Regulation of the intestinal mucin MUC2 gene expression in vivo: evidence for the role of promoter methylation

In the present work we investigated the in vivo regulation of the mucin gene MUC2, which is overexpressed in all mucinous colorectal carcinomas. The inhibition of methylation by 5-azadeoxycytidine induces de novo expression of MUC2 in the colon carcinoma cell line COLO 205. The expression is retained in xenograft tissue and the cells give rise to MUC2-expressing tumours in nude mice. The strong expression of MUC2 in the normal human goblet cells and in the tissue of human mucinous colorectal carcinomas is associated with the average methylation of about 50% at every investigated CpG site of the MUC2 promoter. In contrast, MUC2 promoter in the non-expressing normal columnar cells and in the non-mucinous carcinoma tissue is methylated to nearly 100%. These data show that (i) low methylation of MUC2 promoter is associated with MUC2 expression in vivo and (ii) the pattern of MUC2 promoter methylation in the normal goblet or columnar cells most closely resembles that in mucinous or non-mucinous colorectal carcinomas, respectively. They indicate that MUC2 expression in vivo is regulated by promoter methylation and support the hypothesis that cells with goblet-like differentiation give rise to mucinous colonic carcinomas.     

Expression of mucin core proteins, trefoil factors, APC and p21 in subsets of colorectal polyps and cancers suggests a distinct pathway of pathogenesis of mucinous carcinoma of the colorectum

Mucin core proteins are expressed in a tissue and cell type specific manner in the normal gastrointestinal tract. Aberrant expression of mucin core proteins have been reported in colorectal neoplasms. To examine the relationship between subsets of colorectal polyps and non-mucinous and mucinous adenocarcinomas of the colorectum, we evaluated the frequency of the expression of cell lineage associated mucin core proteins (MUC5AC and MUC2), trefoil factors (TFF1 and TFF3), and APC and p21 in these tissues. An immunohistochemical study was performed in 10 normal rectal mucosa samples (NM) 21 hyperplastic polyps (HP), 20 serrated adenomas (SA), 25 tubular adenomas (TA), 13 tubulovillous adenomas (TVA), 7 villous adenomas (VA), 42 non-mucinous colorectal cancers (NMC), and 19 mucinous colorectal cancers (MC). A higher frequency of ectopic expression of gastric foveolar mucin, MUC5AC, and the expression of intestinal goblet cell mucins, MUC2, was observed respectively in HP (100%, 100%), SA (85%, 85%), TVA (85%, 85%), and VA (100%, 100%), compared to TA (32%, p<0.002; 36%, p<0.01). MC (68%, 100%) also showed a higher frequency of the expression of MUC5AC and MUC2 compared to NMC (31%, p=0.001; 38%, p<0.001), and TFF1 showed similar patterns of expression. APC protein and p21 were also expressed at a higher frequency in HP (100%, 100%), and SA (67%, 83%), than in TA (29%, p<0.03; 46%, p<0.05). MC (68%, 100%) showed a higher frequency of expression of APC protein and p21 than NMC (19%, p<0.001; 45%, p<0.01). Our results showed that MUC2 expression and de novo ectopic expression of MUC5AC and TFF1 are more frequent in HP, SA, TVA, VA, and MC than in TA and NMC. These results suggest that simultaneous activation of differentiation pathways of goblet cells and gastric foveolar cells may occur predominantly in the pathogenesis of HP, SA, TVA, VA, and MC, while the pathogenesis of TA and NMC are less likely to involve these processes.     

