Colonic Mucosa Samples Research Articles

Novel specific microRNA biomarkers in idiopathic inflammatory bowel disease unrelated to disease activity

The diagnosis of idiopathic inflammatory bowel disease can be challenging. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate protein synthesis through post-transcriptional suppression. This study is to identify new miRNA markers in inflammatory bowel disease, and to examine whether miRNA biomarkers might assist in the diagnosis of inflammatory bowel disease. Illumina small RNA sequencing was performed on non-dysplastic fresh-frozen colonic mucosa samples of the distalmost colectomy tissue from 19 patients with inflammatory bowel disease (10 ulcerative colitis and 9 Crohn disease) and 18 patients with diverticular disease serving as controls. To determine differentially expressed miRNAs, the USeq software package identified 44 miRNAs with altered expression (fold change ≥2 and false discovery rate ≤0.10) compared with the controls. Among them, a panel of nine miRNAs was aberrantly expressed in both ulcerative colitis and Crohn disease. Validation assays performed using quantitative reverse transcription PCR (qRT-PCR) on additional frozen tissue from ulcerative colitis, Crohn disease, and control groups confirmed specific differential expression in inflammatory bowel disease for miR-31, miR-206, miR-424, and miR-146a (P<0.05). The expression of these four miRNAs was further evaluated on formalin-fixed, paraffin-embedded tissue of the distalmost colectomy mucosa from cohorts of diverticular disease controls (n=29), ulcerative colitis (n=36), Crohn disease (n=26), and the other diseases mimicking inflammatory bowel disease including infectious colitis (n=12) and chronic ischemic colitis (n=19), again confirming increased expression specific to inflammatory bowel disease (P<0.05). In summary, we demonstrate that miR-31, miR-206, miR-424, and miR-146a are novel specific biomarkers of inflammatory bowel disease. Furthermore, miR-31 is universally expressed in both ulcerative colitis and Crohn disease not only in fresh-frozen but also in formalin-fixed, paraffin-embedded tissues.

The Expression and Prognostic Significance of Retinoic Acid Metabolising Enzymes in Colorectal Cancer

Colorectal cancer is one of the most common types of cancer with over fifty percent of patients presenting at an advanced stage. Retinoic acid is a metabolite of vitamin A and is essential for normal cell growth and aberrant retinoic acid metabolism is implicated in tumourigenesis. This study has profiled the expression of retinoic acid metabolising enzymes using a well characterised colorectal cancer tissue microarray containing 650 primary colorectal cancers, 285 lymph node metastasis and 50 normal colonic mucosal samples. Immunohistochemistry was performed on the tissue microarray using monoclonal antibodies which we have developed to the retinoic acid metabolising enzymes CYP26A1, CYP26B1, CYP26C1 and lecithin retinol acyl transferase (LRAT) using a semi-quantitative scoring scheme to assess expression. Moderate or strong expression of CYP26A1was observed in 32.5% of cancers compared to 10% of normal colonic epithelium samples (p<0.001). CYP26B1 was moderately or strongly expressed in 25.2% of tumours and was significantly less expressed in normal colonic epithelium (p<0.001). CYP26C1 was not expressed in any sample. LRAT also showed significantly increased expression in primary colorectal cancers compared with normal colonic epithelium (p<0.001). Strong CYP26B1 expression was significantly associated with poor prognosis (HR = 1.239, 95%CI = 1.104–1.390, χ2 = 15.063, p = 0.002). Strong LRAT was also associated with poorer outcome (HR = 1.321, 95%CI = 1.034–1.688, χ2 = 5.039, p = 0.025). In mismatch repair proficient tumours strong CYP26B1 (HR = 1.330, 95%CI = 1.173–1.509, χ2 = 21.493, p<0.001) and strong LRAT (HR = 1.464, 95%CI = 1.110–1.930, χ2 = 7.425, p = 0.006) were also associated with poorer prognosis. This study has shown that the retinoic acid metabolising enzymes CYP26A1, CYP26B1 and LRAT are significantly overexpressed in colorectal cancer and that CYP26B1 and LRAT are significantly associated with prognosis both in the total cohort and in those tumours which are mismatch repair proficient. CYP26B1 was independently prognostic in a multivariate model both in the whole patient cohort (HR = 1.177, 95%CI = 1.020–1.216, p = 0.026) and in mismatch repair proficient tumours (HR = 1.255, 95%CI = 1.073–1.467, p = 0.004).

