatory phenotype. The role of IL-10 in anti-TNF-induced macrophage repolarisation has not been examined. DESIGN: We used human peripheral blood monocytes and mouse bone marrow-derived macrophages to study IL-10 production and CD206+ regulatory macrophage differentiation. To determine whether the efficacy of anti-TNF was dependent on IL-10 signalling in vivo and in which cell type, we used the CD4+CD45Rbhigh T-cell transfer model in combination with several genetic mouse models. RESULTS: Anti-TNF therapy increased macrophage IL-10 production in an FcγR-dependent manner, which caused differentiation of macrophages to a more regulatory CD206+ phenotype in vitro. Pharmacological blockade of IL-10 signalling prevented the induction of these CD206+ regulatory macrophages and diminished the therapeutic efficacy of anti-TNF therapy in the CD4+CD45Rbhigh T-cell transfer model of IBD. Using cell type-specific IL-10 receptor mutant mice, we found that IL-10 signalling in macrophages but not T cells was critical for the induction of CD206+ regulatory macrophages and therapeutic response to anti-TNF. CONCLUSION: The therapeutic efficacy of anti-TNF in resolving intestinal inflammation is critically dependent on IL-10 signalling in macrophages.

Anti-Tumor Necrosis Factor With a Glyco-Engineered Fc-Region Has Increased Efficacy in Mice With Colitis.

BACKGROUND & AIMS: Although tumor necrosis factor (TNF) antagonists reduce many clinical features of inflammatory bowel disease, complete mucosal healing occurs in fewer than 50% of patients. The Fc-region of monoclonal antibodies against TNF has immunosuppressive properties via effects on macrophage polarization. We examined the interaction between the anti-TNF Fc-region and Fcγ receptors (FcγR), and whether the absence of the Fc core fucose (which increases binding to FcγRIIIa) increases the efficacy of anti-TNF in mice with colitis. METHODS: We generated Rag1-/- mice that lack all activating FcγRs (FcγRI, FcγRIII, and FcγRIV; called FcγR-/-Rag1-/- mice). We produced hypo-fucosylated antibodies against mouse and human TNF (adalimumab). Colitis was induced in mice by transfer of CD4+CD45RBhi to FcγR-/-Rag1-/- or Rag1-/- littermates; mice were given different antibodies against TNF or isotype (control) antibodies and disease activity index scores were determined. Colon tissues were collected and analyzed by histology. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy donors. T-cell proliferation and proportions of CD206+ (immune regulatory) macrophages were measured in mixed lymphocyte reactions. Human PBMCs were genotyped for FCGR3A158 (the FcγRIIIa-158F allotype displays a lower Fc binding affinity) using the TaqMan single nucleotide polymorphism genotype assay. RESULTS: Rag1-/- mice with colitis given anti-TNF had near complete mucosal healing and Rag1-/- mice given an isotype control antibody developed severe colitis. In contrast, FcγR-/-Rag1-/- mice were refractory to the effects of anti-TNF: their histological colitis scores were as severe as those from FcγR-/-Rag1-/- mice given a control antibody. Colons from Rag1-/- mice that received anti-TNF had an increased number of CD206+ macrophages compared with Rag1-/- mice given control antibody; in FcγR-/-Rag1-/- mice given anti-TNF these numbers were as low as FcγR-/-Rag1-/- given the control antibody. In human PBMCs, anti-TNF increased the number of CD206+ macrophages: this required expression of FcγRIIIa; numbers of these cells were reduced in PBMCs with the low-affinity FcγRIIIa-158F genotype. A hypo-fucosylated form of adalimumab bound human FcγRIIIa with a higher affinity than control adalimumab. When hypo-fucosylated adalimumab was added to PBMCs, a larger number of CD206+ macrophages formed and T-cell proliferation was reduced, compared with addition of a control adalimumab. Hypo-fucosylated adalimumab increased the number of CD206+ macrophages in PMBCs that expressed the low-affinity FcγRIIIa. In mice with colitis, hypo-fucosylated anti-TNF significantly increased the number of CD206+ macrophages in the colon compared with control anti-TNF and was more effective in reducing colitis severity as measured by histology. CONCLUSIONS: In a study of the in vitro and in vivo mechanisms of anti-TNF, we found FcγR engagement by anti-TNF to be required for reduction of colitis in mice and development of CD206+ macrophages. A hypo-fucosylated form of anti-TNF binds FcγRIIIa with higher affinity and induces development of CD206+ macrophages in human PBMCs, especially PBMCs that express low-affinity FcγRIIIa. Hypo-fucosylated anti-TNF might be more effective in patients with inflammatory bowel disease.