HLA antigens in colorectal tumours--low expression of HLA class I antigens in mucinous colorectal carcinomas

Expression of HLA antigens and beta 2-microglobulin was studied by immunoperoxidase staining of frozen sections of 9 mucinous and 10 nonmucinous colorectal adenocarcinomas, 1 cloacogenic carcinoma, 12 colorectal adenomas and 4 samples of normal colorectal mucosa using monoclonal antibodies (MAbs). Staining results were related to histopathological features. HLA Class I antigens were strongly expressed in morphologically normal colorectal epithelium, in all adenomas tested and in all non-mucinous carcinomas. In contrast, expression of HLA class I antigens by the majority of tumour cells was present in only 2 of the 9 mucinous carcinomas, whereas 2 of these mucinous carcinomas were completely negative. In the mucinous carcinomas a striking scarcity of mononuclear inflammatory infiltrate, especially around the mucus accumulations, was observed. HLA class II antigen expression was not detected in normal epithelium and was only focally present in 1 of the 12 adenomas. In 6 out of the 20 carcinomas tested between 20% and 90% of the tumour cells were stained by MAbs against HLA class II antigens. Apart from the low expression of HLA class I antigens in mucinous carcinomas no relationship was found between expression of HLA antigens and histological features of the tumours. The relative poor prognosis of mucinous colorectal carcinoma as reported in the literature may be associated with low expression of HLA class I antigens and scant mononuclear inflammatory infiltrate, which may be a reflection of a weak immune response to the tumour cells.     

Mucin expression in mucinous carcinoma and other invasive carcinomas of the breast.

To investigate mucin expression in breast cancer, immunohistochemical staining was performed on 30 mucinous carcinomas and 95 non-mucinous invasive carcinomas. MUC2 expression was detected in all mucinous carcinomas, but only in 11.1% of invasive ductal carcinomas, and in none of the invasive lobular carcinomas and medullary carcinomas. MUC1 is often expressed in invasive breast carcinoma, but not in medullary carcinoma. Strong cytoplasmic staining was seen in invasive ductal carcinoma, in contrast to surface membrane staining in mucinous carcinoma and intracytoplasmic vacuole staining in invasive lobular carcinoma. CA19-9 and CA50 expression in more than 25% of tumor cells was seen in 17.2 and 16.0% of invasive ductal carcinomas, respectively, but not in mucinous carcinomas. CA125 and human gastric mucin were rarely expressed in breast cancer, irrespective of histologic type.     

Expression of p53 protein in invasive colorectal carcinomas of different histologic types.

BACKGROUND. This study was performed to determine whether morphologic differences of colonic cancer types can be related to different genotypes of these tumors. METHODS. Paraffin sections of 76 human invasive colorectal carcinomas were examined for the overexpression of p53 oncoprotein with the avidin-biotin complex-peroxidase staining procedure and CM-1 antiserum, which detects p53 protein in paraffin-embedded material. The tumors were categorized as mucinous (22 cases), most of which originated from adenomas, and nonmucinous, which were subdivided into carcinomas originating from adenoma-carcinoma sequence (ACS) (29 cases) and de novo (DN) carcinomas (25 cases). RESULTS. Nineteen DN carcinomas (76%), 21 ACS carcinomas (72%), and 8 mucinous carcinomas (36%) exhibited detectable amounts of p53 protein in the tumor cell nuclei. Strong overexpression of p53 protein coincided with a high percentage (> 40%) of stained nuclei in 40% of ACS and 48% of DN carcinomas versus 9% of mucinous tumors. The percentage of stained nuclei, intensity of staining, and distribution of the stained areas did not correlate with the grade of differentiation or the invasive edge of the tumors. Along with nuclear staining of the tumor area, a distinct perinuclear staining of normal epithelial cells adjacent to the tumor was observed in 48% of DN, 7% of ACS, and 9% of mucinous carcinomas. CONCLUSIONS. The current results, in combination with the recently published data on Ki-ras and c-myc alterations, indicate that mucinous carcinomas differ from nonmucinous colorectal carcinomas in their genetic lesions.     

Prognostic and diagnostic significance of beta-catenin nuclear immunostaining in colorectal cancer.

