HETEROGENEOUS EXPRESSION OF CARCINOEMBRYONIC ANTIGEN IN THE NORMAL COLON AND UPREGULATION IN ACTIVE ULCERATIVE COLITIS

Using a technique of tolerization, a murine monoclonal antibody (MAb 2E8) has been raised which displays regional differences in reactivity in the epithelium of the normal human colon and increased reactivity in active ulcerative colitis. MAb 2E8 (IgG₁) was highly colon-specific and gave higher immunoperoxidase staining scores in the proximal colonic mucosa compared with paired rectal sections ( P <0·02). Expression of the antigen reactive with MAb 2E8 was enhanced in active ulcerative colitis compared with quiescent ulcerative colitis ( P <0·05) and normal controls ( P <0·001). Western blotting and indirect immunofluorescent screening on transfected cell lines established that MAb 2E8 was reactive with carcinoembryonic antigen (CEA). This is the first demonstration of regional differences in the expression of CEA in the normal colon and indicates upregulation of this molecule in active ulcerative colitis.     

KISS1 expression in colorectal cancer

Kisspeptins, the products of the KISS1 gene, are involved in cancer invasion, migration, metastasis and angiogenesis, while they induce apoptosis in various cancers. Herein, we studied KISS1 expression in colorectal cancer. We analyzed KISS1 expression using immunohistochemistry and image analysis in normal and malignant tissue samples from 60 patients with colorectal adenocarcinoma. The results correlated with various clinicopathological parameters. The expression of KISS1 was much higher in normal than in malignant colonic mucosa. However, among malignant tissues, KISS1 expression was higher in larger tumors (>4 cm) than in smaller ones (≤4 cm) and in stages III and IV than in stages I and II. In addition, it was higher in patients with lymph node metastases. Moreover, KISS1 levels in the normal mucosa and their difference from those in the malignant mucosa were higher in the right part of the large intestine than in the left one. KISS1 expression is reduced during the malignant transformation of the colonic mucosa and there is a difference in the expression pattern between the right and the left part of the large intestine. However, larger and advanced colorectal tumors express higher KISS1 levels than smaller and localized ones.     

Reciprocity between membranous and nuclear expression of β-catenin in colorectal tumours

 β-Catenin has a central role not only in linking the cadherin-mediated cell adhesion system but also in the intercellular signalling pathway. To investigate alterations of β-catenin in the development of colorectal carcinoma, the pattern of β-catenin expression was studied using immunohistochemistry in 74 sporadic colorectal adenomas, in histologically normal mucosa adjacent to 65 of these adenomas, and in 52 carcinomas arising in adenomas. All normal epithelia displayed cell boundary staining for β-catenin. Adenomas and carcinomas showed varying degrees of membranous staining. However, some tumours also showed nuclear staining of β-catenin protein. Decreased membranous and increased nuclear β-catenin staining were associated with increasing degrees of dysplasia in adenomas (P < 0.005, P < 0.05, respectively). Carcinomas manifested significantly reduced membranous, but enhanced nuclear β-catenin expression compared with their associated adenomas (P < 0.001, P < 0.005, respectively). An inverse correlation was found between decreased membranous and increased nuclear staining of β-catenin in both adenomas and carcinomas (P < 0.025, P < 0.05, respectively). The data confirm that reduced membranous and increased nuclear expression of β-catenin is associated with the progression of colorectal adenomas to carcinomas. Our results also suggest that decreased membranous expression of β-catenin may result from aberrant localisation of the protein in the cell nucleus. Received: 1 January 1997 / Accepted: 26 February 1997     

Intestinal mucin antigens in ulcerative colitis and their relationship with malignancy