High Frequency of Fusion Transcripts Involving TCF7L2 in Colorectal Cancer: Novel Fusion Partner and Splice Variants

VTI1A-TCF7L2 was reported as a recurrent fusion gene in colorectal cancer (CRC), found to be expressed in three out of 97 primary cancers, and one cell line, NCI-H508, where a genomic deletion joins the two genes [1]. To investigate this fusion further, we analyzed high-throughput DNA and RNA sequencing data from seven CRC cell lines, and identified the gene RP11-57H14.3 (ENSG00000225292) as a novel fusion partner for TCF7L2. The fusion was discovered from both genome and transcriptome data in the HCT116 cell line. By triplicate nested RT-PCR, we tested both the novel fusion transcript and VTI1A-TCF7L2 for expression in a series of 106 CRC tissues, 21 CRC cell lines, 14 normal colonic mucosa, and 20 normal tissues from miscellaneous anatomical sites. Altogether, 42% and 45% of the CRC samples expressed VTI1A-TCF7L2 and TCF7L2-RP11-57H14.3 fusion transcripts, respectively. The fusion transcripts were both seen in 29% of the normal colonic mucosa samples, and in 25% and 75% of the tested normal tissues from other organs, revealing that the TCF7L2 fusion transcripts are neither specific to cancer nor to the colon and rectum. Seven different splice variants were detected for the VTI1A-TCF7L2 fusion, of which three are novel. Four different splice variants were detected for the TCF7L2-RP11-57H14.3 fusion. In conclusion, we have identified novel variants of VTI1A-TCF7L2 fusion transcripts, including a novel fusion partner gene, RP11-57H14.3, and demonstrated detectable levels in a large fraction of CRC samples, as well as in normal colonic mucosa and other tissue types. We suggest that the fusion transcripts observed in a high frequency of samples are transcription induced chimeras that are expressed at low levels in most samples. The similar fusion transcripts induced by genomic rearrangements observed in individual cancer cell lines may yet have oncogenic potential as suggested in the original study by Bass et al.

P227 Evaluation of a method to detect cytomegalovirus-DNA in intestinal fluid samples of patients with ulcerative colitis

Background: A severe flare and refractory against steroid in patients with ulcerative colitis (UC) may be complicated by underlying cytomegalovirus (CMV) infection. Pathological detection of the cells infected by CMV in colonic mucosal biopsies is golden standard in diagnosis, however; pathological diagnosis has certain false negative cases. Although CMV antigenemia and polymerase chain reaction (PCR) for detecting CMV in mucosal samples have been widely applied, PCR for CMV in intestinal fluids has not been evaluated. The aim of the study was to investigate the usefulness of PCR for CMV in intestinal fluids of patients with UC. Methods: From 2012 to 2013, 36 patients (16 men, 20 women) with UC were enrolled in this study. Intestinal fluid samples and biopsies were collected during colonoscopy. We evaluated clinical characteristics, CMV antigenemia, real-time PCR for CMV in intestinal fluids and colonic mucosal biopsies, and histopathological examination of colonic mucosal biopsies. Results: Twenty-three of 53 intestinal fluid samples (43%) had detectable CMV DNA, and five of 28 colonic mucosal samples (18%) were positive for CMV DNA. In both examinations, the positive rates of CMV DNA were higher in patients with colonic deep ulcer than in patients without deep ulcer. All 5 cases with positive CMV DNA in mucosal biopsies had all detectable CMV DNA in intestinal fluids, whereas 8 cases with detectable CMV DNA in intestinal fluid samples had negative CMV DNA in mucosal biopsies. In these 8 cases, 7 cases were refractory to steroid, and two patients underwent colectomy. The average of amount of CMV DNA in intestinal fluid samples positive/ mucosal biopsies negative case was 14.3 copies, whereas the other average which is both positive case was 56.9 copies. Conclusions: PCR for detecting CMV in intestinal fluids is useful and easy for diagnosis of CMV infection in patients with UC. This method has a higher sensitivity compared with mucosal biopsies. Most cases of negative results in pathology and detected by intestinal fluids were refractory cases to steroid. This method enables prompt and appropriate treatment.

The expression of STAT3, BCL-XL and MMP-2 proteins in colon adenocarcinomas and their relationship with prognostic factors.