Sex disparity in colonic adenomagenesis involves promotion by male hormones, not protection by female hormones.

It recently has been recognized that men develop colonic adenomas and carcinomas at an earlier age and at a higher rate than women. In the Apc(Pirc/+) (Pirc) rat model of early colonic cancer, this sex susceptibility was recapitulated, with male Pirc rats developing twice as many adenomas as females. Analysis of large datasets revealed that the Apc(Min/+) mouse also shows enhanced male susceptibility to adenomagenesis, but only in the colon. In addition, WT mice treated with injections of the carcinogen azoxymethane (AOM) showed increased numbers of colonic adenomas in males. The mechanism underlying these observations was investigated by manipulation of hormonal status. The preponderance of colonic adenomas in the Pirc rat model allowed a statistically significant investigation in vivo of the mechanism of sex hormone action on the development of colonic adenomas. Females depleted of endogenous hormones by ovariectomy did not exhibit a change in prevalence of adenomas, nor was any effect observed with replacement of one or a combination of female hormones. In contrast, depletion of male hormones by orchidectomy (castration) markedly protected the Pirc rat from adenoma development, whereas supplementation with testosterone reversed that effect. These observations were recapitulated in the AOM mouse model. Androgen receptor was undetectable in the colon or adenomas, making it likely that testosterone acts indirectly on the tumor lineage. Our findings suggest that indirect tumor-promoting effects of testosterone likely explain the disparity between the sexes in the development of colonic adenomas.

The SCFA butyrate stimulates the epithelial production of retinoic acid via inhibition of epithelial HDAC.

In the intestinal mucosa, retinoic acid (RA) is a critical signaling molecule. RA is derived from dietary vitamin A (retinol) through conversion by aldehyde dehydrogenases (aldh). Reduced levels of short-chain fatty acids (SCFAs) are associated with pathological microbial dysbiosis, inflammatory disease, and allergy. We hypothesized that SCFAs contribute to mucosal homeostasis by enhancing RA production in intestinal epithelia. With the use of human and mouse epithelial cell lines and primary enteroids, we studied the effect of SCFAs on the production of RA. Functional RA conversion was analyzed by Adlefluor activity assays. Butyrate (0-20 mM), in contrast to other SCFAs, dose dependently induced aldh1a1 or aldh1a3 transcript expression and increased RA conversion in human and mouse epithelial cells. Epithelial cell line data were replicated in intestinal organoids. In these organoids, butyrate (2-5 mM) upregulated aldh1a3 expression (36-fold over control), whereas aldh1a1 was not significantly affected. Butyrate enhanced maturation markers (Mucin-2 and villin) but did not consistently affect stemness markers or other Wnt target genes (lgr5, olfm4, ascl2, cdkn1). In enteroids, the stimulation of RA production by SCFA was mimicked by inhibitors of histone deacetylase 3 (HDAC3) but not by HDAC1/2 inhibitors nor by agonists of butyrate receptors G-protein-coupled receptor (GPR)43 or GPR109A, indicating that butyrate stimulates RA production via HDAC3 inhibition. We conclude that the SCFA butyrate inhibits HDAC3 and thereby supports epithelial RA production.

Pharmacokinetic Features and Presence of Antidrug Antibodies Associate With Response to Infliximab Induction Therapy in Patients With Moderate to Severe Ulcerative Colitis.