The expression of two intestinal mucin-associated antigens large intestine mucin antigen (LIMA) and small intestine mucin antigen (SIMA) were investigated by indirect immunoperoxidase staining of rectal mucosa from patients suffering from ulcerative colitis with (n = 6) and without (n = 31) associated carcinoma and in noncolitic controls (n = 40). The aim was to assess the relationship between antigen patterns and malignant change. SIMA, which is localised predominantly in the small intestine, is virtually undetectable in the normal adult colonic mucosa. However, this antigen is present in the foetal colon and colonic carcinoma. LIMA is expressed in normal colonic mucosa, but absent from the small intestine. LIMA staining patterns were not significantly different among the three groups. In contrast, expression of SIMA was significantly higher in the patients who had developed carcinoma (6/6) than in the noncancer group (7/71) (P less than 0.001). The presence of SIMA was also significantly related to areas of dysplasia compared to normal (P = .03) or inflammation (P less than .05), but it did not differ from mucosa showing "indefinite" atypia. The finding of 31% SIMA-positive biopsies associated with severe inflammation in colitis with active disease, but no evidence of malignancy, is difficult to explain at the present stage. A followup study would be necessary to determine its significance. Perhaps the most important finding is the increased frequency of SIMA-positive foci in histologically normal mucosa in carcinoma patients compared with the noncancer group (P less than .001), suggesting a field change. These observations may be prove useful for the identification of patients who may be at risk of developing carcinoma.     

A clinicopathological analysis of KISS1 and KISS1R expression in colorectal cancer

Kisspeptins, the products of the KISS1 gene have tumor suppressing and antimetastatic properties. We aimed to study KISS1 and KISS1R expression in colorectal cancer. We analyzed KISS1 and KISS1R expression using immunohistochemistry and image analysis in normal and malignant tissue samples from 111 patients with colorectal adenocarcinoma. KISS1 expression was much higher in the normal than in the malignant colonic mucosa. Regarding malignant tissues, KISS1 levels were higher in larger tumors, in stage III and IV cancers, in cancers with lymph node metastasis and in tumors located in the distal part of the large intestine. Patients with greater KISS1 levels had worse prognosis. No KISS1R expression was detected in normal or malignant tissues or in liver metastases. KISS1 expression is reduced during the malignant transformation of the colonic mucosa. However, larger and advanced colorectal cancers express more KISS1, without reaching the former normal levels, and increased KISS1 levels are associated with worse prognosis. Finally, neither the normal nor the malignant colonic epithelial cells produce KISS1R.     

Differential expression of microRNA 181b and microRNA 21 in hyperplastic polyps and sessile serrated adenomas of the colon

This study was designed to analyse the potential diagnostic value of miR-181b and miR-21 for discriminating hyperplastic polyps (HP) from sessile serrated adenomas (SSA) without cytologic dysplasia. Using real-time polymerase chain reaction expression levels of miR-181b and miR-21 in 18 HPs, 19 SSAs without cytologic dysplasia and 20 normal colonic mucosal specimens were examined. In addition, 20 colorectal cancers specimen were analysed for miR-181b expression. Data were normalised to RNU48 as an internal control. A differential expression of miR-181b and miR-21 was found in HPs, SSAs, and normal colonic mucosa with highest expression levels in SSAs. Levels of miR-181b but not miR-21 differed in HPs and normal mucosa. SSAs exhibited both significantly higher miR-181b levels (up to 2.01-fold; P < 0.001) and miR-21 levels (up to 1.82-fold; P = 0.011) than HPs. In contrast to HPs, SSAs are characterised by high levels of miR-181b and miR-21 expression. However, due to the overlap of values, miR-181b and miR-21 evaluation did not allow discrimination of the two lesions in every case.     

