| MUC1 is a type I transmembrane glycoprotein expressed by ductal epithelial cells of many organs including pancreas, breast, gastrointestinal tract, and airway. It has been demonstrated that MUC1 may influence cell-to-cell adhesion, diminish the immune response, and be involved in intracellular signaling. Many studies have shown that aberrant expression of MUC1 was associated with invasion, metastasis, and worse prognosis of many cancers. KL-6 mucin is one type of MUC1, recognized by a murine monoclonal antibody, KL-6 (Krebs von den Lungen-6) antibody. Since elevated serum KL-6 mucin level was closely correlated to the interstitial pnuemonitis activity, KL-6 mucin has been shown as an effective marker for diagnosing various intestinal pneumonitis and is currently uses in clinical stage. The epitope recognized by the KL-6 antibody exists in sialylated carbohydrate moieties of the MUC1 molecule, which are exposed on the mucin molecules. Thus, KL-6 antibody could effectively recognize MUC1 without epitope masking compared with other antibodies. KL-6 mucin may be a more selective biomarker/target for diagnosis, detection, and therapy of these cancers. Recent reports have indicated that interaction between MUC1 andβ-catenin could affect the intracellular distribution of P-catenin and may be important factor for cancer promotion and progression. In this study, we analyzed KL-6 mucin expression in gastric cancer, pancreatic cancer, colorectal cancer, and various liver cancer tissues, and examined the relationships between KL-6 mucin/β-catenin expression and clinicopathological parameters in gastric cancer and colorectal cancer. We also investigated the role of carbohydrate moiety of KL-6 mucin using glycosylation inhibitors, as well as the biochemical role of KL-6 mucin in proliferation, invasion and metastasis of pancreatic cancer using siRNA.The main contents and results obtained were summarized as follows:1 KL-6 mucin/p-catenin expression in gastric cancer and its clinical significanceThe expression of KL-6 mucin in gastric cancer tissues was detected using immunohistochemical staining and its clinicopathological significance was evaluated. Furthermore, subcellular localization ofβ-catenin, as well as the clinicopathological significance of KL-6 mucin/β-catenin colocalization was also analyzed.The cases with circumferential membrane and/or cytoplasmic expression of KL-6 mucin in both the gastric cancer and invasion front showed higher incidences of deeper invasion, lymphatic vessel invasion, venous invasion, lymph node metastasis, and advanced TNM stage. Further analysis was performed to evaluate subcellular localization ofβ-catenin in invasion front and its relationship to abnormal localization of KL-6 mucin. Circumferential membranous and/or cytoplasmic localization of KL-6 mucin was significantly related to the cytoplasmic and nuclear-accumulated localization ofβ-catenin in invasion front of gastric cancer tissues. Univariate analysis showed that localization of KL-6 mucin and P-catenin in invasion front was significantly related to prognosis. Multivariate analysis showed that positive cytoplasmic or nuclear-accumulated localization ofβ-catenin in invasion front was nominated as an independent prognostic factor.2 KL-6 mucin expression and its possible mechanism in pancreatic cancerThe expressions of KL-6 mucin in pancreatic ductal carcinoma tissues, intraductal papillary mucinous tumor tissues, and islet cell tumor tissues were detected by immunohistochemical staining. Pancreatic carcinoma cells were treated by N-glycosylation inhibitor (tunicamycin) and O-glycosylation inhibitor (GalNAc-O-bn), and the expression profile of KL-6 mucin was detected by Western blotting and immunocytochemical staining. Adhesive and invasive properties of cells was evaluated by adhesion test and transwell chamber assay. To knockdown KL-6 mucin expression, a siRNA targeting MUC1 was synthesized and transfected into Panc-1 and Capan-1 cells in optimal transfection conditions. The down-regulation of KL-6 mucin expression at both mRNA and protein levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. E-cadherin and KL-6 mucin coexpression was detected by immunofluorescence. E-cadherin/β-catenin comlex was determined by immunoprecipitation. The expressions of other Wnt signal pathway related proteins,β-catenin, cyclin D1, and c-myc, were detected by Western blotting.Positive KL-6 mucin staining was observed in all the pancreatic ductal carcinoma tissues, and 1 metastatic intraductal papillary mucinous tumor tissue. The result suggested that KL-6 mucin overexpression might associated with worse tumor behaviors such as invasion and metastasis, though the cases of pancreatic cancer tissues are limited. Western blotting and immunocytochemical staining showed that the expression profile of KL-6 mucin was significantly altered after the inhibitors treatment, especially GalNAc-O-bn. Also, adhesive and invasive potentials of the cells were significantly reduced after GalNAc-O-bn treatment. Further study is needed to understand clinical significance of KL-6 expression and biochemical role of carbohydrate moiety of KL-6 mucin in pancreatic carcinoma. RT-PCR and Western blotting showed that both KL-6 mucin mRNA and protein can be effectively silenced after transfection in optimal transfection conditions. E-cadherin and E-cadherin/β-catenin complex expressions were increased and the invasive ability of the cells was decreased. Nuclearβ-catenin, cyclin D1, and c-myc expressions were decreased after KL-6 mucin knockdown. Down-regulation of KL-6 mucin also resulted in slower proliferation and increased