Koji Matsuzaki

The Relationship between Global Methylation Level, Loss of Heterozygosity, and Microsatellite Instability in Sporadic Colorectal Cancer

Purpose: The relationship between global hypomethylation, chromosomal instability (CIN), and microsatellite instability (MSI) remains unclear in colorectal cancer. The aim of this study was to investigate the relationship between global methylation status, loss of heterozygosity (LOH), and MSI in sporadic colorectal cancer. Experimental Design: We determined global methylation levels in 80 sporadic colorectal cancers, 51 adjacent normal tissues, and 20 normal tissues using the long interspersed nucleotide elements–combined bisulfite restriction analysis method. We also analyzed 80 colorectal cancers for MSI status and LOH at chromosomes 5q21, 8p12-22, 17p13, and 18q21. Results: We identified 14 cases of MSI (17.5%) and 58 cases of LOH (72.5%). LOH was observed more frequently in microsatellite stable (MSS) cancers than in MSI cancers at all loci. Colorectal cancers showed significantly lower global methylation levels than did normal tissues (41.0 ± 9.7% versus 54.3 ± 6.5%; P < 0.001). MSS cancers showed significantly lower global methylation levels when compared with MSI cancers (39.5 ± 9.4% versus 48.2 ± 8.2%; P = 0.003). Tumors with global hypomethylation (with ≤40% of methylation levels) had a significantly increased number of chromosomal loci with LOH than did tumors without global hypomethylation (1.9 versus 0.9; P < 0.001); 11 tumors (13.9%) lacked both MSI and LOH. This subgroup had significantly higher global methylation levels (46.8 ± 8.7%) than did MSS cancers with LOH (38.0 ± 9.0%; P = 0.006). Conclusions: These data showed a significant association between global hypomethylation and chromosomal instability in sporadic colorectal cancer. This suggests that global hypomethylation plays an important role in inducing genomic instability in colorectal carcinogenesis.

BRAF mutation, CpG island methylator phenotype and microsatellite instability occur more frequently and concordantly in mucinous than non-mucinous colorectal cancer.

Mucinous colorectal cancer (CRC) has been reported to have distinct clinicopathological and genetic characteristics. However, the incidence and the relationship among microsatellite instability (MSI), CpG island methylator phenotype (CIMP) and BRAF and KRAS mutations in mucinous and non‐mucinous CRC are not known. Activating mutations of BRAF and KRAS and their relationship with MSI and CIMP were examined in 83 sporadic CRC specimens (26 mucinous and 57 non‐mucinous CRC). MSI, CIMP, BRAF and KRAS mutation were observed in 17, 24, 25 and 36% of the tumors, respectively. BRAF mutation was highly correlated with MSI (p < 0.001) and CIMP (p < 0.001). A higher incidence of MSI (27% vs. 12%), CIMP (38% vs. 18%, p < 0.05) and BRAF mutation (46% vs. 16%, p < 0.01) was observed in mucinous CRC. KRAS mutation (27% vs. 40%) was observed more frequently in non‐mucinous CRC. Significantly higher percentages of mucinous CRC (54%, p < 0.05) had MSI or CIMP or BRAF mutations. Concordant occurrence of 2 or more of these alterations was observed in 39% of mucinous CRC and only 11% of non‐mucinous CRC (p < 0.01). The more frequent occurrence and closer association among MSI, CIMP and BRAF mutation in mucinous CRC observed in our study further supports the idea that its pathogenesis may involve distinct genetic and epigenetic changes.

Reduced sensitivity of inducible nitric oxide synthase-deficient mice to chronic colitis.