Colon adenocarcinomas are the most common malignancy of the gastrointestinal tract. Many factors are described in the prognosis. Herein we aimed to determine the relationship between signal transducer and activator of transcription 3(STAT3), B-cell lymphoma-extra large (Bcl-xL) and matrix metalloproteinase-2 (MMP-2) expression in colon adenocarcinomas and their relationship with prognostic factors. A total of 45 colon adenocarcinomas (15 well, 15 moderately, 15 poorly differentiated) and 15 normal colon mucosa samples were selected randomly. The STAT3, Bcl-xL and MMP-2 immunohistochemicals were applied to the samples. There was a significant difference between the normal mucosa and carcinoma group regarding the STAT3 staining diffuseness and intensity (p < 0.05), but not for Bcl-xL (p > 0.05). The intensity of MMP-2 significantly differed between the normal mucosa and the carcinoma group (p < 0.05). The diffuseness of STAT3 and the intensity of MMP2 showed a significant association with the degree of differentiation (p < 0.05). The intensity and diffuseness of BcL-xL did not show a relationship with the degree of differentiation (p > 0.05). Evaluation of the diffuseness and intensity of the three markers with lymph node metastasis and tumor size revealed that only MMP2 diffuseness had a negative correlation with tumor size (r= -0.318). We observed that Bcl-xL does not have a role in the diagnosis of colon adenocarcinomas or the detection of the degree of differentiation, a prognostic factor, but such a relationship was observed for STAT3 and MMP-2.

Common Fusion Transcripts Identified in Colorectal Cancer Cell Lines by High-Throughput RNA Sequencing

Colorectal cancer (CRC) is the third most common cancer disease in the Western world, and about 40% of the patients die from this disease. The cancer cells are commonly genetically unstable, but only a few low-frequency recurrent fusion genes have so far been reported for this disease. In this study, we present a thorough search for novel fusion transcripts in CRC using high-throughput RNA sequencing. From altogether 220 million paired-end sequence reads from seven CRC cell lines, we identified 3391 candidate fused transcripts. By stringent requirements, we nominated 11 candidate fusion transcripts for further experimental validation, of which 10 were positive by reverse transcription-polymerase chain reaction and Sanger sequencing. Six were intrachromosomal fusion transcripts, and interestingly, three of these, AKAP13-PDE8A, COMMD10-AP3S1, and CTB-35F21.1-PSD2, were present in, respectively, 18, 18, and 20 of 21 analyzed cell lines and in, respectively, 18, 61, and 48 (17%-58%) of 106 primary cancer tissues. These three fusion transcripts were also detected in 2 to 4 of 14 normal colonic mucosa samples (14%–28%). Whole-genome sequencing identified a specific genomic breakpoint in COMMD10-AP3S1 and further indicates that both the COMMD10-AP3S1 and AKAP13-PDE8A fusion transcripts are due to genomic duplications in specific cell lines. In conclusion, we have identified AKAP13-PDE8A, COMMD10-AP3S1, and CTB-35F21.1-PSD2 as novel intrachromosomal fusion transcripts and the most highly recurring chimeric transcripts described for CRC to date. The functional and clinical relevance of these chimeric RNA molecules remains to be elucidated.

Thymosin ß4 expression in colorectal polyps and adenomas

OBJECTIVE:Thymosin beta 4 (Tβ4) is a ubiquitous peptide that plays pivotal roles in the cytoskeletal system and in cell differentiation. Recently, a role for Tβ4 has been proposed in experimental and human carcinogenesis, including gastrointestinal cancer. This study was aimed at evaluating the relationship between Tβ4 immunoreactivity and the initial steps of carcinogenesis.METHODS:In total, 60 intestinal biopsies, including 10 hyperplastic polyps, 10 sessile serrated adenomas/polyps, 15 colorectal adenomas with low-grade dysplasia, 15 adenomas with high-grade dysplasia, 15 adenocarcinomas and 10 samples of normal colon mucosa, were analyzed for Tβ4 expression by immunohistochemistry.RESULTS:Weak cytoplasmic reactivity for Tβ4 was detected in the normal colon mucosa. No reactivity for Tβ4 was found in hyperplastic and sessile serrated polyps/adenomas. Tβ4 expression was observed in 10/15 colorectal adenocarcinomas. In adenomas with low-grade dysplasia, Tβ4 immunoreactivity was mainly detected in dysplastic glands but was absent in hyperplastic glands. Tβ4 immunoreactivity was characterized by spot-like perinuclear staining. In high-grade dysplastic polyps, immunostaining for Tβ4 appeared diffuse throughout the entire cytoplasm of dysplastic cells. Spot-like perinuclear reactivity was detected in adenocarcinoma tumor cells.CONCLUSIONS:Our study shows for the first time that Tβ4 is expressed during different steps of colon carcinogenesis. The shift of Tβ4 immunolocalization from low-grade to high-grade dysplastic glands suggests a role for Tβ4 in colorectal carcinogenesis. However, the real meaning of Tβ4 reactivity in dysplastic intestinal epithelium remains unknown.

