MUC1 Promoter Research Articles

Mucin Expression Profiles in Ulcerative Colitis: New Insights on the Histological Mucosal Healing.

A structural weakness of the mucus barrier (MB) is thought to be a cause of ulcerative colitis (UC). This study aims to investigate the mucin (MUC) composition of MB in normal mucosa and UC. Ileocolonic biopsies were taken at disease onset and after treatment in 40 patients, including 20 with relapsing and 20 with remitting UC. Ileocolonic biopsies from 10 non-IBD patients were included as controls. Gut-specific MUC1, MUC2, MUC4, MUC5B, MUC12, MUC13, MUC15, and MUC17 were evaluated immunohistochemically. The promoters of mucin genes were also examined. Normal mucosa showed MUC2, MUC5B, and MUC13 in terminal ileum and colon, MUC17 in ileum, and MUC1, MUC4, MUC12, and MUC15 in colon. Membranous, cytoplasmic and vacuolar expressions were highlighted. Overall, the mucin expression was abnormal in UC. Derangements in MUC1, MUC4, and MUC5B were detected both at onset and after treatment. MUC2 and MUC13 were unaffected. Sequence analysis revealed glucocorticoid-responsive elements in the MUC1 promoter, retinoic-acid-responsive elements in the MUC4 promoter, and butyrate-responsive elements in the MUC5B promoter. In conclusion, MUCs exhibited distinct expression patterns in the gut. Their expression was disrupted in UC, regardless of the treatment protocols. Abnormal MUC1, MUC4, and MUC5B expression marked the barrier dysfunction in UC.

The function of long non-coding RNA SNHG11 and its working mechanism in triple-negative breast cancer

Triple-negative breast cancer (TNBC) seriously affects woman’s health. The present work is to study the working mechanism of lncRNA SNHG11 in TNBC. The expressions of SNHG11, microRNA (miR)− 7–5p, specificity protein 2 (SP2) and mucin 1 (MUC-1) in TNBC tissues and cells were detected. SNHG11, miR-7–5p and SP2 expressions were then evaluated for TNBC cell malignant behaviors. The relationships among SNHG11, miR-7–5p and SP2 were predicted and verified. Finally, the binding of the transcription factor SP2 to MUC-1 promoter was detected. Abnormally elevated SNHG11, SP2 and MUC-1 expressions were observed in cultured TNBC cells and tumor tissues. SNHG11 knockdown in TNBC cells. Silencing SP2 weakened the promoting effect of SNHG11 on TNBC progression. SNHG11 negatively regulated miR-7–5p expression and positively regulated SP2 expression. SP2 bound to the P2 site of MUC-1 promoter, and SP2 knockdown suppressed MUC-1 expression. It was demonstrated that lncRNA SNHG11 promoted TNBC cell malignant behaviors to facilitate TNBC progression. The study is first of its kinds to unravel the potential of lncRNA SNHG11 in relation to TNBC.

Predicted Prognosis of Patients with Pancreatic Cancer by Machine Learning.

Pancreatic cancer remains a disease of high mortality despite advanced diagnostic techniques. Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in pancreatic cancers. MUC1 and MUC4 expression are related to the aggressive behavior of human neoplasms and a poor patient outcome. In contrast, MUC2 is a tumor suppressor, and we have previously reported that MUC2 is a favorable prognostic factor in pancreatic neoplasia. This study investigates whether the methylation status of three mucin genes from postoperative tissue specimens from patients with pancreatic neoplasms could serve as a predictive biomarker for outcome after surgery. We evaluated the methylation status of MUC1, MUC2, and MUC4 promoter regions in pancreatic tissue samples from 191 patients with various pancreatic lesions using methylation-specific electrophoresis. Then, integrating these results and clinicopathologic features, we used support vector machine-, neural network-, and multinomial-based methods to develop a prognostic classifier. Significant differences were identified between the positive- and negative-prediction classifiers of patients in 5-year overall survival (OS) in the cross-validation test. Multivariate analysis revealed that these prognostic classifiers were independent prognostic factors analyzed by not only neoplastic tissues but also nonneoplastic tissues. These classifiers had higher predictive accuracy for OS than tumor size, lymph node metastasis, distant metastasis, and age and can complement the prognostic value of the TNM staging system. Analysis of epigenetic changes in mucin genes may be of diagnostic utility and one of the prognostic predictors for patients with pancreatic ductal adenocarcinoma.