Abstract Background: Overproduction of nitric oxide by the inducible form of nitric oxide synthase (iNOS) has been implicated in colitis. Different authors have postulated both toxic and protective effects of nitric oxide (NO) in the pathophysiology of active inflammation. The objective of this study was to examine the role of iNOS in experimental chronic colitis using iNOS-deficient mice. Methods: For induction of colitis, mice received three cycles of 2% of dextran sodium sulfate (DSS) (M.W. 40,000) treatment in drinking water. The degree of colonic inflammation, leukocyte infiltration, and the expression of cell adhesion molecules were determined. INOS expression and nitrotyrosine were also determined by immunohistochemistry. Results: After DSS treatment, a moderate colitis with marked cell infiltration was observed. Intense expression of iNOS was observed on infiltrating cells as well as on the colonic mucosal epithelium in these animals. In the iNOS-deficient mice, tissue damage was significantly diminished. No iNOS or nitrotyrosine staining was found in iNOS-deficient mice. The number of infiltrating cells and the expression of mucosal adressin cell adhesion molecule-1 were significantly attenuated in the DSS-treated colon of iNOS-deficient mice. Conclusion: Induction of iNOS seems to act as a critical toxic effector molecule in the pathogenesis of chronic colonic inflammation.

Proximal and distal colorectal cancers show distinct gene-specific methylation profiles and clinical and molecular characteristics

INTRODUCTION Accumulation of genetic and epigenetic alterations contribute to malignant transformation of normal colonic mucosa to cancer. However, the frequency and the pattern of these alterations in proximal and distal colon cancers have not been examined in detail. METHODS In this study, we examined methylation frequencies and patterns using 14 marker genes in 31 proximal and 43 distal colorectal cancers. We also analysed the relationship between these parameters and clinical characteristics, MSI, mutations of BRAF, KRAS and p53, LOH and global hypomethylation. RESULTS Three groups of tumours with varying degrees of methylation frequencies were identified: very high (9%), high (22%) and low (69%) methylation. Tumours with very high and high methylation showed more frequent proximal location, MSI, BRAF mutations and less frequent LOH and global hypomethylation. The methylation markers could be classified into 3 types based on methylation frequencies, MSI status and location. Proximal tumours showed more frequent methylation of Type 2 markers, CIMP+, MSI, BRAF mutations and lower frequencies of LOH and global hypomethylation, whilst Type 3 marker, MGMT methylation was more frequently associated with distal tumours, better survival and G to A mutation in non-CpG sites in KRAS and p53 genes. CONCLUSION These data showed that proximal and distal colorectal cancers have distinct gene-specific methylation profiles and molecular and clinical characteristics.

Regional hypermethylation and global hypomethylation are associated with altered chromatin conformation and histone acetylation in colorectal cancer.

Regional DNA hypermethylation and global DNA hypomethylation are 2 epigenetic alterations associated with colorectal cancers. However, their correlation with microsatellite instability (MSI) and chromosomal instability (CIN) in colorectal cancer, and their relationship with chromatin conformation and histone modification are not clear. In this study, we analyzed regional and global methylation in 16 cell lines and 64 primary colorectal cancers. We found that MSI and CIN are 2 alternative events in most cell lines and tumors. Furthermore, regional hypermethylation and global hypomethylation are also alternative events in most cases. We also observed a strong correlation between MSI and regional hypermethylation and between CIN and global hypomethylation. We further analyzed chromatin conformation and histone acetylation in cell lines with CIN or MSI. CIN cancers had open chromatin conformation and enriched histone acetylation in repetitive as well as in gene‐specific regions. MSI cancers, on the other hand, had closed chromatin conformation and low levels of histone acetylation. After a MSI cell line was treated with 5‐aza‐2′‐deoxycytidine or trichostatin A, the closed chromatin conformation became open, and histone acetylation was enriched. These observations support our hypothesis that in colorectal cancer, regional hypermethylation and global hypomethylation are associated with altered chromatin conformation and histone acetylation, which might have a causal correlation with MSI and CIN, respectively.