In the present study, we investigated the prognostic and diagnostic significance of beta-catenin nuclear immunostaining in 60 specimens of normal colorectal tissue; 180 specimens of colorectal polyps, adenomas, and carcinomas; and 40 specimens from patients with the simultaneous occurrence of polyps, adenomas, and carcinomas. Additional specimens from 59 patients with colorectal carcinoma and 14 patients with adenoma who subsequently developed carcinoma were examined for possible survival study. Immunohistochemical staining showed that the occurrence of nuclear beta-catenin correlated with the sequential stages in colorectal carcinogenesis, in which positive staining was observed in 0% of normal tissues, 8% of polyps, 92% of adenomas, and 100% of carcinomas. High immunohistochemical scores in colorectal carcinoma were significantly associated with lymph node metastasis and poor survival. Adenomas associated with synchronous or metachronous carcinomas showed significantly higher levels of nuclear beta-catenin compared with adenomas without associated carcinomas. Nuclear translocation of beta-catenin was rare or absent in other types of cytokeratin 20 positive adenocarcinomas examined (99 cases). Thus, it was positive in only 7% of colonic mucinous adenocarcinomas, 3% of pancreatic adenocarcinomas, 8% of ovarian mucinous cystadenocarcinomas, and 0% of gastric adenocarcinomas. However, 100% of primary and metastatic colorectal adenocarcinomas were positive for nuclear staining for beta-catenin. Thus, nuclear staining for beta-catenin may serve as an additional parameter to help distinguish colorectal adenocarcinomas from adenocarcinomas of other tissue sites. Collectively, the present large-scale study has clearly addressed the clinical significance of beta-catenin nuclear translocation with respect to tumor progression, survival, and differential diagnosis.     

大肠肿瘤中bcl—2及PCNA表达及其意义

目的：研究ｂｃｌ－２及ＰＣＮＡ在大肠肿瘤肆生中的作用及其临床意义。方法：应用免疫组化Ｓ－Ｐ法分别检测大肠正常粘膜、腺瘤及腺癌中ｂｃ；－２及ＰＣＮＡ表达。结果：Ｂｃｌ－２在正常粘膜基底部上皮细胞表达，在腺瘤（７７．５％）和腺癌中（５６．３％）ｂｃｌ－２表达差异显（Ｐ〈０．０５）。高分化腺癌ｂｃｌ－２表达率（６８．４％）显高于差分化腺癌（４１．７％），ＰＣＮＡ表达在正常粘膜，腺瘤及腺癌中依次递增，     

Expression of beta-catenin and full-length APC protein in normal and neoplastic colonic tissues

Mutations of the APC gene are thought to be early events in the process of colorectal carcinogenesis. Although the complete function(s) of the APC gene product is not known, it has been shown that the APC protein interacts with beta-catenin in a multi-protein complex to regulate the level of expression of beta-catenin. Loss of normal APC protein function can lead to an accumulation of beta-catenin in the cytosol and the nucleus. Immunohistochemical methods were used to determine the relationship between APC and beta-catenin protein expression in human colonic tissues (150 normal, 9 hyperplastic, 58 adenomas and 83 carcinomas) and 12 paired samples of normal and cancer tissue in mouse colon. In all samples of normal human and mouse colonic mucosa and in human hyperplastic polyps both APC and beta-catenin immunoreactivity were present in colonocytes. APC expression was cytoplasmic, with maximal immunoreactivity in the goblet cells. beta-Catenin expression was predominantly localized to the plasma membrane, with no nuclear immunoreactivity. APC immunoreactivity was absent in all of the mouse adenocarcinomas and 83% of the human colon cancers. All of the human and mouse carcinomas had nuclear and cytoplasmic beta-catenin expression. In contrast, only 29% of the 58 colonic adenomas were completely negative for APC immunoreactivity. Regardless of the presence or absence of APC, all of the adenomas had cytoplasmic and nuclear beta-catenin immunoreactivity. Many colonic adenomas retain expression of full-length APC protein whereas it is usually lost in colorectal cancers. Regardless of the status of APC protein expression, beta-catenin protein was found in the cytoplasm and nucleus of all neoplastic colonic mucosa. The dissociation between loss of expression of APC and accumulation of beta-catenin in the nucleus suggests that inactivation of both alleles of the APC gene may not be required for beta-catenin nuclear accumulation in colonic adenomas.     

Clinicopathological and genetic characteristics of mucinous carcinomas in the colorectum.