Melatonin restores Muc2 depletion induced by V. vulnificus VvpM via melatonin receptor 2 coupling with G\u03b1q

BackgroundMelatonin (5-methoxy-N-acetyltryptamine), a hormone produced in the pineal gland, has a variety of biological functions as an antioxidant, but a functional role of melatonin in the regulation of intestinal mucin (Muc) production during bacterial infection has yet to be described in detail. In this study, we investigate the effects of melatonin during Muc2 repression elicited by the Gram-negative bacterium V. vulnificus.MethodsMucus-secreting human HT29-MTX cells were used to study the functional role of melatonin during Muc2 depletion induced by the recombinant protein (r) VvpM produced by V. vulnificus. The regulatory effects of melatonin coupling with melatonin receptor 2 (MT2) on the production of reactive oxygen species (ROS), the activation of PKCδ and ERK, and the hypermethylation of the Muc2 promoter as induced by rVvpM were examined. Experimental mouse models of V. vulnificus infection were used to study the role of melatonin and how it neutralizes the bacterial toxin activity related to Muc2 repression.ResultsRecombinant protein (r) VvpM significantly reduced the level of Muc2 in HT29-MTX cells. The repression of Muc2 induced by rVvpM was significantly restored upon a treatment with melatonin (1 μM), which had been inhibited by the knockdown of MT2 coupling with Gαq and the NADPH oxidase subunit p47 phox. Melatonin inhibited the ROS-mediated phosphorylation of PKCδ and ERK responsible for region-specific hypermethylation in the Muc2 promoter in rVvpM-treated HT29-MTX cells. In the mouse models of V. vulnificus infection, treatment with melatonin maintained the level of Muc2 expression in the intestine. In addition, the mutation of the VvpM gene from V. vulnificus exhibited an effect similar to that of melatonin.ConclusionsThese results demonstrate that melatonin acting on MT2 inhibits the hypermethylation of the Muc2 promoter to restore the level of Muc2 production in intestinal epithelial cells infected with V. vulnificus.

The Antioxidant Alpha-Lipoic Acid Inhibits Proliferation and Invasion of Human Gastric Cancer Cells via Suppression of STAT3-Mediated MUC4 Gene Expression.

Background Metastasis and invasion are the main causes of mortality in gastric cancer. To improve the treatment of gastric cancer, the development of effective and innovative antitumor agents toward invasion and proliferation is needed. Alpha-lipoic acid (ALA), a naturally occurring thiol antioxidant, showed antiproliferative and cytotoxic effects on several cancers. So it is feasible to explore whether ALA can be used to inhibit proliferation and invasion in human gastric cancer. Methods The expression of MUC4 in human gastric cancer tissues was assayed by immunohistochemistry. Then, we performed in vitro cell proliferation and invasion analysis to explore the antitumor effect of ALA using AGS, BGC-823, and MKN-28 cells. To further explore the mechanism of ALA-mediated downregulation of MUC4, we cotransfected human gastric cancer cells with STAT3 siRNA and STAT3 overexpression construct. ChIP assays were carried out to find the relationship between MUC4 and STAT3. Results We found that the MUC4 gene was strongly expressed in human gastric cancer tissues. Meanwhile, ALA reduced proliferation and invasion of human gastric cancer cells by suppressing MUC4 expression. We also found that STAT3 was involved in the inhibition of MUC4 by ALA. Mechanistically, ALA suppressed MUC4 expression by inhibiting STAT3 binding to the MUC4 promoter region. Conclusion ALA inhibits both proliferation and invasion of gastric cancer cells by suppression of STAT3-mediated MUC4 gene expression.

Effects of high progesterone in in-vitro fertilization cycle on DNA methylation and gene expression of adhesion molecules on endometrium during implantation window.

