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Objectives: The aim of this study was to further examine the utility of mucin (MUC) expression profiles as prognostic factors in pancreatic ductal adenocarcinoma (PDAC)., Methods: Mucin expression was examined by immunohistochemistry analysis in endoscopic ultrasound-guided fine-needle aspiration specimens obtained from 114 patients with PDAC. The rate of expression of each MUC was compared with clinicopathologic features., Results: The expression rates of MUCs in cancer lesions were MUC1, 87.7%; MUC2, 0.8%; MUC4, 93.0%; MUC5AC, 78.9%; MUC6, 24.6%; and MUC16, 67.5%. MUC1 and MUC4 were positive, and MUC2 was negative in most PDACs. Patients with advanced stage of PDAC with MUC5AC expression had a significantly better outcome than those who were MUC5AC-negative (P = 0.002). With increasing clinical stage, total MUC6 expression decreased (P for trend = 0.001) and MUC16 cytoplasmic expression increased (P for trend = 0.02). The prognosis of patients with MUC16 cytoplasmic expression was significantly poorer than those without this expression. Multivariate survival analysis revealed that MUC16 cytoplasmic expression was a significant independent predictor of a poor prognosis after adjusting for the effects of other prognostic factors (P = 0.002)., Conclusions: Mucin expression profiles in ultrasound-guided fine-needle aspiration specimens have excellent diagnostic utility and are useful predictors of outcome in patients with PDAC.

Background: Mucins are implicated in survival in various cancers, but there have been no report addressed on survival in appendiceal carcinoma, an uncommon disease with different clinical and pathological features from those of other colon cancers. We aimed to investigate the clinical implications of expression of mucins in appendiceal carcinoma., Methods: Expression profiles of MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC6, MUC16 and MUC17 in cancer tissue were examined by immunohistochemistry in 108 cases of surgically resected appendiceal carcinoma., Results: The following relationships of mucins with clinicopathologic factors were identified: MUC1 with positive lymphatic invasion (p = 0.036); MUC2 with histological type (mucinous carcinoma, p<0.001), superficial invasion depth (p = 0.007), negative venous invasion (p = 0.003), and curative resection (p = 0.019); MUC3 with non-curative resection (p = 0.017); MUC5AC with histological type (mucinous carcinoma, p = 0.002), negative lymphatic invasion (p = 0.021), and negative venous invasion (p = 0.022); and MUC16 with positive lymph node metastasis (p = 0.035), positive venous invasion (p<0.05), and non-curative resection (p = 0.035). A poor prognosis was related to positive lymph node metastasis (p = 0.04), positive lymphatic invasion (p = 0.02), positive venous invasion (p<0.001), non-curative resection (p<0.001), and positive expression of MUC3 (p = 0.004). In multivariate analysis, positive venous invasion (HR: 6.93, 95% CI: 1.93-24.96, p = 0.003), non-curative resection (HR: 10.19, 95% CI: 3.05-34.07, p<0.001) and positive MUC3 expression (HR: 3.37, 95% CI: 1.13-10.03, p = 0.03) were identified as significant independent prognostic factors in patients with appendiceal carcinoma., Conclusions: Expression of MUC3 in appendiceal carcinoma is an independent factor for poor prognosis and a useful predictor of outcome in patients with appendiceal carcinoma after surgery.

