Background

Tumor cell death generates products that can be measured in the circulation of cancer patients. CK18-Asp396 (M30 antigen) is a caspase-degraded product of cytokeratin 18 (CK18), produced by apoptotic epithelial cells, and is elevated in breast and lung cancer patients.

Methods

We determined the CK18-Asp396 and total CK18 levels in plasma of 49 colorectal cancer patients, before and after surgical resection of the tumor, by ELISA. Correlations with patient and tumor characteristics were determined by Kruskal-Wallis H and Mann-Whitney U tests. Disease-free survival was determined using Kaplan-Meier methodology with Log Rank tests, and univariate and multivariate Cox proportional hazard analysis.

Results

Plasma CK18-Asp396 and total CK18 levels in colorectal cancer patients were related to disease stage and tumor diameter, and were predictive of disease-free survival, independent of disease-stage, with hazard ratios (HR) of patients with high levels (> median) compared to those with low levels (< or = median) of 3.58 (95% CI: 1.17-11.02) and 3.58 (95% CI: 0.97-7.71), respectively. The CK18-Asp396/CK18 ratio, which decreased with tumor progression, was also predictive of disease-free survival, with a low ratio (< or = median) associated with worse disease-free survival: HR 2.78 (95% CI: 1.06-7.19). Remarkably, the plasma CK18-Asp396 and total CK18 levels after surgical removal of the tumor were also predictive of disease-free survival, with patients with high levels having a HR of 3.78 (95% CI: 0.77-18.50) and 4.12 (95% CI: 0.84-20.34), respectively, indicating that these parameters can be used also to monitor patients after surgery.

Conclusion

CK18-Asp396 and total CK18 levels in the circulation of colorectal cancer patients are predictive of tumor progression and prognosis and might be helpful for treatment selection and monitoring of these patients.

The ability of mesenchymal stromal cells (MSCs) to suppress immune responses combined with their potential to actively participate in tissue repair provides a strong rationale for the use of MSCs as a new treatment option in diseases characterized by inflammation and severe tissue damage, such as Crohn's disease (CD) and perianal fistulas. Multiple studies have shown that MSCs suppress a range of immune cells, such as dendritic cells (DC), naïve and effector T cells, and natural killer (NK) cells. Recently published papers attribute the immunosuppressive capacity of MSCs to soluble factors produced by MSCs, such as prostaglandin E2 (PGE(2)), inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase (IDO). Promising results are obtained from phase I and II clinical trials with autologous and allogeneic MSCs as treatment for refractory CD and perianal fistulas; however the question remains: what are the molecular mechanisms underlying the immunomodulating properties of MSCs? This paper highlights the present knowledge on the immunosuppressive effects of MSCs and its complexity in relation to CD and perianal fistulas.

Background & aims

Chronic inflammation promotes development and progression of colorectal cancer (CRC). We explored the distribution of Corticotropin-Releasing-Hormone (CRH)-family of receptors and ligands in CRC and their contribution in tumor growth and oncogenic EMT.

Methods

mRNA expression of CRH-family members was analyzed in CRC (N=56) and control (N=46) samples, 7 CRC cell lines and normal NCM460 cells. Immunohistochemical detection of CRHR2 was performed in 20 CRC and 5 normal tissues. Cell proliferation, migration and invasion were compared between Urocortin-2 (Ucn2)-stimulated parental and CRHR2-overexpressing (CRHR2+) cells in absence or presence of IL-6. CRHR2/Ucn2-targeted effects on tumor growth and EMT were validated in SW620-xenograft mouse models.