Propionibacterium freudenreichii component 1.4‐dihydroxy‐2‐naphthoic acid (DHNA) attenuates dextran sodium sulphate induced colitis by modulation of bacterial flora and lymphocyte homing

Background and aim: 1.4-Dihydroxy-2-naphthoic acid (DHNA), a bifidogenic growth stimulator from Propionibacterium freudenreichii, is thought to have a beneficial effect as a prebiotic; however, its in vivo effect on intestinal inflammation remains unknown. The aim of this study was to determine whether oral administration of DHNA can ameliorate dextran sodium sulphate (DSS) induced colitis and to determine the possible underlying mechanisms. Method: Colitis was induced in mice by treatment with 2.0% DSS for seven days. DHNA (0.6 or 2.0 mg/kg) was given in drinking water prior to (preventive study) or after (therapeutic study) DSS administration. Colonic damage was histologically scored, and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expression and β7 positive cell infiltration were determined by immunohistochemistry. mRNA levels of proinflammatory cytokines (interleukin (IL)-1β, IL-6 and tumour necrosis factor α (TNF-α)) were determined by quantitative real time polymerase chain reaction. In addition, bacterial flora in the caecum, concentrations of short chain acids, and luminal pH were examined. Results: DHNA improved survival rate and histological damage score in mice administered DSS in both the preventive and therapeutic studies. DHNA significantly attenuated the enhanced expression of MAdCAM-1, the increased β7 positive cell number, and the increased mRNA levels of IL-1β, IL-6, and TNF-α in DSS treated colon. In addition, the decreased number of Lactobacillus and Enterobacteriaceae induced by DSS was recovered by DHNA. Preventive effects on decrease in butyrate concentration and decrease in pH level in mice administered DSS were also observed in the DHNA preventive study. Conclusion: DHNA, a novel type of prebiotic, attenuates colonic inflammation not only by balancing intestinal bacterial flora but also by suppressing lymphocyte infiltration through reduction of MAdCAM-1.

Blockade of PSGL-1 attenuates CD14+ monocytic cell recruitment in intestinal mucosa and ameliorates ileitis in SAMP1/Yit mice.

The pathogenesis of Crohn’s disease (CD) is not known. However, monocytes and macrophages are thought to play important roles in the development of mucosal inflammation. Therefore, in this study, we examined the role of monocyte‐endothelial cell interactions in senescence‐accelerated mouse P1 (SAMP1)/Yit mice, a murine model of spontaneous ileitis. Fluorescence‐labeled CD14+ monocytic cells isolated from the spleen and mesenteric lymph nodes of AKR/J (control) mice were injected into the tail veins of recipient (AKR/J and SAMP1/Yit) mice, and migration in the postcapillary venules (PCV) of Peyer’s patches, submucosal venules, and villus microvessels of the terminal ileum was monitored by using an intravital microscope. Rolling and adhesion of CD14+ monocytic cells in the PCV of Peyer’s patches and microvessels of the terminal ileum were increased in SAMP1/Yit mice. An imunohistochemical study showed increased expression of P‐selectin glycoprotein‐1 (PSGL‐1), P‐selectin, and vascular cell adhesion molecule‐1 in the terminal ileum of SAMP1/Yit mice. Antibodies against these three adhesion molecules significantly inhibited adhesion of CD14+ monocytic cells to the PCV of Peyer’s patches and microvessels of the terminal ileum, treatment with an anti‐PSGL‐1 monoclonal antibody (mAb) showing the strongest suppressive effect. Anti‐PSGL‐1 mAb also attenuated T cell adhesion in microvessels of intestinal mucosa. In addition, periodical administration of an anti‐PSGL‐1 mAb for 7 weeks significantly ameliorated ileitis of SAMP1/Yit mice. The results suggest that PSGL‐1‐P‐selectin interaction plays an important role in monocyte‐endothelial cell interactions and the development of ileitis in a murine model of CD and that the blockade of this adhesion molecule may be a novel strategy for treating CD.

Expression of bone morphogenetic proteins in colon carcinoma with heterotopic ossification.