Impact of microRNAs on regulatory networks and pathways in human colorectal carcinogenesis and development of metastasis

BackgroundQualitative alterations or abnormal expression of microRNAs (miRNAs) in colon cancer have mainly been demonstrated in primary tumors. Poorly overlapping sets of oncomiRs, tumor suppressor miRNAs and metastamiRs have been linked with distinct stages in the progression of colorectal cancer. To identify changes in both miRNA and gene expression levels among normal colon mucosa, primary tumor and liver metastasis samples, and to classify miRNAs into functional networks, in this work miRNA and gene expression profiles in 158 samples from 46 patients were analysed.ResultsMost changes in miRNA and gene expression levels had already manifested in the primary tumors while these levels were almost stably maintained in the subsequent primary tumor-to-metastasis transition. In addition, comparing normal tissue, tumor and metastasis, we did not observe general impairment or any rise in miRNA biogenesis. While only few mRNAs were found to be differentially expressed between primary colorectal carcinoma and liver metastases, miRNA expression profiles can classify primary tumors and metastases well, including differential expression of miR-10b, miR-210 and miR-708. Of 82 miRNAs that were modulated during tumor progression, 22 were involved in EMT. qRT-PCR confirmed the down-regulation of miR-150 and miR-10b in both primary tumor and metastasis compared to normal mucosa and of miR-146a in metastases compared to primary tumor. The upregulation of miR-201 in metastasis compared both with normal and primary tumour was also confirmed. A preliminary survival analysis considering differentially expressed miRNAs suggested a possible link between miR-10b expression in metastasis and patient survival. By integrating miRNA and target gene expression data, we identified a combination of interconnected miRNAs, which are organized into sub-networks, including several regulatory relationships with differentially expressed genes. Key regulatory interactions were validated experimentally. Specific mixed circuits involving miRNAs and transcription factors were identified and deserve further investigation. The suppressor activity of miR-182 on ENTPD5 gene was identified for the first time and confirmed in an independent set of samples.ConclusionsUsing a large dataset of CRC miRNA and gene expression profiles, we describe the interplay of miRNA groups in regulating gene expression, which in turn affects modulated pathways that are important for tumor development.

Genetic variants of membrane metallopeptidase genes in inflammatory bowel diseases

The substance P pathway modulates neuroimmune interactions during intestinal inflammation. To analyse mucosal expression and genetic variants of the genes coding for substance P, neurokinin-1 receptor and neutral endopeptidase in patients with inflammatory bowel disease. qRT-PCR was used to analyse mRNA levels in matched, paired samples of inflamed colonic mucosa and adjacent non-inflamed endoscopic tissue from 26 Crohn's disease and 25 ulcerative colitis patients. Allele and genotype frequencies of tag-SNPs were determined in 908 Crohn's disease, 929 ulcerative colitis, and 853 controls. Expression levels and genotype distributions were examined within patients' clinical sub-phenotypes. All 3 evaluated genes were overexpressed in inflamed tissues from Crohn's disease (P=0.033, P=4×10(-5), P=0.001), while in ulcerative colitis only higher levels of the gene coding for neutral endopeptidase were statistically significant (P=2.5×10(-5)). Smoking habit and perianal disease were significantly associated with substance P (P=0.002) and neurokinin-1 receptor levels (P=0.02) in Crohn's disease. Neutral endopeptidase rs701109 variant was associated with inflammatory bowel disease (Crohn's disease: P=0.022; ulcerative colitis: P=0.045), and with the need for colectomy in ulcerative colitis (P=0.008, OR=2.46, 95% CI=1.27-4.76). Genetic variants of the gene coding for neutral endopeptidase might affect the neuroimmune interaction during intestinal inflammation and influence clinical sub-phenotypes in patients with inflammatory bowel disease.

Hypermethylation of the KEAP1 gene in colorectal cancer and association with disease progression.

e14655 Background: Aberrant methylation of the KEAP1 (Kelch-like ECH-associated protein 1) gene promoter is emerging as a main mechanism of dysregulation of the NrF2 (nuclear factor-erythroid 2-related factor 2 ) which plays a pivotal role in the cellular response to oxidative stress. Under basal conditions, Nrf2 is retained in the cytoplasm by the binding with Keap1 and it is maintained at a reduced level by the Keap1-dependent ubiquitination and proteasomal degradation systems. Reduced Keap1 expression by promoter aberrant methylation allows Nrf2 to translocate in the nucleus and to activate the detoxification pathway leading ultimately to drug resistance. Methods: We determined KEAP1 promoter methylation status in 50 metastatic colorectal cancer (CRC) and 9 normal colonic mucosa samples by using quantitative methylation specific PCR in real time (QMSP). The median age of the patients cohort was 65 (IQR 57-74) years. At diagnosis 12 patients were staged Duke D and 38 were staged as Duke C. As first line treatment the FOLFOX or FOLFIRI therapeutical schemes were used. Results: Methylation was detected in 20 out of 50 CRC (40%) and methylation levels were significantly higher in tumour samples (Median 0.65, IQR 0-8.28) as compared with normal colonic mucosa (Median 0, IQR 0-0.08) (P=0.03). Tumours from patients who experieced disease progression had significantly higher methylation levels (Median 0.53; IQR 0-7.61) as compared with patients showing partial response (Median 0, IQR 0-2.21) or disease stability (Median 0, IQR 0-0) (P=0.03 Kruskall Wallis Test). Conclusions: Our results suggest that Keap1 aberrant methylation is a frequent event in colorectal cancer and is associated with response to chemotherapeutical treatments.

