Expression of MUC17 is regulated by HIF1α-mediated hypoxic responses and requires a methylation-free hypoxia responsible element in pancreatic cancer.

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\u27-RCGTG-3\u27), 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\u27-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

MUC4 and MUC1 expression in adenocarcinoma of the stomach correlates with vessel invasion and lymph node metastasis: an immunohistochemical study of early gastric cancer.

We have previously reported that MUC4 expression is a poor prognostic factor in various carcinomas. Our previous study also showed that MUC1 expression in gastric cancers, including the early and advanced stages is a poor prognostic factor. In the present study, the expression profiles of MUC4 and MUC1 were examined by immunohistochemistry (IHC) using two anti-MUC4 monoclonal antibodies (MAbs), 8G7 and 1G8, and anti-MUC1 MAb DF3 in 104 gastrectomy specimens of early gastric adenocarcinoma with submucosal invasion (pT1b2), including 197 histological subtype lesions. Before the IHC study of the human specimens, we evaluated the specificity of the two MAbs by Western blotting and IHC of two MUC4 mRNA expressing gastric cancer cell lines. MAb 8G7 reacted clearly, whereas MAb 1G8 did not show any reactivity, in either Western blotting or IHC. In the IHC of the gastric cancers, the expression rates of MUC4/8G7 detected by MAb 8G7, MUC4/1G8 detected by MAb 1G8 and MUC1/DF3 detected by MAb DF3 in well differentiated types (70%, 38/54; 67%, 36/54; 52%, 28/54) were significantly higher than those in poorly differentiated types (18%, 10/55; 36%, 20/55; 13%, 7/55) (

The application of methylation specific electrophoresis (MSE) to DNA methylation analysis of the 5' CpG island of mucin in cancer cells

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Result</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Pathobiological implications of mucin (MUC) expression in the outcome of small bowel cancer.

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 (

Ki-67 is a strong prognostic marker of non-small cell lung cancer when tissue heterogeneity is considered

Background: Ki-67 expression is a well-established prognostic marker in various cancers. However, Ki-67 expression is also known as being heterogeneous. We investigated the prognostic significance of Ki-67 from the view of staining heterogeneity by the technique of Spiral Array. Methods. 100 cases of resected lung cancer from Toyama university hospital archive were collected. Spiral Array blocks were generated out of 100 cases using 100 μm thick paraffin sections. Four μm thick sections of the Array block were stained for Ki-67. Staining results in each reel were scored for areas with lowest (LS), highest (HS), and average (AS) expression, exclusively in the cancer cells. Heterogeneity score (HeS) was designed as the difference between HS and LS. The scores were divided into four grades (0-3). Clinical information was collected, and the prognostic significance of Ki-67 was analyzed. Results: Pathological stage was available for 91 patients (43 stage IA, 22 stage IB, 2 stage IIA, 9 stage IIB, 13 stage IIIA, 1 stage IIIB, and 1 stage IV). The HS of Ki-67 score in non-small cell lung cancer was 3 in 17 cases, 2 in 27 cases, 1 in 28 cases, 0 in 21 cases, and 4 reels were lost. 78 cases had clinical follow up. 74 cases had all the information available and were analyzed for correlation between Ki-67 expression and survival. Cases with score 2 and 3 of HS and HeS showed significant poorer prognosis (both P < 0.001), whereas LS or AS did not show significance. The results were identical when analyzing adenocarcinoma and squamous cell carcinoma, separately. Cox multivariate analysis of Ki-67 showed that HS was an independent risk factor affecting overall survival. Conclusions: Ki-67 is a strong prognostic marker for non-small cell lung cancer when the degree of highest staining frequency or heterogeneity is considered

Aberrant overexpression of membrane-associated mucin contributes to tumor progression in adult T-cell leukemia/lymphoma cells.

Aberrant overexpression of membrane-associated mucin (MUC1) is implicated in the pathogenesis of cancer, particularly of adenocarcinomas. Adult T-cell leukemia/lymphoma (ATL), an aggressive neoplasm etiologically associated with human T-lymphotropic virus type-1 (HTLV-1), exhibits invasive tropism into various organs, resulting in disease progression and resistance to treatment. In the present study, we showed that MUC1 is overexpressed exclusively in cells of ATL among hematological malignancies. Furthermore, increased expression of MUC1 correlated with a poor prognosis, suggesting MUC1 to be a prognostic marker in ATL. Various functional analyses with knockdown experiments using a specific siRNA for MUC1 revealed that MUC1 is involved in cell growth, cell aggregation, and resistance to apoptosis. Although it has been shown that the anti-adhesive properties of MUC1 facilitate migration and metastasis of tumor cells, our findings indicated that MUC1 contributes to cell-cell adhesion. Mucins thus seem to play a role in the pathogenesis and/or progression of ATL