We investigated the clinicopathological and genetic characteristics including patients' gender, age, tumour location, growth pattern, Dukes' stage, DNA ploidy, S-phase fraction, PCNA, apoptosis, c-erbB-2, bcl-2, K-ras, p53, DCC and heat shock protein in 32 mucinous carcinomas versus 261 non-mucinous carcinomas in the colorectum. Sixty percent of mucinous carcinomas were located in the right colon, 13% in the left colon and 27% in the rectum (p=0.01). More mucinous carcinomas grew in expanding pattern than non-mucinous carcinomas (66% vs 39%, p=0.005). Compared with non-mucinous carcinoma, mucinous carcinoma had more K-ras mutations (50% vs 25%, p=0.02), but less p53 expression (72% vs 49%, p=0.02) and less apoptotic activity (19% vs 51%, p=0.01). We further confirm that the mucinous carcinoma in the colorectum represents a distinct clinicopathologic and genetic features as compared to non-mucinous tumour, and may have a different biological behaviour.     

Altered Calreticulin expression in human colon cancer: maintenance of Calreticulin expression is associated with mucinous differentiation

Calreticulin is an endoplasmic reticulum luminal calcium-binding chaperone involved in various cellular functions and is a ligand for the scavenger receptor CD91. Recent studies, based on proteomic approaches on whole tissue samples containing both neoplastic and non-neoplastic cells, have shown alterations of Calreticulin expression in colon carcinomas, albeit with divergent results. The aims of this study were: 1) to assess the expression of Calreticulin and its receptor CD91 in 58 human colon adenocarcinomas, compared with paired normal mucosa, using a semi-quantitative immunohistochemical analysis, and 2) to examine associations between the tumour phenotypic features, and Calreticulin and/or CD91 expressions. Calreticulin expression was down-regulated in 51.7% human colon adenocarcinomas. Accordingly, quantitative immunoblot analysis showed that Calreticulin expression was significantly lower in human colonic cancer cell lines than in preparations of isolated human normal colonic epithelial cells. CD91 was co-expressed with Calreticulin in both normal colonic epithelial cells and pericryptic myofibroblasts. Calreticulin and CD91, that characterize the 'amateur phagocyte' function of epithelial cells, were both down-regulated in 48% of adenocarcinomas. Finally, Calreticulin expression was significantly associated with the mucinous differentiation of the tumour. Collectively, these results show that Calreticulin is likely to play a pivotal role in the differentiation of human colonic adenocarcinomas.     

Evidence of reciprocity of bcl-2 and p53 expression in human colorectal adenomas and carcinomas.

Evidence of accumulating for the failure of apoptosis as an important factor in the evolution of colorectal cancer and its poor response to adjuvant therapy. The proto-oncogene bcl-2 suppresses apoptosis. Its expression could provide an important survival advantage permitting the development of colorectal cancer. The expression of bcl-2 and p53 was determined by immunohistochemistry in 47 samples of histologically normal colonic mucosa, 19 adenomas and 53 adenocarcinomas. Expression of bcl-2 in colonic crypts > 5 cm from the tumours was confined to crypt bases but was more extensive and intense in normal crypts < 5 mm from cancers. A higher proportion of adenomas (63.2%) than carcinomas (36.5%) expressed bcl-2 (P < 0.05). A lower proportion of adenomas (31.6%) than carcinomas (62.3%) expressed p53 (P < 0.02). A total of 26.3% of adenomas and 22% of carcinomas expressed both bcl-2 and p53. To determine whether these samples contained cells which expressed both proteins, a dual staining technique for bcl-2 and p53 was used. Only 1/19 adenomas and 2/53 carcinomas contained cells immunopositive for both bcl-2 and p53. Moreover there was evidence of reciprocity of expression of bcl-2 and p53 in these three double staining neoplasms. We suggest that bcl-2 provides a survival advantage in the proliferative compartment of normal crypts and colorectal neoplasms. However, its expression is lost during the evolution from adenoma to carcinoma, whereas p53 expression is increased, an event generally coincident with the expression of stabilised p53, which we presume to represent the mutant form.     