Inhibition of arginine ADP-ribosyltransferase 1 reduces the expression of poly(ADP-ribose) polymerase-1 in colon carcinoma

Poly(ADP-ribose) polymerase-1 (PARP-1) which mediates poly-ADP-ribosylation, has been extensively investigated in carcinoma compared to arginine ADP-ribosyltransferase 1 (ART1), which mediates mono‑ADP‑ribosylation. Previous studies have demonstrated that these enzymes promote proliferation and tumor development in colon carcinoma. However, whether there is any association between PARP-1 and ATR1 in colon carcinoma, remains unelucidated. In this study, using immunohistochemical analysis, we detected a higher expression of PARP-1 and ART1 in 63 samples from patients with colon carcinoma compared to 10 samples of normal colonic mucosa; our results revealed a positive correlation between the expression of PARP-1 and ART1 in the 63 human colon carcinoma tissue samples. To determine the correlation between PARP-1 and ART1, inhibitors of PARP-1 and ART1 and lentivirus vector‑mediated ART1 short‑hairpin RNA (shRNA) were used to culture CT26 murine colon adenocarcinoma cells separately. Using double‑label immunofluorescence assay, we detected the expression of PARP-1 in the CT26 cells, which was decreased following treatment with 5‑aminoisoquinolinone (5-AIQ, a PARP-1 inhibitor) or meta‑iodobenzylguanidine (MIBG, an ART1 inhibitor). However, the expression of ART1 only decreased when the CT26 cells were treated with MIBG. Furthermore, our results demonstrated that silencing ART1 inhibited PARP‑1 expression by decreasing the expression of nuclear factor-κB (NF-κB), inhibiting ras homolog A (RhoA). Hence, our data demonstrate the positive correlation between ART1 and PARP-1; the inhibition of ART1 activity downregulates PARP-1 expression by decreasing the activity of NF-κB in CT26 colon carcinoma cells.

Alterations in mucosal neuropeptides in patients with irritable bowel syndrome and ulcerative colitis in remission: A role in pain symptom generation?

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by chronic abdominal pain. The transient receptor potential vanilloid 1 (TRPV1) channel, which is involved in visceral pain signalling, has been shown to be up-regulated in IBS. Activation of TRPV1 leads to the release of neuropeptides, such as somatostatin and substance P (SP). We hypothesized that increased pain perception in IBS could be explained by increased transcription in TRPV1 and/or altered levels of neuropeptides. We therefore assessed the transcription of TRPV1 and the mucosal concentration of somatostatin and SP in IBS in comparison to healthy volunteers and patients with ulcerative colitis (UC) in remission as disease controls, and to ascertain their relationship to pain symptoms. Sigmoid colonic mucosal samples were collected from 12 patients with IBS, 34 patients with UC in remission and 9 healthy volunteers, in which groups TRPV1 mRNA levels were determined using quantitative polymerase chain reaction and neuropeptide concentrations by radioimmunoassay. Pain symptom intensity was determined by questionnaires. Transcription of TRPV1 as well as the concentration of neuropeptides were significantly higher in IBS, but only the former correlated with pain symptom severity. Increased transcription of TRPV1 may provide a possible explanation for pain generation in IBS. While the neuropeptides SP and somatostatin were both found to be increased in IBS, these changes are not sufficient to explain pain generation. Pain generation in IBS is probably explained by a complex redundancy in the regulation of local nociceptive mechanisms, which remains a subject of intensive investigation.