High progesterone is associated with low implantation rate. Our previous study demonstrated that DNA methylation in endometrium was increased in women with high progesterone in IVF cycles. However, the DNA methylation status is still not yet confirmed, and how it affects endometrial receptivity in high progesterone is still unknown. Current study investigated the effects of high progesterone on DNA methylation and gene expression of adhesion molecules on endometrium during implantation window. A cohort study included 20 women with high progesterone (HP) and 20 with normal progesterone (NP) on the day of human chorionic gonadotropin (hCG) administration after controlled ovarian hyperstimulation in IVF cycle. Endometrial tissues were collected on the 7th day after hCG administration. Immunohistochemical staining of DNA methyltransferases (DNMT1 and DNMT3B) and adhesion molecules (MUC1, CDH1 and CTNNB1) were performed. Methylation of MUC1, CDH1, and CTNNB1 promoter regions was detected by Sequenom MassARRAY or bisulfite sequencing PCR. RT-qPCR was used to quantify mRNA expression levels, and correlation of methylation and gene expression level of the adhesion molecules were determined. DNMT3B, but not DNMT1, in nucleus of luminal and glandular epithelial cells in HP group was significantly higher than that in NP group. Promoter regions of CDH1 and CTNNB1, but not MUC1, in endometrium of HP group were hypermethylated. Protein and mRNA expression of MUC1, CDH1, and CTNNB1 in endometrium of HP group was significantly lower than that in NP group. Level of DNA methylation was negatively correlated with the gene expression of CDH1 and CTNNB1, but not MUC1. DNA hypermethylation and low expression of adhesion molecules on endometrium were associated with high progesterone during implantation window, which may contribute to the underlying epigenetic mechanism in the failure of IVF treatment.

Polyethyleneimine-polyethylene glycol copolymer targeted by anti-HER2 nanobody for specific delivery of transcriptionally targeted tBid containing construct

The present research seeks to investigate the process of mixing targeted gene delivery and transcriptional targeting. We have conjugated Polyethylenimine polymers (PEI) and molecules of poly (ethylene glycol). The next step was covalent attachment of anti-HER2 variables domains of camelid heavy chains antibodies (VHHs) or nanobodies (Nbs) to the distal terminals of NHS-PEG3500 in PEI-PEG nanoparticles. The whole procedure yielded PEI-PEG-Nb immunoconjugates. Having determined the properties of polyplexes, steps were taken to investigate the most efficient ratio of PEI polymers to pDNA molecules (N/P) so that the greatest rate of transfection may be obtained. This immune targeted nano biopolymer could condense the gene constructs that coded a transcriptionally targeted truncated –Bid (tBid) killer gene which was controlled by the breast cancer-specific MUC1 promoter. The favourable physicochemical properties matching both the size and zeta potential were observed in engineered polyplexes. Elevated transfection efficiency in HER2 positive cell lines using Nb-modified polyplexes were shown by the results of flow cytometry as compared against non-modified particles. 1.6 and 4.8 fold higher transfection efficiencies were observed in in vitro gene expression researches which used PEI-PEG-Nb/pGL4.50 compared to the situation when native PEI polymers were utilized in both BT-474 and SK-BR-3, respectively. A 2.22 and 3.62 fold rise in the level of tBid gene expression in BT-474 and SK-BR-3 cell lines relative to unmodified PEI treated cells was the result of transfection with PEI-PEG-Nb/pMUC1-tBid, respectively. In those HER2-positive cells which were transfected by targeted polyplexes, higher levels of cell death were observed. This fact points not only to the effective targeted delivery, but it is also indicative of transcriptional targeting efficiency of tBid killer gene when its expression is controlled by MUC1 promoter.

Nuclear orphan receptor NR2F6 as a safeguard against experimental murine colitis

ObjectiveNuclear receptors are known to regulate both immune and barrier functions in the GI tract. The nuclear orphan receptor NR2F6 has been shown to suppress the expression of proinflammatory cytokines...