Fhit, Mlh1, P53 and phenotypic expression in the early stage of colorectal neoplasms

There are two different pathways for the development of colorectal carcinoma (CRC), adenoma-carcinoma sequence (ACS) and de novo (DN) carcinogenesis. To clarify the molecular and clinicopathological characteristics in colorectal carcinogenesis, we examined endoscopically resected specimens of 30 adenomas, 30 carcinoma in adenomas (CIAs), and 18 early pure colorectal carcinomas without any adenoma component (EPCs, so called DN carcinoma) and compared the expression of Fhit, Mlh1, Msh2, P53 and cellular phenotype (HGM, MUC2 and CD10). Markedly reduced or absent Fhit expression was noted in 8 (44%) of 18 EPCs, but none of the adenomas or CIAs (p<0.0001). Six (33%) of 18 EPCs showed loss of Mlh1 expression, but rarely in adenomas and CIAs (p=0.008). This altered Fhit expression was significantly higher in submucosal invasive cancers (p=0.001), lymphatic or venous invasive cancers (p=0.0018), and tumors with altered expression of Mlh1 (p=0.01). The incidence of P53 overexpression was significantly higher in EPCs (39%) and CIAs (27%) than in adenomas (3.3%) (p<0.05). There were significant differences in phenotypic expression between the adenomatous and carcinomatous areas. Moreover, in CIAs and EPCs, the rate of P53 overexpression was significantly higher in the CD10-positive cases (53%) than CD10-negative cases (19%) (p=0.04). The present findings suggested that aberrant Fhit and Mlh1 expression could be related to DN carcinogenesis and that P53 overexpression and changes in phenotypic expression could contribute to the malignant transformation of colorectal precursor lesions.     

Immunohistochemical study of mucin carbohydrates and core proteins in hepatolithiasis and cholangiocarcinoma

The expression of mucin carbohydrates [Tn, sialosyl-Tn(STn), and T antigens] and core proteins [MUCI-apomucin-related antigen (ARA) and MUC2-ARA] was examined immunohisto-chemically in tissues from 40 patients with hepatolithiasis and 26 patients with intrahepatic bile-duct carcinoma. Tn and STn antigens were expressed in most of the carcinomas, and were also often expressed in the atypical bile-duct epithelium of the patients with hepatolithiasis or carcinoma, whereas they were rarely or never expressed in the normal bile duct, suggesting that they are effective tumor markers. T antigen was less useful as a marker for intrahepatic bile-duct carcinoma or the atypical epithelium, because it was expressed in normal bile-duct of some cases. Regarding the expression of ARAs in the carcinomas, non-invasive bile-duct cyst adenocarcinomas with favorable prognosis either expressed no MUCI-ARA with [DF3(-), MUSE11(-) and 139H2(-)] staining pattern or expressed MUCI-ARA with [DF3(-), MUSE11(+) and 139H2(+)] staining pattern. However these tumors often expressed MUC2-ARA with [anti-MRP(+) and CCP58(+)] staining pattern. In contrast, most invasive non-papillary cholangiocarcinomas with poor prognosis expressed MUCI-ARA with [DF3( + ), MUSE 11(+) and 139H2(+)] staining pattern, but expressed no MUC2-ARA with [anti-MRP(-) and CCP58(-)] staining pattern. These results suggests that different apomucins are produced by bile-duct cystadenocarcinomas and cholangiocarcinomas with differing prognosis. Furthermore, expression of Tn and STn antigens is a useful indicator of malignancy in the intrahepatic duct. © 1993 Wiley-Liss, Inc.     