Normal colon tissue and colon carcinoma show no difference in heparanase promoter methylation

IntroductionHeparanase, the sole heparan sulfate degrading enzyme, has a role in cellular invasion. Accordingly, a large number of studies have demonstrated an association between heparanase expression and tumor stage and patients' prognosis. In colon carcinoma, heparanase shows increased expression in tumor compared to normal tissue and its expression correlates with the presence of metastasis. One of the regulatory mechanisms of heparanase expression is de-methylation on its promoter. In the present study we evaluated the role of heparanase promoter methylation in colon carcinoma. Material and methodsAnalysis of heparanase promoter methylation was done on 32 samples of colon carcinoma as well as 30 samples of normal colonic mucosa. DNA was extracted from FFPE tissue and subjected to bisulfite conversion. The relative fraction of methylated and unmethylated DNA was evaluated using quantitative real-time PCR. ResultsThe fraction of methylated DNA was 1±3.4% in the colon carcinoma group, and 2.5±3.3% in the normal colon group (P=0.11). Only one case in the normal group and one case in the tumor group showed more than 10% methylation in the heparanase promoter. ConclusionWe did not find any significant difference in heparanase promoter methylation between colon carcinoma and normal colonic mucosa, suggesting that heparanase overexpression in colon carcinoma is mediated by other mechanisms.

Expression Profiling of Proliferation and Apoptotic Markers along the Adenoma-Carcinoma Sequence in Familial Adenomatous Polyposis Patients

Introduction. Familial adenomatous polyposis (FAP) patients have a germline mutation in the adenomatous polyposis coli (APC) gene. The APC protein interacts with beta-catenin, resulting in the activation of the Wnt signalling pathway. This results in alterations in cell proliferation and apoptosis. We investigated the expression of beta-catenin and related proliferation and apoptotic factors in FAP patients, exploring the expression along the adenoma-carcinoma sequence. Methods. The expression of beta-catenin, p53, bcl-2, cyclin-D1, caspase-3, CD10, and Ki-67 proteins was studied by immunohistochemistry in samples of colonic nonneoplastic mucosa (n = 71), adenomas (n = 152), and adenocarcinomas (n = 19) from each of the16 FAP patients. Results. The expression of beta-catenin, caspase-3, cyclin-D1, and Ki-67 was increased in both adenomas and carcinomas in FAP patients, compared with normal mucosa. p53 and CD10 expression was only slightly increased in adenomas, but more frequently expressed in carcinomas. Bcl-2 expression was increased in adenomas, but decreased in carcinomas. Conclusion. This is the first study investigating collectively the expression of these molecules together in nonneoplastic mucosa, adenomas, and carcinomas from FAP patients. We find that beta-catenin and related proliferative and apoptotic factors (cyclin-D1, bcl-2, caspase-3, and Ki-67) are expressed early in the sequence, in adenomas. However, p53 and CD10 are often expressed later in the sequence, in carcinomas.

Prevention of Familial Adenomatous Polyp Development in APCmin Mice and Azoxymethane-Induced Colon Carcinogenesis in F344 Rats by ω-3 Fatty Acid Rich Perilla Oil

The present study explored the preventive effects of perilla oil, rich in α-linolenic acid, in rodent models of colon tumorigenesis. Six-week-old male F344 rats were fed diets containing 5% corn oil or 10 or 20% perilla oil. Colonic aberrant crypt foci (ACF) were induced by azoxymethane (AOM) and colonic ACF were evaluated. In familial adenomatous polyposis mode, APCmin mice fed with 20% corn oil or perilla oil for 80 days and intestines were evaluated for polyps. Multiple colonic mucosal and polyp samples were assayed for the expression and activity of cyclooxygenase COX-isoforms. Dietary perilla oil produced a dose-dependent inhibition of AOM-induced colonic ACF formation (by 35–53%, P < 0.01–0.005) and reduced the number of foci with ≥4 crypts/focus (by 38–50%, P < 0.01–0.001) in F344 rats. Dietary perilla oil significantly inhibited development of small intestinal (>69%, P < 0.0001) and colon tumors (>52%, P < 0.03) in APCmin mice. Administration of perilla oil produced lower levels of type-2 prostaglandins (38–53%) from COX-activities in polyps of APCmin mice. These observations demonstrate that dietary perilla oil rich in ω-3 fatty acids possesses preventive activity against intestinal neoplastic lesions, both in FAP in genetically-predisposed tissues, as well as against chemically induced preneoplastic lesions in the colon.