BACKGROUND & AIMS: The pharmacokinetics of infliximab during induction treatment for ulcerative colitis (UC) have not been studied. We investigated serum concentrations of infliximab and the early appearance of antibodies to infliximab (ATI) during induction treatment in patients with moderate-to-severe UC. METHODS: We performed a prospective analysis of 19 consecutive patients with moderate-severe UC (endoscopic Mayo ≥ 2) receiving induction therapy with infliximab (5 mg/kg at weeks 0, 2, and 6) at 2 centers in Amsterdam, The Netherlands, from July 2012 through March 2014. Serial serum and fecal samples were collected for 6 weeks and concentrations of infliximab, ATI, c-reactive protein (CRP), albumin, and fecal calprotectin were measured. Treatment success was defined as endoscopic response (≥ 1 point reduction in the endoscopic Mayo score) at week 8. RESULTS: Eleven patients (58%) had an endoscopic response. The median serum concentrations of infliximab at week 6 were 8.1 µg/mL in responders (interquartile range, 3.0-13.7 µg/mL) and 2.9 µg/mL in nonresponders (interquartile range, 0.01-5.8 µg/mL) (P = .03). ATIs were detected in 7 patients as early as day 18 (median, 28 d; interquartile range, 18-42 d). Six of the 8 nonresponders tested positive for ATIs vs 1 of 11 responders (P < .01; odds ratio, 30.0; 95% CI, 2.2-406.2). Patients with a baseline concentration of CRP greater than 50 mg/L had lower drug exposure from weeks 0 to 6 (587 mg/L/d in patients with high levels of CRP vs 1361 mg/L/day in patients with low CRP; P = .001). The median area under the curve for serum concentration of infliximab during induction therapy was 1230 mg/L/d in nonresponders vs 1352 mg/L/d in responders (P = .65). CONCLUSIONS: There is a significant difference in serum concentration of infliximab at week 6 of treatment between responders and nonresponders. Early development of ATIs during induction therapy reduces the serum concentration of infliximab and is associated with nonresponse to treatment. Patients with high baseline serum levels of CRP had lower serum concentrations of infliximab. CLINICAL TRIAL NUMBER: NL39626.018.12.

Loss of Infliximab Into Feces Is Associated With Lack of Response to Therapy in Patients With Severe Ulcerative Colitis.

BACKGROUND & AIMS: It is not clear why some patients with ulcerative colitis (UC) do not respond to treatment with anti-tumor necrosis factor (TNF) agents, such as infliximab. It could be that some patients have high level of inflammation, with large quantities of TNF to be neutralized by the drug. We investigated whether loss of anti-TNF agents through ulcerated intestinal mucosa reduces the efficacy of these drugs in patients with severe UC. METHODS: We collected fecal samples from 30 consecutive patients with moderate to severely active UC during the first 2 weeks of infliximab therapy at the University of Amsterdam hospital. Infliximab concentrations were measured in serum and supernatants of fecal samples using an enzyme-linked immunosorbent assay (Sanquin Biologicals Laboratory, Amsterdam, The Netherlands). Clinical and endoscopic responses were assessed 2 and 8 weeks and 3 months after treatment began. RESULTS: Infliximab was detected in 129 of 195 fecal samples (66%); the highest concentrations were measured in the first days after the first infusion. Patients that were clinical nonresponders at week 2 had significantly higher fecal concentrations of infliximab after the first day of treatment than patients with clinical responses (median concentration, 5.01 µg/mL in nonresponders vs 0.54 µg/mL in responders; P = .0047). We did not observe a correlation between fecal and serum concentrations of infliximab. CONCLUSIONS: Infliximab is lost into stools of patients with UC. High fecal concentrations of infliximab in the first days after therapy begins are associated with primary nonresponse. Additional studies are needed to determine how therapeutic antibodies are lost through the intestinal mucosa and how this process affects treatment response. Clinical trial ID: NL41310.018.12.

Findings From a Randomized Controlled Trial of Fecal Transplantation for Patients With Ulcerative Colitis.