OVEREXPRESSION OF THE 67-kD LAMININ RECEPTOR CORRELATES WITH TUMOUR PROGRESSION IN HUMAN COLORECTAL CARCINOMA

The high affinity 67-kD laminin receptor (67LR) is a cell surface protein whose expression is increased in a number of human carcinoma models. To date, 67LR expression in colorectal carcinomas has been examined in a small number of cases. 67LR expression has been immunohistochemically analysed in a large series of human colorectal neoplasms, using the MLuC5 monoclonal antibody. The study included 59 samples of non-neoplastic mucosa, 45 polyps (11 hyperplastic, 34 adenomas), 196 carcinomas, and lymph node metastases of 87 carcinomas. Epithelial cells of normal mucosa and hyperplastic polyps were negative or showed weak positivity in the paranuclear and apical areas of the cytoplasm. In adenomas and carcinomas, the staining was stronger, with a membranous or cytoplasmic pattern. The expression of 67LR correlated significantly with the progression from normal mucosa (22 per cent) to adenoma (44 per cent), carcinoma (61 per cent), and lymph node metastasis (75 per cent) ( P <0·0001). Expression of the laminin receptor showed a tendency to be more frequently positive in advanced stage (III+IV; 67 per cent) when compared with early stage (I+II) carcinomas (54 per cent). The difference, however, was not statistically significant ( P =0·058). In addition, 14 out of 28 (50 per cent) primary carcinomas without 67LR expression became positive in lymph node metastases, while most (86 per cent) of the MLuC5-positive primary carcinomas were also immunoreactive in metastases. In conclusion, these results indicate that 67LR is up-regulated in the progression of human colorectal carcinomas and may play a role in the local and metastatic progression of these tumours.     

Expression of insulin-like growth factor-1 receptor in human colorectal cancer.

The activation of the insulinlike growth factor 1/IGF-1 receptor system (IGF1/IGF1-R) has recently emerged as critical event in transformation and tumorigenicity of several murine and human tumors. Expression of IGF1 and of IGF1-R has been demonstrated in normal and neoplastic intestinal cell lines of rats and humans. However, the modulation of IGF1-R expression during the progression from normal colonic mucosa to adenoma, to carcinoma, and to metastasis, has not been evaluated. In this retrospective study, we investigated the expression of IGF1-R in 12 colonic adenomas (AD), 36 primary colorectal adenocarcinomas (CA), and in 27 corresponding metastases (MT). Normal colonic mucosa (N) was adjacent to the CA in 34 cases. Formalin-fixed, paraffin-embedded tissues of each case were immunostained using the avidin-biotin-peroxidase method. We used an anti-IGF1-R rabbit polyclonal antibody (Santa Cruz Biotechnology, CA; dilution 1:100). Positive staining was quantitated by CAS-200. Moderate to strong cytoplasmic immunostaining was observed in 34 of 36 CA (96%), and in 25 of 27 MT (93%). In all of the positive MTs, the intensity of the staining was always strong. In 10 of 12 ADs (83%), only a faint cytoplasmic stain was identified. Normal mucosa when present was negative. Strong IGF1-R positivity correlated with higher grade and higher-stage tumors (P < .01). These data suggest a role of IGF1-R expression during the progression of colorectal adenoma to carcinoma. An increased number of IGF1-R receptors may favor the metastasis of colorectal cancer.     

Malignant and other properties of human colon carcinoma cells after suppression of sulfomucin production in vitro

Although the loss of sulfomucins was known as an indicator of carcinogenesis and malignant progression of colonic epithelia, it was not known whether the loss was directly related to the malignant behavior of colon carcinoma cells. We have studied the biological properties of LS174T human colon carcinoma cells before and after suppression of sulfomucin production. Incorporation of [³⁵S]-sulfate into high molecular weight mucins decreased after carcinoma cell treatment with 1.5% dimethylsulfoxide (DMSO) for 8 days. The amounts of sulfomucin determined using a sulfomucin-specific monoclonal antibody (mAb 91.9H), in Western blot and flowcytometric analyses, also decreased. In addition, the levels of MUC2 and MUC5B mucin gene expression measured by RT-PCR were reduced after DMSO-treatment, whereas the levels of MUC1, MUC5AC, and MUC6 mucin gene expression were not. The DMSO-treated cells were tested in vitro and in vivo for their properties. Differences were not detected in their anchorage-independent growth, anchorage-dependent growth, E-selectin-dependent cell adhesion or sensitivity to interleukin (IL)-2-activated lymphocyte cytolysis. When untreated or DMSO-treated LS174T cells were injected intrasplenically into nude mice, the treated cells lacking certain cell surface sulfomucins formed fewer metastatic colonies in the liver. These results suggest that the loss of sulfomucins by colonic epithelial cells during progression is not directly related to the enhanced malignant behavior.     