Neurotensin receptor 1 overexpression in inflammatory bowel diseases and colitis-associated neoplasia

To explore the association of neurotensin receptor 1 (NTSR1) with inflammatory bowel diseases (IBD) and colitis-associated neoplasia. NTSR1 was detected by immunohistochemistry in clinical samples of colonic mucosa with IBD colitis, colitis-associated raised low-grade dysplasia (LGD) including dysplasia-associated lesions or masses (DALMs, n = 18) and adenoma-like dysplastic polyps (ALDPs, n = 4), colitis-associated high-grade dysplasia (HGD, n = 11) and colitis-associated colorectal carcinoma (CACRC, n = 13), sporadic colorectal adenomatous polyp (SAP, n = 17), and sporadic colorectal carcinoma (SCRC, n = 12). The immunoreactivity of NTSR1 was semiquantitated (as negative, 1+, 2+, and 3+) and compared among different conditions. NTSR1 was not detected in normal mucosa but was expressed similarly in both active and inactive colitis. LGD showed a significantly stronger expression as compared with non-dysplastic colitic mucosa, with significantly more cases showing > 2+ intensity (68.75% in LGD vs 32.26% in nondysplastic mucosa, P = 0.001). However, no significant difference existed between DALMs and ALDPs. CACRC and HGD showed a further stronger expression, with significantly more cases showing 3+ intensity than that in LGD (61.54% vs 12.50% for CACRC vs LGD, P = 0.022; 58.33% vs 12.50% for CACRC/HGD vs LGD, P = 0.015). No significant difference existed between colitis-associated and non-colitic sporadic neoplasia. NTSR1 in colonic epithelial cells is overexpressed in IBD, in a stepwise fashion with sequential progress from inflammation to dysplasia and carcinoma.

Diagnostic utility of MS-MLPA in DNA methylation profiling of adenocarcinomas and neuroendocrine carcinomas of the colon–rectum

Methylation-specific multiple ligation-dependent probe amplification (MS-MLPA) is a fast, new, inexpensive method that has rarely been exploited in DNA methylation profiling of colorectal cancers (CRCs). The aim of this study was to test the diagnostic utility of MS-MLPA to evaluate the methylation status of 34 genes in normal colonic mucosa samples and in a well-characterized series of 83 adenocarcinomas and 21 neuroendocrine carcinomas of colon-rectum. Two commercial MS-MLPA kits (SALSA MS-MLPA ME001-C1 Tumor suppressor-1 Kit and SALSA MS-MLPA ME002-B1 Tumor suppressor-2 Kit) were used to perform promoter methylation analysis on formalin-fixed and paraffin-embedded tissues. MS-MLPA analysis was validated by bisulfite pyrosequencing, bisulfite cycle sequencing, and methylation-specific PCR. MS-MLPA analysis identified a subset of 27 CRCs (26% of cases) showing high levels of gene methylation involving a mean percentage of 34% of the promoters examined. These tumors exhibited all the main clinicopathological and genetic features described for CRCs with CpG island Methylator Phenotype-High. High levels of methylation were observed with similar frequency in adenocarcinomas and in neuroendocrine carcinomas (25% versus 29%, respectively), but different methylation profiles were observed in the two tumor types. In both groups, tumors with microsatellite instability and widespread methylation represented a homogeneous clinicopathological entity. MS-MLPA assay is an easy and reliable system for epigenetic characterization of tumor tissues and leads to a rapid identification of CRCs with the highest levels of gene methylation. Aberrant gene methylation is a common abnormality in CRC initiation and may be observed in tumors with very different genetic and clinicopathological profiles.

Chicken Skin Mucosa (CSM): An Endoscopic Marker of Colonic Intestinal Metaplasia

Purpose: An endoscopic finding of pale yellow-speckled mucosa adjacent to both benign and colonic neoplasms has been previously reported as “chicken skin mucosa” (CSM). This case report is unique in that the patient's colonic mucosa had this unusual “chicken skin” finding covering half of the entire colonic mucosa; not just in the basal area near polyps as previously reported. A 63-year-old white male presented to the open access colonoscopy clinic for his first “screening colonoscopy.” Optical colonoscopy revealed colonic mucosa with contiguous non-elevated pale yellow speckles beginning abruptly in the mid transverse colon and extending into the cecum. These pale- yellow speckles were consistently 1-2 mm in diameter and were evenly distributed and circumferential on the background of a pinkish-red colonic mucosa. The typical mucosal colonic vascular pattern remained intact. The visual impression was one of looking at “chicken skin.” Narrow band imaging accentuated the contrast of these yellow speckles against the darker background colonic mucosa. Numerous sessile polyps were found in this patient only in this “chicken-skin” area of colonic mucosa, the remainder of the colonic mucosa appeared normal and contained no polyps. The polyps within the CSM were free of this yellow-speckled pattern and visually were quite distinct from the surrounding CSM. All of the polyps were tubular adenomas with no high grade dysplasia. Samples of colonic mucosa taken from the surrounding “chicken skin appearing” mucosa revealed mild hyperplastic changes with pockets (the yellow plaques seen endoscopically) of numerous lipid-filled macrophages contained within the lamina propria. Chicken skin mucosa is a xanthelasma-like endoscopic finding of pale yellow-speckled mucosa adjacent to colonic neoplasms described in adults and children. CSM gets its endoscopic appearance from lipid-laden macrophages in the colonic lamina propria. Previously CSM has been reported to be seen only within a few centimeters of the base of colonic polyps. This case is unique in that this individual CSM covered at least half of the colonic mucosa. Tubular adenomas occurred only in this half of the colon. The visual appearance of CSM may alert the endoscopist to the presence of nearby metaplasia as has been previously noted or as in this case, be more extensive. This may be a visual clue to help lower the 5% miss rate for colonic neoplasms.