062 Identification of the MUC2 promoter as a strong promoter for intestinal gene expression through generation of transgenic quail expressing GFP in gut epithelial cells

062 Identification of the MUC2 promoter as a strong promoter for intestinal gene expression through generation of transgenic quail expressing GFP in gut epithelial cells

The hypermethylation of MUC2 promoter associated with mRNA and metastasis in esophageal squamous cell carcinoma

Background: We evaluated the association between MUC2 methylation status and mRNA level in esophageal squamous cell carcinoma (ESCC) patients. Methods: The methylation index was examined by methylation-specific PCR and MUC2 mRNA was examined in paired tissue samples of 310 ESCC patients by real-time PCR. Results: The mean of MUC2 methylation index was 0.68 in ESCCs, and only 0.45 in non-tumor samples. There was a significant association between MUC2 methylation and lymph node metastasis, distant metastasis in ESCC. The lower level of MUC2 mRNA were in ESCC tissues (Mean −∆Ct =−6.65), and higher in Non-tumor tissues (Mean −∆Ct =−4.11). The lower MUC2 mRNA (−ΔΔCt MUC2 ≤−2.54) was 112/185 (60.54%) in ESCC with AJCC stage III-IV, 105/162 (64.81%) with N2–3 Lymph node metastasis, and 99/142 (69.72%) with differentiation G2. Meanwhile, the level of MUC2 mRNA were lower in ESCC patients with methylated promoter (Mean −∆∆Ct =−3.58) than those with hypomethylation (Mean −∆∆Ct =−0.32). And the ESCC patients with MUC2 silencing and hypermethylation in promoter had an unfavorable outcome. Conclusions: The results implied that the hypermethylation could be an important factor for silencing of MUC2 mRNA and a potential biomarker for poor survival in ESCC.

The hypermethylation of MUC2 promoter associated with mRNA and metastasis in esophageal squamous cell carcinoma

The hypermethylation of MUC2 promoter associated with mRNA and metastasis in esophageal squamous cell carcinoma

Abstract 3009: Targeting G-protein coupled receptor-related pathway in mucinous tumors

Abstract Introduction: Pseudomyxoma peritonei (PMP) is an indolent malignancy characterized by excessive production of Mucin 2 (MUC2; a gel-forming secreted mucin). We have previously demonstrated mucinous tumor growth inhibition in a patient-derived murine xenograft model of PMP using the cyclooxygenase-2 (COX-2) inhibitor celecoxib. We evaluated the downstream mechanism for celecoxib-mediated MUC2-inhibition. Methods: In vitro and in vivo studies were conducted using MUC2-secreting LS174T cells, PMP explant tissue and a unique intraperitoneal patient-derived murine xenograft model of PMP (PDX model of PMP). Results: LS174T cells treated with COX-2 inhibitor celecoxib (5-20 μM) for 24 hours demonstrated a significant reduction in MUC2 protein expression measured by flow cytometric analysis. Similarly, in the PDX model of PMP chronic oral therapy with celecoxib (20 mg/kg/d) for 28 days significantly inhibited MUC2 protein expression in harvested tumor tissue. In the LS174T cells, PGE2 (a COX-2 product) induced MUC2 mRNA (real-time PCR) and protein expression (flow cytometry) and this was inhibited by the G-protein coupled-EP4 receptor inhibitor AH23848 as well as siRNA for CREB (cAMP response element-binding protein). Using chromatin immunoprecipitation assay in LS174T cells, we found that treatment with celecoxib or protein kinase A inhibitor (fragment 6-22 amide) significantly decreased CREB-transcription factor binding to the MUC2 promoter. Conclusions: These data suggest that celecoxib inhibits MUC2 expression via the G-protein coupled receptor/ cAMP/ Protein kinase A/ CREB pathway. Since activating G-protein (GNAS) mutations are commonly found in PMP and may be responsible for its unique mucinous phenotype, our data provide a rationale for targeting this pathway to control mucin production in this malignancy. Citation Format: Ashok K. Dilly, Yong J. Lee, Herbert J. Zeh, David L. Bartlett, Mohammad Haroon A. Choudry. Targeting G-protein coupled receptor-related pathway in mucinous tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3009.

MUC4 is negatively regulated through the Wnt/β-catenin pathway via the Notch effector Hath1 in colorectal cancer.