Results

CRC tissues and cell lines showed decreased mRNA and protein CRHR2 expression compared to controls and NCM460, respectively. The opposite trend was shown for Ucn2. CRHR2/Ucn2 signaling inhibited cell proliferation, migration, invasion and colony formation in CRC-CRHR2+ cells. In vivo, SW620-CRHR2+ xenografts showed decreased growth, reduced expression of EMT-inducers and elevated levels of EMT-suppressors. IL-1b, IL-6 and IL-6R mRNAs where diminished in CRC-CRHR2+ cells, while CRHR2/Ucn2 signaling inhibited IL-6-mediated Stat3 activation, invasion, migration and expression of downstream targets acting as cell cycle- and EMT-inducers. Expression of cell cycle- and EMT-suppressors was augmented in IL-6/Ucn2-stimulated CRHR2+ cells. In patients, CRHR2 mRNA expression was inversely correlated with IL-6R and vimentin levels and metastasis occurrence, while positively associated with E-cadherin expression and overall survival.

Conclusions

CRHR2 downregulation in CRC supports tumor expansion and spread through maintaining persistent inflammation and constitutive Stat3 activation. CRHR2^low CRC phenotypes are associated with higher risk for distant metastases and poor clinical outcomes.

In gastric cancer, a new epigenetic mechanism of tumour suppressor loss has been suggested where the histone methyltransferase enhancer of zeste homolog 2 (EZH2) is responsible for loss of expression of RUNX3. This is consistent with EZH2 upregulation in multiple cancer types being associated with poor prognosis. We investigated whether EZH2 influences the expression of RUNX3 in colorectal cancer (CRC) and whether this is independent of methylation. We determined protein and messenger RNA (mRNA) levels of EZH2 and RUNX3 and assessed RUNX3 methylation with methylation-specific polymerase chain reaction using 72 human CRCs and 8 CRC cell lines. We assessed the effect of efficient RNA interference-mediated knockdown of EZH2 on RUNX3 levels, cell viability and H3K27 trimethylation of the RUNX3 promoter using chromatin immunoprecipitation. Despite higher levels of EZH2 and lower levels of RUNX3 in CRC specimens in general, no inverse correlation between EZH2 and RUNX3 in paired samples was found arguing against a major role for histone methylation in silencing RUNX3 in CRC. Conversely, downregulation of RUNX3 mRNA in the same tumours was associated with RUNX3 DNA methylation (P < 0.05). In cell lines, knockdown of EZH2 removed the repressive chromatin marks from RUNX3 but did not result in RUNX3 re-expression. However, it prevented the re-silencing of RUNX3 after the removal of demethylating agents. In conclusion, DNA methylation is primarily responsible for the transcriptional silencing of RUNX3 in CRC, but EZH2 and histone methylation are necessary for its methylation-dependent re-silencing after the removal of demethylating agents. These results would predict that inhibitors of EZH2 and histone methylation would enhance the effects of demethylating agents in cancer therapy.

Background

We previously showed that activation of the bile salt nuclear receptor Farnesoid X Receptor (FXR) protects against intestinal inflammation in mice. Reciprocally, these inflammatory mediators may decrease FXR activation. We investigated whether FXR activation is repressed in the ileum and colon of inflammatory bowel disease (IBD) patients in remission. Additionally, we evaluated whether genetic variation in FXR is associated with IBD.

Methods

mRNA expression of FXR and FXR target gene SHP was determined in ileal and colonic biopsies of patients with Crohn's colitis (n = 15) and ulcerative colitis (UC; n = 12), all in clinical remission, and healthy controls (n = 17). Seven common tagging SNPs and two functional SNPs in FXR were genotyped in 2355 Dutch IBD patients (1162 Crohn's disease (CD) and 1193 UC) and in 853 healthy controls.

Results

mRNA expression of SHP in the ileum is reduced in patients with Crohn's colitis but not in patients with UC compared to controls. mRNA expression of villus marker Villin was correlated with FXR and SHP in healthy controls, a correlation that was weaker in UC patients and absent in CD patients. None of the SNPs was associated with IBD, UC or CD, nor with clinical subgroups of CD.

Conclusions

FXR activation in the ileum is decreased in patients with Crohn's colitis. This may be secondary to altered enterohepatic circulation of bile salts or transrepression by inflammatory signals but does not seem to be caused by the studied SNPs in FXR. Increasing FXR activity by synthetic FXR agonists may have benefit in CD patients.