The mRNA and protein expression of A-kinase anchor proteins 13 in human colorectal cancer

There was no literature reporting the relationship between AKAP13 and colorectal carcinoma. This study is aim to investigate the expression and role of AKAP13 in human colorectal cancers. This study investigated 94 pair-matched human colorectal cancers and adjacent normal mucosa, as well as 36 adenomas, of which mRNA expression of AKAP13 was detected by relative Quantitative-Real-Time RT-PCR and protein expression by immunohistochemical staining. AKAP13 gene was upregulated in colorectal cancer group by 2.259 times compared to control group without significant difference (P = 0.081), and no expression was detected in adenoma by RT-PCR. The positive expression rate of AKAP13 protein in colorectal carcinoma (52.3%) was significantly higher than those in adenoma (9.1%) and normal tissue (34.7%) (P = 0.006) by immunohistochemical staining. Either the mRNA or protein expressions of AKAP13 were correlated with histological types and differentiation grade (P < 0.05). Our results suggest AKAP13 protein may be related to the carcinogenesis of human colorectal cancer. However, more deeply and larger scale research are required to prove the correlation.     

Cathepsin B expression in colorectal carcinomas correlates with tumor progression and shortened patient survival.

Cathepsin B is a lysosomal cysteine proteinase that has the ability to degrade several extracellular matrix components at both neutral and acidic pH and has been implicated in the progression of several human and rodent tumors. We have studied the expression of cathepsin B in human colorectal tissues using a monospecific polyclonal rabbit antibody raised against human liver cathepsin B. In immunoblots of normal and neoplastic colorectal tissues this antibody specifically recognized only cathepsin B. We studied 101 cases of formalin-fixed, paraffin-embedded tissue (15 normal mucosa, 17 adenomas, and 69 carcinomas). Epithelial cells of normal mucosa and adenomas were either negative or showed a weak granular reactivity located in the paranuclear and apical cytoplasm of superficial cells. Small clusters of histiocytes were also positive in the region of the superficial area of the lamina propria. In carcinomas, increased expression of cathepsin B correlated with advanced stage of the disease. Increased immunoreactivity of cathepsin B in malignant cells was associated with either a diffuse cytoplasmic staining or was polarized to the basal pole of the cells. This is in contrast to the punctate paranuclear staining pattern observed in normal colonic mucosal cells. In tumor stromal cells, increased expression of the enzyme correlated with neoplastic progression. Expression of high levels of cathepsin B in the tumor epithelial cells was associated with a significantly shorter survival of the patients. In conclusion, our results indicate that cathepsin B expression is up-regulated in human colorectal carcinomas compared with normal mucosa and adenomas and correlates with tumor progression.     

Immunoblot analysis of c-Met expression in human colorectal cancer: Overexpression is associated with advanced stage cancer

c-Met, the receptor of hepatocyte growth factor is known to be responsible for the motility and mitogenesis of epithelial cells including cancer cells. To investigate the significance of c-Met expression in human colorectal cancer (CRC), total cellular protein, extracted from 130 CRCs were examined by Western blot analysis. The signal was quantitated by ChemiImager™ 4000 Low Light Imaging System. c-Met expression was analyzed as the ratio of tumor to matched normal tissue (T/N) and expressed as fold-increase. The cellular localization of c-Met was assessed by immunohistochemistry. The T/N fold increase of c-Met varied from 0.2 to 10.7 with a mean of 3.41 ± 0.23 (mean ± SE). 69% primary CRC showed overexpression (T/N >2.0) of c-Met. Significantly higher c-Met levels were found in CRC with blood vessel invasion (P = 0.04), and in advanced stage (P = 0.04). No relationship was noted between c-Met expression and age, tumor size, location, differentiation. C-Met immunoreactivity was observed in the membrane and cytoplasm of cancer cells. Positive staining of endothelial cells of blood vessels within normal submucosa and tumor was also evident. C-Met protein is expressed at levels significantly higher than adjacent mucosa in most primary adenocarcinomas of the colon. Our results support an important role for c-Met in human CRC progression and metastasis.     