BACKGROUND & AIMS: Several case series have reported the effects of fecal microbiota transplantation (FMT) for ulcerative colitis (UC). We assessed the efficacy and safety of FMT for patients with UC in a double-blind randomized trial. METHODS: Patients with mild to moderately active UC (n = 50) were assigned to groups that underwent FMT with feces from healthy donors or were given autologous fecal microbiota (control); each transplant was administered via nasoduodenal tube at the start of the study and 3 weeks later. The study was performed at the Academic Medical Center in Amsterdam from June 2011 through May 2014. The composite primary end point was clinical remission (simple clinical colitis activity index scores ≤2) combined with ≥1-point decrease in the Mayo endoscopic score at week 12. Secondary end points were safety and microbiota composition by phylogenetic microarray in fecal samples. RESULTS: Thirty-seven patients completed the primary end point assessment. In the intention-to-treat analysis, 7 of 23 patients who received fecal transplants from healthy donors (30.4%) and 5 of 25 controls (20.0%) achieved the primary end point (P = .51). In the per-protocol analysis, 7 of 17 patients who received fecal transplants from healthy donors (41.2%) and 5 of 20 controls (25.0%) achieved the primary end point (P = .29). Serious adverse events occurred in 4 patients (2 in the FMT group), but these were not considered to be related to the FMT. At 12 weeks, the microbiota of responders in the FMT group was similar to that of their healthy donors; remission was associated with proportions of Clostridium clusters IV and XIVa. CONCLUSIONS: In this phase 2 trial, there was no statistically significant difference in clinical and endoscopic remission between patients with UC who received fecal transplants from healthy donors and those who received their own fecal microbiota, which may be due to limited numbers. However, the microbiota of responders had distinct features from that of nonresponders, warranting further study. ClinicalTrials.gov Number: NCT01650038.

Stromal Indian hedgehog signaling is required for intestinal adenoma formation in mice.

BACKGROUND & AIMS: Indian hedgehog (IHH) is an epithelial-derived signal in the intestinal stroma, inducing factors that restrict epithelial proliferation and suppress activation of the immune system. In addition to these rapid effects of IHH signaling, IHH is required to maintain a stromal phenotype in which myofibroblasts and smooth muscle cells predominate. We investigated the role of IHH signaling during development of intestinal neoplasia in mice. METHODS: Glioma-associated oncogene (Gli1)-CreERT2 and Patched (Ptch)-lacZ reporter mice were crossed with Apc(Min) mice to generate Gli1CreERT2-Rosa26-ZSGreen-Apc(Min) and Ptch-lacZ-Apc(Min) mice, which were used to identify hedgehog-responsive cells. Cyp1a1Cre-Apc (Apc(HET)) mice, which develop adenomas after administration of ß-naphthoflavone, were crossed with mice with conditional disruption of Ihh in the small intestine epithelium. Apc(Min) mice were crossed with mice in which sonic hedgehog (SHH) was overexpressed specifically in the intestinal epithelium. Intestinal tissues were collected and analyzed histologically and by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction. We also analyzed levels of IHH messenger RNA and expression of IHH gene targets in intestinal tissues from patients with familial adenomatous polyposis (n = 18) or sessile serrated adenomas (n = 15) and normal colonic tissue from control patients (n = 12). RESULTS: Expression of IHH messenger RNA and its targets were increased in intestinal adenomas from patients and mice compared with control colon tissues. In mice, IHH signaling was exclusively paracrine, from the epithelium to the stroma. Loss of IHH from Apc(HET) mice almost completely blocked adenoma development, and overexpression of SHH increased the number and size of adenomas that developed. Loss of IHH from Apc(HET) mice changed the composition of the adenoma stroma; cells that expressed α-smooth muscle actin or desmin were lost, along with expression of cyclooxygenase-2, and the number of vimentin-positive cells increased. CONCLUSIONS: Apc mutant epithelial cells secrete IHH to maintain an intestinal stromal phenotype that is required for adenoma development in mice.

HDAC1 and HDAC2 collectively regulate intestinal stem cell homeostasis.

Histone deacetylases (HDACs) are posttranslational modifiers that deacetylate proteins. Despite their crucial role in numerous biological processes, the use of broad-range HDAC inhibitors (HDACi), has shown clinical efficacy. However, undesired side effects highlight the necessity to better understand the biology of different HDACs and target the relevant HDACs. Using a novel mouse model, in which HDAC1 and HDAC2 can be simultaneously deleted in the intestine of adult mice, we show that the simultaneous deletion of HDAC1 and HDAC2 leads to a rapid loss of intestinal homeostasis. Importantly, this deletion cannot be sustained, and 8 days after initial ablation, stem cells that have escaped HDAC1 or HDAC2 deletion swiftly repopulate the intestinal lining. In vitro ablation of HDAC1 and HDAC2 using intestinal organoid cultures resulted in a down-regulation of multiple intestinal stem cell markers and functional loss of clonogenic capacity. Importantly, treatment of wild-type organoids with class I-specific HDACi MS-275 also induced a similar loss of stemness, providing a possible rationale for the gastrointestinal side effects often observed in HDACi-treated patients. In conclusion, these data show that HDAC1 and HDAC2 have a redundant function and are essential to maintain intestinal homeostasis.