MUC4 is a transmembrane mucin lining the normal colonic epithelium. The aberrant/de novo over-expression of MUC4 is well documented in malignancies of the pancreas, ovary and breast. However, studies have reported the loss of MUC4 expression in the majority of colorectal cancers (CRCs). A MUC4 promoter analysis showed the presence of three putative TCF/LEF sites, implying a possible regulation by the Wnt/β-catenin pathway, which has been shown to drive CRC progression. Thus, the objective of our study was to determine whether MUC4 is regulated by β-catenin in CRC. We first knocked down (KD) β-catenin in three CRC cell lines; LS180, HCT-8 and HCT116, which resulted in increased MUC4 transcript and MUC4 protein. Additionally, the overexpression of stabilized mutant β-catenin in LS180 and HCT-8 resulted in a decrease in MUC4 expression. Immunohistochemistry (IHC) of mouse colon tissue harboring tubular adenomas and high grade dysplasia showed dramatically reduced Muc4 in lesions relative to adjacent normal tissue, with increased cytosolic/nuclear β-catenin. Luciferase assays with the complete MUC4 promoter construct p3778 showed increased MUC4 promoter luciferase activity in the absence of β-catenin (KD). Mutation of all three putative TCF/LEF sites showed that MUC4 promoter luciferase activity was increased relative to the un-mutated promoter. Interestingly, it was observed that MUC4 expressing CRC cell lines also expressed high levels of Hath1, a transcription factor repressed by both active Wnt/β-catenin and Notch signaling. The KD of β-catenin and/or treatment with a Notch γ-secretase inhibitor, Dibenzazepine (DBZ) resulted in increased Hath1 and MUC4 in LS180, HCT-8 and HCT116. Furthermore, overexpression of Hath1 in HCT-8 and LS180 caused increased MUC4 transcript and MUC4 protein. Taken together, our results indicate that the Wnt/β-catenin pathway suppresses the Notch pathway effector Hath1, resulting in reduced MUC4 in CRC.

Aberrant methylation of MUC1 and MUC4 promoters are potential prognostic biomarkers for pancreatic ductal adenocarcinomas.

Pancreatic cancer is still a disease of high mortality despite availability of diagnostic techniques. Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in pancreatic neoplasms. MUC1 and MUC4 are high molecular weight transmembrane mucins. These are overexpressed in many carcinomas, and high expression of these molecules is a risk factor associated with poor prognosis. We evaluated the methylation status of MUC1 and MUC4 promoter regions in pancreatic tissue samples from 169 patients with various pancreatic lesions by the methylation specific electrophoresis (MSE) method. These results were compared with expression of MUC1 and MUC4, several DNA methylation/demethylation factors (e.g. ten-eleven translocation or TET, and activation-induced cytidine deaminase or AID) and CAIX (carbonic anhydrase IX, as a hypoxia biomarker). These results were also analyzed with clinicopathological features including time of overall survival of PDAC patients. We show that the DNA methylation status of the promoters of MUC1 and MUC4 in pancreatic tissue correlates with the expression of MUC1 and MUC4 mRNA. In addition, the expression of several DNA methylation/demethylation factors show a significant correlation with MUC1 and MUC4 methylation status. Furthermore, CAIX expression significantly correlates with the expression of MUC1 and MUC4. Interestingly, our results indicate that low methylation of MUC1 and/or MUC4 promoters correlates with decreased overall survival. This is the first report to show a relationship between MUC1 and/or MUC4 methylation status and prognosis. Analysis of epigenetic changes in mucin genes may be of diagnostic utility and one of the prognostic predictors for patients with PDAC.

Bile acids-mediated overexpression of MUC4 via FAK-dependent c-Jun activation in pancreatic cancer

The majority of pancreatic cancer (PC) patients are clinically presented with obstructive jaundice with elevated levels of circulatory bilirubin and alkaline phosphatases. In the current study, we examined the implications of bile acids (BA), an important component of bile, on the pathophysiology of PC and investigated their mechanistic association in tumor-promoting functions. Integration of results from PC patient samples and autochthonous mouse models showed an elevated levels of BA (p < 0.05) in serum samples compared to healthy controls. Similarly, an elevated BA levels was observed in pancreatic juice derived from PC patients (p < 0.05) than non-pancreatic non-healthy (NPNH) controls, further establishing the clinical association of BA with the pathogenesis of PC. The tumor-promoting functions of BA were established by observed transcriptional upregulation of oncogenic MUC4 expression. Luciferase reporter assay revealed distal MUC4 promoter as the primary responsive site to BA. In silico analysis recognized two c-Jun binding sites at MUC4 distal promoter, which was biochemically established using ChIP assay. Interestingly, BA treatment led to an increased transcription and activation of c-Jun in a FAK-dependent manner. Additionally, BA receptor, namely FXR, which is also upregulated at transcriptional level in PC patient samples, was demonstrated as an upstream molecule in BA-mediated FAK activation, plausibly by regulating Src activation. Altogether, these results demonstrate that elevated levels of BA increase the tumorigenic potential of PC cells by inducing FXR/FAK/c-Jun axis to upregulate MUC4 expression, which is overexpressed in pancreatic tumors and is known to be associated with progression and metastasis of PC.