Here we report the case of a 50‐year‐old woman with adenocarcinoma of the colon, showing heterotopic ossification. The patient was referred to our hospital for investigation of anemia secondary to occult gastrointestinal blood loss. By colonoscopy, an irregular polypoid mass was found in the ascending colon. A biopsy of the lesion revealed moderately to poorly differentiated adenocarcinoma with heterotopic ossification. A right hemicolectomy was done and revealed areas of heterotopic bone within the tumor, but no ossification was evident in the metastatic lesions within the mesenteric lymph nodes. The formation of heterotopic bone in gastrointestinal tumors is rare and its exact mechanism is unknown. Immunohistochemical localization of bone morphogenetic proteins (BMP), known to be primary inducers of new bone formation, was determined. BMP‐5 and ‐6 were prominent in the cytoplasm of tumor cells, and they stained weakly in osteoblast‐like cells adjacent to newly formed bone. Cytoplasmic staining for BMP‐2 and ‐4 was weak in tumor cells, osteoblast‐like cells, and stromal fibroblast cells. BMP may play an important role in heterotopic ossification in colon adenocarcinoma.

Involvement of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the pathogenesis of granulomatous colitis in rats

Although increased expression of mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1) has been demonstrated in inflammatory sites of various diseases, its role in colitis remains unknown. In this study, we examined whether MAdCAM‐1 is involved in the pathogenesis of granulomatous colitis induced by peptidoglycan‐polysaccharide (PG‐PS). Experimental colitis was induced by intramural injection of PG‐PS to rat colon. After 3 weeks the colon was removed and the mucosal inflammation was assessed. The area of MAdCAM‐1‐positive venules and the subsets of infiltrating cells were determined in colonic mucosa by immunohistochemistry. In another experiment, monoclonal antibody against MAdCAM‐1 was administered intraperitoneally to examine its attenuating effect on colitis. The intramural injection of PG‐PS induced significant colonic inflammation with granuloma formation. The submucosa was drastically thickened with the infiltration of CD4 positive lymphocytes and ED‐1 positive macrophages. Intense MAdCAM‐1 expression was observed on endothelium of the submucosal venules in inflamed mucosa. Administration of anti‐MAdCAM‐1 antibody significantly attenuated the PG‐PS‐induced colonic damage and cell infiltration. Enhanced expression of MAdCAM‐1 was demonstrated in venular endothelium of the inflamed colon in PG‐PS‐induced colitis. The attenuating effect of anti‐MAdCAM‐1 suggests the importance of the MAdCAM‐1‐dependent process in the formation of chronic granulomatous colitis.

Clinicopathologic Characteristics, CpG Island Methylator Phenotype, and BRAF Mutations in Microsatellite-Stable Colorectal Cancers Without Chromosomal Instability

CONTEXT The 2 chief pathways implicated in colorectal carcinogenesis, microsatellite instability and chromosomal instability, are not present in 20% to 37% of cases. OBJECTIVE To determine whether the CpG island methylator phenotype (CIMP) pathway, characterized by simultaneous methylation of several known tumor suppressor genes, is the principal underlying mechanism in cases without chromosomal or microsatellite instability, and to determine the significance of CIMP pathway and BRAF mutations in microsatellite-stable (MSS) cases. DESIGN Clinicopathologic features and chromosomal instability status by loss of heterozygosity analysis were determined in 83 cases of colorectal cancer in which microsatellite instability, CIMP status, BRAF mutations, and KRAS mutations were previously known. RESULTS Microsatellite instability was present in 14 cases (17%). Of the 69 MSS cases (83%), chromosomal instability manifested by LOH involving at least one locus was observed in 53 cases (64%). Hence, 16 (19%) of 83 colorectal cancer cases showed neither microsatellite instability nor chromosomal instability. These cases had a low incidence of CIMP (3/16; 19%) and BRAF mutation (1/16; 6%). The 5-year survival in these cases was significantly better compared with MSS cases with chromosomal instability (80% vs 54%, P = .02). BRAF mutations were identified in 10 MSS cases (15%). BRAF mutation in MSS cases correlated significantly with high-level chromosomal instability (P = .009) and poor 5-year survival (0% vs 70%, P < .001). CONCLUSIONS CIMP does not appear to play a key role in colorectal cancer without microsatellite instability and chromosomal instability. These cases have a better survival, probably related to absence of significant chromosomal instability. BRAF mutations in MSS cases are associated with high levels of chromosomal instability that are likely responsible for the adverse outcomes in these cases.