Background & aims

Persistent activation of the inflammatory response contributes to the development of inflammatory bowel diseases, which increase the risk of colorectal cancer. We aimed to identify microRNAs that regulate inflammation during the development of ulcerative colitis (UC) and progression to colitis-associated colon cancer (CAC).

Methods

We performed a quantitative polymerase chain reaction analysis to measure microRNAs in 401 colon specimens from patients with UC, Crohn's disease, irritable bowel syndrome, sporadic colorectal cancer, or CAC, as well as subjects without these disorders (controls); levels were correlated with clinical features and disease activity of patients. Colitis was induced in mice by administration of dextran sodium sulfate (DSS), and carcinogenesis was induced by addition of azoxymethane; some mice also were given an inhibitor of microRNA214 (miR214).

Results

A high-throughput functional screen of the human microRNAome found that miR214 regulated the activity of nuclear factor-κB. Higher levels of miR214 were detected in colon tissues from patients with active UC or CAC than from patients with other disorders or controls and correlated with disease progression. Bioinformatic and genome-wide profile analyses showed that miR214 activates an inflammatory response and is amplified through a feedback loop circuit mediated by phosphatase and tensin homolog (PTEN) and PDZ and LIM domain 2 (PDLIM2). Interleukin-6 induced signal transducer and activator of transcription 3 (STAT3)-mediated transcription of miR214. A miR214 chemical inhibitor blocked this circuit and reduced the severity of DSS-induced colitis in mice, as well as the number and size of tumors that formed in mice given azoxymethane and DSS. In fresh colonic biopsy specimens from patients with active UC, the miR214 inhibitor reduced inflammation by increasing levels of PDLIM2 and PTEN.

Conclusions

Interleukin-6 up-regulates STAT3-mediated transcription of miR214 in colon tissues, which reduces levels of PDLIM2 and PTEN, increases phosphorylation of AKT, and activates nuclear factor-κB. The activity of this circuit correlates with disease activity in patients with UC and progression to colorectal cancer.

Crohn's disease and ulcerative colitis, the two common forms of inflammatory bowel disease (IBD), affect over 2.5 million people of European ancestry, with rising prevalence in other populations. Genome-wide association studies and subsequent meta-analyses of these two diseases as separate phenotypes have implicated previously unsuspected mechanisms, such as autophagy, in their pathogenesis and showed that some IBD loci are shared with other inflammatory diseases. Here we expand on the knowledge of relevant pathways by undertaking a meta-analysis of Crohn's disease and ulcerative colitis genome-wide association scans, followed by extensive validation of significant findings, with a combined total of more than 75,000 cases and controls. We identify 71 new associations, for a total of 163 IBD loci, that meet genome-wide significance thresholds. Most loci contribute to both phenotypes, and both directional (consistently favouring one allele over the course of human history) and balancing (favouring the retention of both alleles within populations) selection effects are evident. Many IBD loci are also implicated in other immune-mediated disorders, most notably with ankylosing spondylitis and psoriasis. We also observe considerable overlap between susceptibility loci for IBD and mycobacterial infection. Gene co-expression network analysis emphasizes this relationship, with pathways shared between host responses to mycobacteria and those predisposing to IBD.

Genome-wide association studies and candidate gene studies in ulcerative colitis have identified 18 susceptibility loci. We conducted a meta-analysis of six ulcerative colitis genome-wide association study datasets, comprising 6,687 cases and 19,718 controls, and followed up the top association signals in 9,628 cases and 12,917 controls. We identified 29 additional risk loci (P < 5 × 10(-8)), increasing the number of ulcerative colitis-associated loci to 47. After annotating associated regions using GRAIL, expression quantitative trait loci data and correlations with non-synonymous SNPs, we identified many candidate genes that provide potentially important insights into disease pathogenesis, including IL1R2, IL8RA-IL8RB, IL7R, IL12B, DAP, PRDM1, JAK2, IRF5, GNA12 and LSP1. The total number of confirmed inflammatory bowel disease risk loci is now 99, including a minimum of 28 shared association signals between Crohn's disease and ulcerative colitis.