Background: Methylation of CpG sites in genomic DNA plays an important role in gene regulation and especially in gene silencing. We have reported mechanisms of epigenetic regulation for expression of mucins, which are markers of malignancy potential and early detection of human neoplasms. Epigenetic changes in promoter regions appear to be the first step in expression of mucins. Thus, detection of promoter methylation status is important for early diagnosis of cancer, monitoring of tumor behavior, and evaluating the response of tumors to targeted therapy. However, conventional analytical methods for DNA methylation require a large amount of DNA and have low sensitivity., Methods: Here, we report a modified version of the bisulfite-DGGE (denaturing gradient gel electrophoresis) using a nested PCR approach. We designated this method as methylation specific electrophoresis (MSE). The MSE method is comprised of the following steps: (a) bisulfite treatment of genomic DNA, (b) amplification of the target DNA by a nested PCR approach and (c) applying to DGGE. To examine whether the MSE method is able to analyze DNA methylation of mucin genes in various samples, we apply it to DNA obtained from state cell lines, ethanol-fixed colonic crypts and human pancreatic juices., Result: The MSE method greatly decreases the amount of input DNA. The lower detection limit for distinguishing different methylation status is < 0.1% and the detectable minimum amount of DNA is 20 pg, which can be obtained from only a few cells. We also show that MSE can be used for analysis of challenging samples such as human isolated colonic crypts or human pancreatic juices, from which only a small amount of DNA can be extracted., Conclusions: The MSE method can provide a qualitative information of methylated sequence profile. The MSE method allows sensitive and specific analysis of the DNA methylation pattern of almost any block of multiple CpG sites. The MSE method can be applied to analysis of DNA methylation status in many different clinical samples, and this may facilitate identification of new risk markers.

MUC17 is a type 1 membrane-bound glycoprotein that is mainly expressed in the digestive tract. Recent studies have demonstrated that the aberrant overexpression of MUC17 is correlated with the malignant potential of pancreatic ductal adenocarcinomas (PDACs); however, the exact regulatory mechanism of MUC17 expression has yet to be identified. Here, we provide the first report of the MUC17 regulatory mechanism under hypoxia, an essential feature of the tumor microenvironment and a driving force of cancer progression. Our data revealed that MUC17 was significantly induced by hypoxic stimulation through a hypoxia-inducible factor 1α (HIF1α)-dependent pathway in some pancreatic cancer cells (e.g., AsPC1), whereas other pancreatic cancer cells (e.g., BxPC3) exhibited little response to hypoxia. Interestingly, these low-responsive cells have highly methylated CpG motifs within the hypoxia responsive element (HRE, 5'-RCGTG-3'), a binding site for HIF1α. Thus, we investigated the demethylation effects of CpG at HRE on the hypoxic induction of MUC17. Treatment of low-responsive cells with 5-aza-2'-deoxycytidine followed by additional hypoxic incubation resulted in the restoration of hypoxic MUC17 induction. Furthermore, DNA methylation of HRE in pancreatic tissues from patients with PDACs showed higher hypomethylation status as compared to those from non-cancerous tissues, and hypomethylation was also correlated with MUC17 mRNA expression. Taken together, these findings suggested that the HIF1α-mediated hypoxic signal pathway contributes to MUC17 expression, and DNA methylation of HRE could be a determinant of the hypoxic inducibility of MUC17 in pancreatic cancer cells.

Mucins are high molecular weight glycoproteins that play important roles in carcinogenesis and tumor invasion. Our immunohistochemical studies demonstrated that MUC1 or MUC4 expression is related to the aggressive behavior and poor outcome of human neoplasms. MUC2 is expressed in indolent pancreatobiliary neoplasms, but these tumors sometimes show invasive growth with MUC1 expression in invasive areas. MUC5AC shows de novo high expression in many types of precancerous lesions of pancreatobiliary cancers and is an effective marker for early detection of the neoplasms. The combination of MUC1, MUC2, MUC4 and MUC5AC expression may be useful for early detection and evaluation of the potential for malignancy of pancreatobiliary neoplasms. Regarding the mechanism of mucin expression, we have recently reported that expression of the mucin genes is regulated epigenetically in cancer cell lines, using quantitative MassARRAY analysis, methylation-specific polymerase chain reaction analysis and chromatin immunoprecipitation analysis, with confirmation by the treatment with 5-aza-2'-deoxycytidine and trichostatin A. We have also developed a monoclonal antibody against the MUC1 cytoplasmic tail domain, which has many biological roles. Based on all of the above findings, we suggest that translational research into mucin gene expression mechanisms, including epigenetics, may provide new tools for early and accurate detection of human neoplasms., (© 2011 The Authors. Pathology International © 2011 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.)