ER stress induces epithelial differentiation in the mouse oesophagus.

OBJECTIVE: Stress in the endoplasmic reticulum (ER) leads to activation of the unfolded protein response (UPR). Xbp1, a key component of the UPR has recently been linked to the risk of developing oesophageal squamous cell carcinoma, suggesting an important role for the UPR in the oesophageal epithelium. Here we examined the role of ER stress and the UPR in oesophageal epithelial homoeostasis. DESIGN: We examined the expression of components of the UPR in the oesophageal epithelium. We used a pharmacological approach and a genetic approach to examine the effects of ER stress in vivo in the mouse oesophagus. The oesophagus of these mice was examined using immunohistochemistry and real-time reverse transcription (RT)-PCR. RESULTS: Components of the UPR were heterogeneously expressed in the basal layer of the epithelium. Induction of ER stress by 24-h treatment with thapsigargin resulted in depletion of proliferating cells in the basal layer of the oesophagus and induced differentiation. We next activated the UPR by inducible deletion of the major ER chaperone Grp78 in Ah1Cre-Rosa26-LacZ-Grp78(-/-) mice in which mutant cells could be traced by expression of LacZ. In these mice LacZ-positive mutant cells in the basal layer lost their proliferative capacity, migrated towards the oesophageal lumen and were replaced by LacZ-negative non-mutant cells. We observed no apoptosis in mutant cells. CONCLUSIONS: These results show that ER stress induces epithelial differentiation in precursor cells in the oesophageal epithelium. This UPR induced differentiation may serve as a quality control mechanism that protects against oesophageal cancer development.

Esophageal development and epithelial homeostasis.

The esophagus is a relatively simple organ that evolved to transport food and liquids through the thoracic cavity. It is the only part of the gastrointestinal tract that lacks any metabolic, digestive, or absorptive function. The mucosa of the adult esophagus is covered by a multilayered squamous epithelium with a remarkable similarity to the epithelium of the skin despite the fact that these tissues originate from two different germ layers. Here we review the developmental pathways involved in the establishment of the esophagus and the way these pathways regulate gut-airway separation. We summarize current knowledge of the mechanisms that maintain homeostasis in esophageal epithelial renewal in the adult and the molecular mechanism of the development of Barrett's metaplasia, the precursor lesion to esophageal adenocarcinoma. Finally, we examine the ongoing debate on the hierarchy of esophageal epithelial precursor cells and on the presence or absence of a specific esophageal stem cell population. Together the recent insights into esophageal development and homeostasis suggest that the pathways that establish the esophagus during development also play a role in the maintenance of the adult epithelium. We are beginning to understand how reflux of gastric content and the resulting chronic inflammation can transform the squamous esophageal epithelium to columnar intestinal type metaplasia in Barrett's esophagus.

Safety and Feasibility of Using the Second-Generation Pillcam Colon Capsule to Assess Active Colonic Crohn's Disease.

BACKGROUND & AIMS: The second-generation Pillcam Colon Capsule Endoscope (PCCE-2; Given Imaging Ltd, Yoqneam, Israel) is an ingestible capsule for visualization of the colon. We performed a multicenter pilot study to assess its safety and feasibility in evaluating the severity of Crohn's disease (CD). METHODS: In a prospective study, 40 patients with active colonic CD underwent PCCE-2 and optical colonoscopy procedures. Using both techniques, we generated values for the Crohn's Disease Endoscopic Index of Severity (CDEIS), the Simple Endoscopic Score for CD, and global evaluation of lesion severity. In the first stage of the study, we calculated the correlation between PCCE-2 and optical colonoscopy scores. In the second stage, we performed interobserver agreement analysis for a random subset of 20 PCCE-2 recordings, graded in duplicate by 2 independent readers. RESULTS: There was substantial agreement between PCCE-2 and optical colonoscopy in the measurement of the CDEIS (intraclass correlation coefficient [ICC], 0.65; 95% confidence interval [CI], 0.43-0.80). There was substantial interobserver agreement between 2 independent PCCE-2 readers for the CDEIS (ICC, 0.67; 95% CI, 0.35-0.86) and the Simple Endoscopic Score for CD (ICC, 0.66; 95% CI, 0.32-0.85). However, the PCCE-2 scoring systematically underestimated the severity of disease compared with optical colonoscopy; based on our results, PCCE-2 detected colonic ulcerations with 86% sensitivity and 40% specificity. No adverse events were observed and PCCE-2 was better tolerated than colonoscopy. CONCLUSIONS: PCCE-2 is feasible, safe, and well tolerated for the assessment of mucosal CD activity in selected populations. Larger studies are needed to assess its operating characteristics further. European clinical trials database number: 2014-003854-15.