Expression profile of mucin-associated sialyl-Tn antigen in Chinese patients with different colorectal lesions (adenomas,carcinomas)

Background: The sialyl-Tn (sTn) antigen is a mucin-associated carbohydrate antigen expressed by numerous human carcinomas, and is also claimed to be a prognostic factor in colorectal cancer. But the associations between sTn and colorectal cancer remain elusive and controversial. Here, we investigated the expression profile of sTn antigen in a series of human colorectal tissue samples including normal colon, colorectal adenomas, and colorectal carcinomas (CRCs), with an aim to analyzing whether sTn plays a role in the progression and development of Chinese patients with CRCs. Methods: Immunohistochemical staining of sTn antigen was performed in formalin-fixed, paraffin-embedded colonic sections from 4 healthy controls, 44 patients with colorectal adenomas, and 186 patients with primary CRCs. Results: No sTn antigen was detected in normal colonic tissues. There were 41 of 44 patients with colorectal adenomas (93.2%), and 141 of 186 patients with CRCs (75.8%) found to express sTn antigen. The patterns of sTn localization were different in adenomas and carcinomas of colonic tissues. Colorectal adenomas showed predominant supranuclear distribution of sTn antigen, while carcinomas revealed apical membrane, mucin droplet and diffuse cytoplasmic localization. Notably, sTn was significantly associated with the degree of differentiation (P = 0.006) and perineural invasion (P = 0.041) of the tumors, but was independent of age, gender, tumor location, depth of penetration, status of lymph nodes, lymphovascular invasion and TNM stage. Conclusions: These results indicate that sTn may play a role in initiating colorectal carcinogenesis and promoting tumor progression. Determination of sTn expression and localization may assist in evaluating malignant status of colorectal lesions.     

The zinc finger protein OZF (ZNF146) is overexpressed in colorectal cancer

Overexpression of the OZF gene has previously been demonstrated in the majority of pancreatic cancers. However, because the stages of tumour progression in this disease are poorly defined, no conclusion could be drawn concerning the relationship between OZF overexpression and the course of tumour progression. In contrast, initiation and progression steps are well defined in colorectal cancer. Most colon cancers are believed to arise from polypoid adenomas as a result of the gradual accumulation of genetic alterations, allowing the study of genetic events in the early stages of neoplasia. Accordingly, we wanted to assess the frequency of OZF overexpression in this tumour type and the relationship between overexpression and disease stage. Twenty-five colon carcinoma specimens from different sites and at various stages were analysed by immunoblotting using a highly specific mouse monoclonal antibody. Each sample was compared with adjacent normal colonic mucosa. Complementary immunohistochemical analysis was also carried out on a commercially available tissue array to identify cells expressing OZF. Of the 25 tumours analysed by immunoblotting, 20 expressed higher levels of OZF protein than their adjacent normal mucosa. Immunohistochemistry revealed OZF expression in tumour cells of 56/59 carcinomas and occasionally in infiltrating lymphocytes but at lower levels. Little or no staining was observed in cells taken from normal or inflammatory colon specimens. In both immunoblot and immunohistochemistry experiments, no correlation was found between OZF expression and clinical parameters such as TNM classification, stage, localization and age. Immunostaining was observed in low-grade adenomas, indicating that OZF expression occurs very early in the course of tumour progression. OZF expression, infrequent or absent in normal colonic mucosa, is present in more than 80% of colorectal cancers. Dysregulation of the OZF gene is an early event that may be implicated in the genesis of colonic carcinoma and may therefore provide a potential therapeutic target.     