Mucins are a family of heavily glycosylated high molecular mass proteins that have great potential as novel clinical biomarkers for the diagnosis of various malignant tumors. Supported molecular matrix electrophoresis (SMME) is a new type of membrane electrophoresis that can be used to characterize mucins. In SMME, mucins migrate in a molecular matrix supported by membrane materials. Here, we have developed an immunostaining method for the identification of SMME-separated mucins. The novel method involves stably fixing the mucins onto the SMME membrane and optimizing the molecular matrix for the fixation process. We applied this technique for the detection of MUC1 produced from three cancer cell lines (T47D, HPAF-II and BxPC3) and also analyzed their O-linked glycans by mass spectrometry. Our results revealed that properties of the MUC1 molecules from the three cell lines are different in terms of migrating position in SMME and glycan profile. The present method allows simple and rapid characterization of mucins in terms of both glycans and core proteins. The method will be a useful tool for the exploration of mucin alterations associated with various diseases such as cancer., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

MUC17 glycoprotein is a membrane-associated mucin that is mainly expressed in the digestive tract. It has been suggested that MUC17 expression is correlated with the malignancy potential of pancreatic ductal adenocarcinomas (PDACs). In the present study, we provided the first report of the MUC17 gene expression through epigenetic regulation such as promoter methylation, histone modification and microRNA (miRNA) expression. Near the transcriptional start site, the DNA methylation level of MUC17-negative cancer cell lines (e.g. PANC1) was high, whereas that of MUC17-positive cells (e.g. AsPC-1) was low. Histone H3-K9 (H3-K9) modification status was also closely related to MUC17 expression. Our results indicate that DNA methylation and histone H3-K9 modification in the 5' flanking region play a critical role in MUC17 expression. Furthermore, the hypomethylation status was observed in patients with PDAC. This indicates that the hypomethylation status in the MUC17 promoter could be a novel epigenetic marker for the diagnosis of PDAC. In addition, the result of miRNA microarray analysis showed that five potential miRNA candidates existed. It is also possible that the MUC17 might be post-transcriptionally regulated by miRNA targeting to the 3'-untranslated region of its mRNA. These understandings of the epigenetic changes of MUC17 may be of importance for the diagnosis of carcinogenic risk and the prediction of outcomes for cancer patients.

Mucins are high molecular weight glycoproteins that play important roles in carcinogenesis and tumor invasion. We have described, for the first time, that pancreatic ductal adenocarcinomas (PDACs) with an aggressive behavior and a poor outcome expressed MUC1 (pan-epithelial membrane-associated mucin) but did not express MUC2 (intestinal-type secreted mucin), whereas intraductal papillary mucinous neoplasms (IPMNs) with indolent behavior and a favorable outcome did not express MUC1 but did express MUC2. These expression profiles of MUC1 and MUC2 related to the prognoses of the patients were also observed in biliary neoplasms such as intrahepatic cholangiocarcinoma (ICC)-mass-forming type (MF), mucin-producing bile duct tumor (MPBT), and extrahepatic bile duct carcinoma (EHBDC). We also found recently that high expression of MUC4 (tracheobronchial membrane-associated mucin) in PDACs, ICCs-MF, and EHBDCs was a new independent poor prognostic factor, although MUC4 was not expressed in normal pancreatobiliary tissue. High de novo expression of MUC5AC (gastric-type secreted mucin) was observed in many types of pancreatobiliary neoplasms, including all grades of pancreatic intraepithelial neoplasia (PanIN) and biliary intraepithelial neoplasia (BilIN), and all types of IPMNs and MPBTs, as well as PDACs and ICCs-MF, although MUC5AC was not expressed in normal pancreatobiliary tissue. The combined status of MUC1, MUC2, MUC4, and MUC5AC expression may be useful for the early detection of pancreatobiliary neoplasms and evaluation of their malignancy. In regard to the mechanism of mucin expression, we have recently reported that MUC1, MUC2, MUC4, and MUC5AC gene expression is regulated by epigenetics (DNA methylation and histone H3 lysine 9 modification) in cancer cell lines, including PDAC cells. Translational research of mucin gene expression mechanisms, including epigenetics, in pancreatobiliary neoplasms may give us new tools for the early and accurate detection of these neoplasms.

Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasms (IPMNs). Our immunohistochemistry (IHC) studies have shown a consensus position on mucin expression profiles in pancreatic neoplasms as follows: MUC1-positive but MUC2-negative expression in PDACs; MUC1-negative but MUC2-positive expression in intestinal-type IPMNs (dangerous type); MUC1-negative and MUC2-negative expression in gastric-type IPMNs (safe type); High MUC4 expression in PDAC patients with a poor outcome; and MUC4-positive expression in intestinal-type IPMNs. We also showed that three mucin genes (MUC1, MUC2 and MUC4) expression in cancer cell line was regulated by DNA methylation. We have developed a novel ‘methylation-specific electrophoresis (MSE)’ method to analyze the DNA methylation status of mucin genes by high sensitivity and resolution. By using the MSE method, we evaluated pancreatic juice samples from 45 patients with various pancreatic lesions. The results were compared with final diagnosis of the pancreatic lesions including IHC of mucin expression in the paired pancreatic tissues. The results indicated that the DNA methylation status of MUC1, MUC2 and MUC4 in pancreatic juice matched with the mucin expression in tissue. Analyses of the DNA methylation status of MUC1, MUC2 and MUC4 were useful for differential diagnosis of human pancreatic neoplasms, with specificity and sensitivity of 87% and 80% for PDAC; 100% and 88% for intestinal-type IPMN; and 88% and 77% for gastric-type IPMN, respectively. In conclusion, MSE analysis of human pancreatic juice may provide useful information for selection of treatment for pancreatic neoplasms. [ABSTRACT FROM AUTHOR]

Mucins have been associated with survival in various cancer patients, but there have been no studies of mucins in small bowel carcinoma (SBC). In this study, we investigated the relationships between mucin expression and clinicopathologic factors in 60 SBC cases, in which expression profiles of MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC6 and MUC16 in cancer and normal tissues were examined by immunohistochemistry. MUC1, MUC5AC and MUC16 expression was increased in SBC lesions compared to the normal epithelium, and expression of these mucins was related to clinicopathologic factors, as follows: MUC1 [tumor location (p = 0.019), depth (p = 0.017) and curability (p = 0.007)], MUC5AC [tumor location (p = 0.063) and lymph node metastasis (p = 0.059)], and MUC16 [venous invasion (p = 0.016) and curability (p = 0.016)]. Analysis of 58 cases with survival data revealed five factors associated with a poor prognosis: poorly-differentiated or neuroendocrine histological type (p<0.001), lymph node metastasis (p<0.001), lymphatic invasion (p = 0.026), venous invasion (p<0.001) and curative resection (p<0.001), in addition to expression of MUC1 (p = 0.042), MUC5AC (p = 0.007) and MUC16 (p<0.001). In subsequent multivariate analysis with curability as the covariate, lymph node metastasis, venous invasion, and MUC5AC and/or MUC16 expression were significantly related to the prognosis. Multivariate analysis in curative cases (n = 45) showed that SBC with MUC5AC and/or MUC16 expression had a significantly independent high hazard risk after adjusting for the effects of venous invasion (hazard ratio: 5.6, 95% confidence interval: 1.8–17). In conclusion, the study shows that a MUC5AC-positive and/or MUC16-positive status is useful as a predictor of a poor outcome in patients with SBC. [ABSTRACT FROM AUTHOR]

Abstract: The characterization of mucins is critically important for gaining insights into the molecular pathology of diseases, including cancers, as well as for the discovery of biomarkers for disease diagnosis. However, no practical method has yet been reported for identifying mucin proteins. Here, we report a technique for immunological identification of mucins separated by supported molecular matrix electrophoresis (SMME), a recently developed membrane electrophoresis method. The technique involves on-membrane deglycosylation of mucins by using mild periodate oxidation/base-catalyzed elimination, followed by immunostaining with an antibody that specifically recognizes the mucin tandem repeat (TR) peptide. We demonstrated the method's feasibility by using MUC1 derived from 2 cancer cell lines, T47D and HPAF-II. The present method shows potential as an alternative approach for the identification of mucins separated by SMME or blotted from conventional gel electrophoresis, on a PVDF membrane. [Copyright &y& Elsevier]