Loss of SMAD4 alters BMP signaling to promote colorectal cancer cell metastasis via activation of Rho and ROCK.

BACKGROUND & AIMS: SMAD4 frequently is lost from colorectal cancers (CRCs), which is associated with the development of metastases and a poor prognosis. SMAD4 loss is believed to alter transforming growth factor ß signaling to promote tumor progression. However, SMAD4 is also a central component of the bone morphogenetic protein (BMP) signaling pathway, implicated in CRC pathogenesis by human genetic studies. We investigated the effects of alterations in BMP signaling on the invasive and metastatic abilities of CRC cells and changes in members in this pathway in human tumor samples. METHODS: We activated BMP signaling in SMAD4-positive and SMAD4-negative CRC cells (HCT116, HT-29, SW480, and LS174T); SMAD4 was stably expressed or knocked down using lentiviral vectors. We investigated the effects on markers of epithelial-mesenchymal transition and on cell migration, invasion, and formation of invadopodia. We performed kinase activity assays to characterize SMAD4-independent BMP signaling and used an inhibitor screen to identify pathways that regulate CRC cell migration. We investigated the effects of the ROCK inhibitor Y-27632 in immunocompromised (CD-1 Nu) mice with orthotopic metastatic tumors. Immunohistochemistry was used to detect BMPR1a, BMPR1b, BMPR2, and SMAD4 in human colorectal tumors; these were related to patient survival times. RESULTS: Activation of BMP signaling in SMAD4-negative cells altered protein and messenger RNA levels of markers of epithelial-mesenchymal transition and increased cell migration, invasion, and formation of invadopodia. Knockdown of the BMP receptor in SMAD4-negative cells reduced their invasive activity in vitro. SMAD4-independent BMP signaling activated Rho signaling via ROCK and LIM domain kinase (LIMK). Pharmacologic inhibition of ROCK reduced metastasis of colorectal xenograft tumors in mice. Loss of SMAD4 from colorectal tumors has been associated with reduced survival time; we found that this association is dependent on the expression of BMP receptors but not transforming growth factor ß receptors. CONCLUSIONS: Loss of SMAD4 from colorectal cancer cells causes BMP signaling to switch from tumor suppressive to metastasis promoting. Concurrent loss of SMAD4 and normal expression of BMP receptors in colorectal tumors was associated with reduced survival times of patients. Reagents that interfere with SMAD4-independent BMP signaling, such as ROCK inhibitors, might be developed as therapeutics for CRC.

Superantigen-induced steroid resistance depends on activation of phospholipase Cß2.

The glucocorticoid receptor is present in a TCR-associated complex, which includes the Src family tyrosine kinase Lck. Glucocorticoids rapidly dissociate this complex, resulting in the inhibition of canonical Lck-phospholipase C (PLC)γ-dependent TCR signaling. The relative importance of this nongenomic role for the glucocorticoid receptor compared with its direct transcriptional effects is not known. Superantigens induce a state of steroid resistance in activated T cells. It was reported that, in addition to canonical Lck-PLCγ signaling, superantigens can activate a noncanonical G protein-PLCß-dependent signaling pathway. In this study, we show that staphylococcal enterotoxin B activates a Gαq and PLCß2-dependent pathway in human T cells. We find that this pathway bypasses the need for canonical Lck-PLCγ signaling in T cell activation and renders superantigen-stimulated T cells insensitive to glucocorticoids in vitro. We show that the PLCß inhibitor U-73122 sensitizes staphylococcal enterotoxin B-treated mice to dexamethasone in vivo. In conclusion, we find that effects of glucocorticoids on TCR-induced T cell proliferation are mainly nongenomic and can be bypassed by the activation of an Lck-independent signaling pathway.