Ki-67表达与结直肠癌Dukes分期关系

目的检测ki-67在正常结直肠组织和结直肠癌中的表达情况,探讨其表达与结直肠癌Dukes分期关系,为临床结直肠癌的诊疗判断提供参考。方法收集临床病理资料完整的结直肠癌54例,正常结直肠黏膜组织28例。应用免疫组化法(SP法)检测正常结直肠组织和结直肠癌中ki-67蛋白的表达。结果Ki-67蛋白在结直肠癌及正常结直肠黏膜中的表达率分别为85.19%和10.71%,在结直肠癌组织中的表达高于正常结直肠黏膜的表达,差异有统计学意义(P<0.01)。ki-67蛋白的表达和Dukes分期无相关性,差异无统计学意义(P>0.05)。结论ki-67蛋白过表达与结直肠癌的发生发展密切相关,但不能作为结直肠癌预后的参考指标。     

Methylation status of T-lymphoma invasion and metastasis 1 promoter and its overexpression in colorectal cancer

T-lymphoma invasion and metastasis 1 has been implicated in tumor invasion and metastasis. However, the regulatory mechanisms underlying aberrant T-lymphoma invasion and metastasis 1 expression in human colorectal cancer have not been well defined. To investigate the relationship between methylation status and expression levels of T-lymphoma invasion and metastasis 1 gene, methylation-specific polymerase chain reaction, and immunohistochemistry staining were performed in 232 matched samples of human colorectal cancer tissue and normal colorectal mucosa. Results showed that T-lymphoma invasion and metastasis 1 protein was overexpressed in colorectal cancer, especially in metastatic cases (P < .001). The degree of T-lymphoma invasion and metastasis 1 promoter methylation was a little lower in cancer tissues than in matched normal mucosa (P < .05), and the expression level of T-lymphoma invasion and metastasis 1 was inversely related to the methylation status in cancer tissues (P < .001). Colon cancer cell lines HT29 and LS174T were treated with demethylating agent 5-aza-2'-deoxycytidine, resulting in promoter hypomethylation accompanied by reexpression of T-lymphoma invasion and metastasis 1 mRNA and protein. In contrast, colon cancer cell lines SW620 and LoVo were treated with hypermethylation agent S-adenosylmethionine, resulting in T-lymphoma invasion and metastasis 1 promoter hypermethylation, accompanied by suppression of T-lymphoma invasion and metastasis 1 expression and inhibition of cell growth, plate colony formation, and migration. The present study demonstrates that overexpression of T-lymphoma invasion and metastasis 1 is associated with hypomethylation status of T-lymphoma invasion and metastasis 1 promoter region in colorectal cancer tissues. It suggests that promotor hypomethylation of T-lymphoma invasion and metastasis 1 may play a role in the progression and metastasis of colorectal cancer. Pharmacologic reversal of T-lymphoma invasion and metastasis 1 promoter hypomethylation may inhibit cell proliferation and migration.     

DCLK1, a promising colorectal cancer stem cell marker,regulates tumor progression and invasion through miR-137 and miR-15a dependent manner

Cancer stem cells (CSCs) are thought to be a major player in tumor initiation, progression, and metastasis. Targeting CSCs for elimination presents a promising therapeutic strategy; however, this approach will require a stronger understanding of CSC biology and identification of CSC-specific markers. The present study was conducted to examine the correlation between DCLK1 and miR-137 and miR-15a levels in colorectal cancer. A total of 222 samples, including 181 colorectal cancer specimens, 24 adenomatosis, and 17 non-adenomatosis colonic polyps, were stained for DCLK1 expression using immunohistochemistry. Also, expression of miR-137 and miR-15a was assessed in colorectal cancer with high and low DCLK1 expression levels. Most colorectal cancer specimens (76%) showed strong expression of DCLK1, whereas only 21% of adenomatous and none of non-adenomatous colonic polyps showed strong DCLK1 expression. A significant difference in DCLK1 expression was found between colorectal cancer, adenomatous, and non-adenomatous colonic polyps (P < 0.001). Higher expression of DCLK1 was more frequently detected in colorectal cases with larger tumor size (P = 0.03), poor differentiation (P = 0.03), and lymph node involvement (P = 0.04). Comparison of miR-137 and miR-15a in colorectal cancer cases revealed a significant inverse correlation with DCLK1 expression (P = 0.03 and P = 0.04, respectively). DCLK1 may act as a candidate marker for colorectal cancer stem cells. The critical role of DCLK1 in colorectal cancer suggests that it may represent an early diagnostic marker and therapeutic target; however, further investigation is warranted.