Aberrant expression of MUC5AC and MUC6 gastric mucin genes in colorectal polyps

Altered mucin glycosylation and the de novo appearance of gastric mucin antigens have been described in colonic adenomas. The purpose of our study was to determine if expression of the gastric mucin genes MUC5AC and MUC6 occurs in colorectal adenomas and whether this correlates with histopathologic criteria of malignant potential. Immunohistochemical staining using antibodies against MUC5AC and MUC6 tandem repeat synthetic peptides was performed on specimens of normal colon mucosa (n = 26), hyperplastic polyps (n = 9) and adenomatous polyps (n = 111). Mucin mRNA levels were determined using RNase protection assays using riboprobes corresponding to unique non‐repetitive sequences. MUC5AC and MUC6 staining were rarely detected and of low intensity in normal colon and hyperplastic polyps. The number of immunoreactive polyps and intensity of MUC5AC and MUC6 staining were greatest in larger adenomas of moderate villous histology and dysplasia. MUC5AC and MUC6 staining tended to decrease in highly villous polyps with severe dysplasia. Increased MUC5AC mRNA levels were found in 26/45 of adenomas tested compared with 0/9 normal colon specimens. MUC6 mRNA levels were found in 20/45 of adenomas compared with 1/9 normal colon specimens. MUC5AC and MUC6 mRNA were present more frequently and at higher levels in polyps with intermediate stages of size, villous histology and dysplasia. We conclude that aberrant expression of MUC5AC and MUC6 mucin genes is likely responsible for an expanded repertoire of mucin antigen expression in colorectal neoplasia. Int. J. Cancer 80:210–218, 1999. Published 1999 Wiley‐Liss, Inc.     

Clinicopathological significance of mucin 2 immunohistochemical expression in colorectal cancer: A meta-analysis

Objective: To evaluate the association between mucin 2 (MUC2) expression and clinicopathological characters of colorectal cancer. Methods: A literature search was performed on December 31, 2010 according to defined selection criteria. We evaluated the correlation between MUC2 (detected by immunohistochemistry) and clinicopathological characters of colorectal cancer. According to the tumor histological type, differentiation, location and TNM staging of colorectal carcinoma, we divided the clinicopathological characteristics into different subgroups. Fixed and random effects models were applied for estimation of the summarized risk ratios (RRs) and 95% confidence intervals (CIs) in different subgroups. Finally, forest plots and funnel plots were created to allow for visual comparison of the results or the effect of publication bias. Results: According with the inclusive criteria, fourteen studies (n=1,558) were eligible for the meta-analysis. We observed a trend towards a correlation of MUC2 higher positivity in mucinous than non-mucinous carcinoma (RR, 2.10; 95% CI, 1.30-3.40; P=0.002) and less positivity in distal than proximal colon (RR, 0.74; 95% CI, 0.64-0.85; P=0.000). There was no statistically significance for the association between MUC2 expression and differentiation or TNM staging of colorectal cancer, but MUC2 overexpression tended to be associated with the presence of T stage tumor (RR, 1.17; P=0.052). Conclusion: MUC2 overexpression was associated with the mucinous and proximal colorectal cancer.     

225例大肠腺瘤癌变的临床病理、免疫组化及其超微结构

目的：探讨大肠腺瘤及其癌变的临床病理、免疫组织化学及其超微结构。方法：随机选择我院近5年间纤维肠镜1804人次，检出大肠腺瘤225枚，其中91例行CEA组织化学染色，15例行电镜观察。结果：腺瘤肠镜检出率为12．47％（225／1804），癌变33例，癌变率为14．67％（33／225），其中管状腺瘤癌变4例，绒毛管状腺瘤癌变8例，绒毛状腺瘤癌变21例。有蒂的管状腺瘤异型程度轻、癌变率低、预后好；宽蒂、无蒂的扁平型、凹陷型、绒毛状腺瘤，异形程度重，癌变率高，浸润深，预后差。CEA染色阳性率：腺瘤癌变者93．33％（14／15），绒毛状腺瘤78．95％（15／19），绒管状腺瘤70．50％（12／17），管状腺瘤67．5％（27／40）。电镜观察显示绒毛状腺瘤部分腺上皮细胞突破基底膜向间质伸长，可出现异形细胞，核大核沟深，常染色质丰富核仁明显，癌变者可见淋巴细胞溶解癌细胞膜现象。结论：大肠腺瘤为癌前病变，不同类型的大肠腺瘤其治疗及预后不同。