A High Degree of LINE-1 Hypomethylation Is a Unique Feature of Early-Onset Colorectal Cancer

ObjectiveEarly-onset colorectal cancer (CRC) represents a clinically distinct form of CRC that is often associated with a poor prognosis. Methylation levels of genomic repeats such as LINE-1 elements have been recognized as independent factors for increased cancer-related mortality. The methylation status of LINE-1 elements in early-onset CRC has not been analyzed previously.DesignWe analyzed 343 CRC tissues and 32 normal colonic mucosa samples, including 2 independent cohorts of CRC diagnosed ≤50 years old (n = 188), a group of sporadic CRC >50 years (MSS n = 89; MSI n = 46), and a group of Lynch syndrome CRCs (n = 20). Tumor mismatch repair protein expression, microsatellite instability status, LINE-1 and MLH1 methylation, somatic BRAF V600E mutation, and germline MUTYH mutations were evaluated.ResultsMean LINE-1 methylation levels (±SD) in the five study groups were early-onset CRC, 56.6% (8.6); sporadic MSI, 67.1% (5.5); sporadic MSS, 65.1% (6.3); Lynch syndrome, 66.3% (4.5) and normal mucosa, 76.5% (1.5). Early-onset CRC had significantly lower LINE-1 methylation than any other group (p<0.0001). Compared to patients with <65% LINE-1 methylation in tumors, those with ≥65% LINE-1 methylation had significantly better overall survival (p = 0.026, log rank test).ConclusionsLINE-1 hypomethylation constitutes a potentially important feature of early-onset CRC, and suggests a distinct molecular subtype. Further studies are needed to assess the potential of LINE-1 methylation status as a prognostic biomarker for young people with CRC.

The Endoplasmic Reticulum Stress Response Activates Tissue Transglutaminase and Intestinal Inflammation by Reducing Levels of IκBα

Background & AimsDefects in resolution of the endoplasmic reticulum (ER) stress response contribute to inflammation and might be involved in pathogenesis of Crohn's disease (CD). We investigated the ability of lipopolysaccharide from Escherichia Coli (EC-LPS) to induce ER stress, and its effects on tissue transglutaminase (TG2), nuclear factor (NF)- κB, and its inhibitor (IκB) κ in cultured intestinal epithelial cells and colonic mucosa samples from patients with CD. MethodsWe investigated effects of EC-LPS on ER stress, calcium release, EC-LPS, and sumoylation and ubiquitination of TG2 in Caco-2 intestinal epithelial cells, cultured intestinal mucosa samples from patients with CD, and mucosal samples from individuals without CD (controls). Mucosa samples were incubated with or without anti-oxidants or the TG2 inhibitor L682777. Levels of TG2 were reduced in Caco-2 cells using small interfering RNAs. Tissues and cells were analyzed by immunoprecipitation, immunoblot, reverse transcriptase-PCR, confocal microscopy, and fluorescence resonance energy transfer microscopy analyses. ResultsEC-LPS induced ER stress in Caco-2 cells, leading to oxidative stress and sumoylation of TG2; this inhibited TG2 ubiquitination and degradation by the proteasome. TG2 induced cross-linking, ubiquitination, and proteasome degradation of IκBα, leading to activation of NF-κB and an inflammatory response in mucosal samples from patients with CD, determined by increased expression of COX-2 and ICAM-1 by lamina propria cells and mucosal expression of inflammatory cytokines such as tumor necrosis factor-α. Knockdown of TG2 in Caco-2 cells, or incubation of CD colon mucosa samples with L682777, increased levels of IkBα following administration of EC-LPS, preventing IκBα crosslinking and degradation. ConclusionInduction of ER stress with EC-LPS activates TG2 in intestinal epithelial cells and tissues from patients with CD. Inhibition of TG2 in colon mucosa from patients with CD reduces activation of NF-kB and the inflammatory response.