The morphogenetic code and colon cancer development.

The initiating genetic lesion in sporadically occurring cancers is impossible to identify. The existence of rare inherited cancer syndromes has helped to uncover some of the mutations that can initiate tumorigenesis. Most of these initiating lesions affect genes belonging to morphogenetic signaling pathways. We review the evidence that the cellular fate of individual epithelial cells in the adult is nonautonomous and depends on extrinsic information, just like cells in a developing embryo. Cancer stem cells need to disrupt these extrinsic restraints to gain an autonomous clonal proliferative advantage over neighboring stem cells.

The ACCURE-trial: the effect of appendectomy on the clinical course of ulcerative colitis, a randomised international multicenter trial (NTR2883) and the ACCURE-UK trial: a randomised external pilot trial (ISRCTN56523019).

BACKGROUND: Over the past 20 years evidence has accumulated confirming the immunomodulatory role of the appendix in ulcerative colitis (UC). This led to the idea that appendectomy might alter the clinical course of established UC. The objective of this body of research is to evaluate the short-term and medium-term efficacy of appendectomy to maintain remission in patients with UC, and to establish the acceptability and cost-effectiveness of the intervention compared to standard treatment. METHODS/DESIGN: These paired phase III multicenter prospective randomised studies will include patients over 18 years of age with an established diagnosis of ulcerative colitis and a disease relapse within 12 months prior to randomisation. Patients need to have been medically treated until complete clinical (Mayo score <3) and endoscopic (Mayo score 0 or 1) remission. Patients will then be randomised 1:1 to a control group (maintenance 5-ASA treatment, no appendectomy) or elective laparoscopic appendectomy plus maintenance treatment. The primary outcome measure is the one year cumulative UC relapse rate - defined both clinically and endoscopically as a total Mayo-score ≥5 with endoscopic subscore of 2 or 3. Secondary outcomes that will be assessed include the number of relapses per patient at 12 months, the time to first relapse, health related quality of life and treatment costs, and number of colectomies in each arm. DISCUSSION: The ACCURE and ACCURE-UK trials will provide evidence on the role and acceptability of appendectomy in the treatment of ulcerative colitis and the effects of appendectomy on the disease course. TRIAL REGISTRATION: NTR2883 ; ISRCTN56523019.

Oestrogens promote tumorigenesis in a mouse model for colitis-associated cancer.

BACKGROUND: Hormone replacement therapy increases the risk of developing ulcerative colitis in postmenopausal women. Chronic intestinal inflammation predisposes to colon cancer development, but effects of female hormones on colitis-associated cancer development have not been examined. AIM: To investigate the role of female hormones in the dextran sodium sulfate (DSS)-azoxymethane (AOM) mouse model for colitis-associated cancer. DESIGN: We performed ovariectomies, or sham operations, on mice, and supplemented these animals with indicated hormones. Additionally, we used oestrogen receptor α or ß (Erα or Erß) mutant mice. To study colitis or colitis-associated cancer, we used DSS only, or DSS and AOM, respectively. RESULTS: Ovariectomy protects female mice against colitis-associated tumour development. Hormone replacement in ovariectomised mice with either oestradiol (E2), medroxyprogesterone acetate or a combination of both suggests that oestrogens are the ovary-derived factor that promotes tumour development in the context of inflammatory damage. E2-treated animals showed increased clinical symptoms and Il-6 production upon DSS-induced colitis and enhanced epithelial proliferation. Treatment with E2 markedly increased the numbers of polyps in ovariectomised mice and also strongly promoted tumour progression with all E2-treated animals developing at least one invasive adenocarcinoma, whereas, placebo-treated animals developed adenomas only. Using Er mutant mice, we find that the protumorigenic effect of oestrogen depends on both Erα and Erß. CONCLUSIONS: Our results suggest that oestrogens promote inflammation-associated cancer development by impairing the mucosal response to inflammatory damage.