apoptosis.3 KL-6 mucin expression in colorectal carcinoma and its metastatic liver cancerImmunohistochemical staining was used to detect KL-6 mucin and P-catenin expression in colorectal carcinoma tissues. KL-6 mucin expression was also detected in the metastatic liver cancer tissues from primary colorectal carcinoma and primary hepatocellular carcinoma tissues.Positive KL-6 mucin expression in circumferential membranous and/or cytoplasmic was detected in about 59.7%(46/77) of the colorectal carcinoma tissues. Preservation of membranousβ-catenin expression was detected in 45.5%(35/77) of the colorectal carcinoma tissues and decreased membranous P-catenin expression was found in 54.5% (42/77) of them. Positive KL-6 mucin circumferential membranous and/or cytoplasmic expression was correlated with decreased membranousβ-catenin expression, while no correlation was found between positive KL-6 mucin circumferential membranous and/or cytoplasmic expression and nuclearβ-catenin expression. Combined positive KL-6 mucin circumferential membranous and/or cytoplasmic expression and decreased membranousβ-catenin expression was found in 30 patients (39.0%), whose survival was significantly worse than that of patients with other expression patterns. Multivariate analysis showed that this combination was as an independent predictor of survival.All the patients with metastatic liver cancer tissues showed positive staining for KL-6 mucin in cancerous tissues but not in non-cancerous tissues. All staining was observed in the circumferential membrane and/or cytoplasm of the cancer cells. In contrast, no staining for KL-6 mucin was observed in either cancerous or non-cancerous tissues of the hepatocellular carcinoma tissues. KL-6 mucin may be an indicator for liver metastasis of colorectal carcinoma. Additionally, detection of KL-6 mucin may be helpful in distinguishing hepatocellular carcinoma tissues from metastatic liver cancer tissues.4 KL-6 mucin expression in various liver cancer tissues and its diagnosis valueEnzyme-linked immunosorbent assay and immunohistochemical staining was used to measure KL-6 mucin expression in serum and tissues from intrahepatic cholangiocarcinoma patients, hepatocellular carcinoma patients, and metastatic liver cancer patients. Furthermore, KL-6 mucin, CK7, and Hep Par 1 expressions in intrahepatic cholangiocarcinoma patients, hepatocellular carcinoma patients, and combined hepatocellular and cholangiocarcinoma (cHCC-CC) patients were detected by immunohistochemical staining.Serum KL-6 mucin levels were significantly higher in intrahepatic cholangiocarcinoma patients than in healthy individuals, hepatocellular carcinoma patients, and metastatic liver cancer patients. KL-6 mucin was positively expressed in all the intrahepatic cholangiocarcinoma and metastatic liver cancer tissues but not in any hepatocellular carcinoma tissue and the surrounding non-cancerous tissues. Thus, KL-6 mucin might be a useful serological marker to distinguish intrahepatic cholangiocarcinoma patients from other liver cancer patients.Staining for CK7 was positive in both intrahepatic cholangiocarcinoma tissues (95.2%) and hepatocellular carcinoma tissues (35.9%), while cHCC-CC specimens also displayed CK7 positive staining in the cholangiocarcinoma areas (58.3%) and hepatocellular carcinoma areas (25.0%). In contrast, Hep Par 1 was positive in only 79.5% of hepatocellular carcinoma and 25.0% of hepatocellular carcinoma areas of cHCC-CC specimens. KL-6 mucin staining was positive in all of the intrahepatic cholangiocarcinoma tissues examined, while it was not positive in any of the hepatocellular carcinoma tissues. Similar selectivity of KL-6 mucin staining was also observed in the cHCC-CC specimens. These results suggested that KL-6 mucin might be a useful tumor marker for distinguishing intrahepatic cholangiocarcinoma tissues from hepatocellular carcinoma tissues.The main conclusions were as follows:1. Sustained localization of KL-6 mucin in circumferential membrane and/or cytoplasm in invasion front of gastric cancer tissues was significantly associated with invasion, metastasis and prognosis of gastric cancer. Aaberrant localization ofβ-catenin in invasion front was proved to be an independent prognosis indicator of gastric cancer.2. KL-6 mucin overexpression was closely associated with worse tumor behaviors such as invasion and metastasis, though the cases of pancreatic cancer tissues in this study are limited. O-glycosylation of KL-6 mucin was closely related with the metastasis of pancreatic cancer. Therapeutic strategies that target O-glycosylation of KL-6 mucin may be a useful for the treatment of aggressive pancreatic cancer. KL-6 mucin can influence the aggressive behaviors of pancreatic cancer, such as proliferation and metastasis, by regulatingβ-catenin subcelluar localization.3. Combined evaluation of KL-6 mucin expression in circumferential membrane and/or cytoplasm and decreased membranous (3-catenin expression was a useful biomarker for distinguishing a subgroup of colorectal carcinoma patients with a worse prognosis.4. KL-6 mucin circumferential membrane and/or cytoplasm expression may be an indicator for liver metastasis of colorectal carcinoma. Detection of KL-6 mucin may be helpful in distinguishing hepatocellular carcinoma from metastatic liver cancer.5. KL-6 mucin might be a useful serological marker to distinguish intrahepatic cholangiocarcinoma patients from other liver cancer patients. Thus, KL-6 mucin may be a useful tumor marker for distinguishing intrahepatic cholangiocarcinoma from hepatocellular carcinoma.
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