Functional interactions of the cystine/glutamate antiporter, CD44v and MUC1-C oncoprotein in triple-negative breast cancer cells.

The xCT light chain of the cystine/glutamate transporter (system XC−) is of importance for the survival of triple-negative breast cancer (TNBC) cells. The MUC1-C transmembrane oncoprotein is aberrantly overexpressed in TNBC and, like xCT, has been linked to maintaining glutathione (GSH) levels and redox balance. However, there is no known interaction between MUC1-C and xCT. Here we show that silencing MUC1-C is associated with decreases in xCT expression in TNBC cells. The results demonstrate that MUC1-C forms a complex with xCT and the CD44 variant (CD44v), which interacts with xCT and thereby controls GSH levels. MUC1-C binds directly with CD44v and in turn promotes stability of xCT in the cell membrane. The interaction between MUC1-C and xCT is further supported by the demonstration that targeting xCT with silencing or the inhibitor sulfasalazine suppresses MUC1 gene transcription by increasing histone and DNA methylation on the MUC1 promoter. In terms of the functional significance of the MUC1-C/xCT interaction, we show that MUC1-C protects against treatment with erastin, an inhibitor of XC− and inducer of ferroptosis, a form of non-apoptotic cell death. These findings indicate that targeting this novel MUC1-C/xCT pathway could represent a potential therapeutic approach for promoting TNBC cell death.

Generation of transgenic cattle expressing human β‐defensin 3 as an approach to reducing susceptibility to Mycobacterium bovis infection

Bovine tuberculosis results from infection with Mycobacterium bovis, a member of the Mycobacterium tuberculosis family. Worldwide, M. bovis infections result in economic losses in the livestock industry; cattle production is especially hard-hit by this disease. Generating M. bovis-resistant cattle may potentially mitigate the impact of this disease by reducing M. bovis infections. In this study, we used transgenic somatic cell nuclear transfer to generate cattle expressing the gene encoding human β-defensin 3 (HBD3), which confers resistance to mycobacteria in vitro. We first generated alveolar epithelial cells expressing HBD3 under the control of the bovine MUC1 promoter, and confirmed that these cells secreted HBD3 and possessed anti-mycobacterial capacity. We then generated and identified transgenic cattle by somatic cell nuclear transfer. The cleavage and blastocyst formation rates of genetically modified embryos provided evidence that monoclonal transgenic bovine fetal fibroblast cells have an integral reprogramming ability that is similar to that of normal cells. Five genetically modified cows were generated, and their anti-mycobacterial capacities were evaluated. Alveolar epithelial cells and macrophages from these cattle expressed higher levels of HBD3 protein compared with non-transgenic cells and possessed effective anti-mycobacterial capacity. These results suggest that the overall risk of M. bovis infection in transgenic cattle is efficiently reduced, and support the development of genetically modified animals as an effective tool to reduce M. bovis infection.

CCL21-CCR7 promotes the lymph node metastasis of esophageal squamous cell carcinoma by up-regulating MUC1.

BackgroundCCR7 and MUC1 are correlated with lymph node metastasis in ESCC, but the role of MUC1 in the CCR7-induced lymphatic metastasis and the underlying molecular mechanism is still unclear.MethodsThe expression of CCR7 and MUC1 was detected in the ESCC samples by IHC, and the clinical significance of CCR7 and MUC1 in ESCC was analyzed. The expression of CCR7 and MUC1 in ESCC cell lines was detected by qRT-PCR and western blot. The effect of CCL21 on the migration and invasion of ESCC cells was determined by transwell assay. The activity of MUC1 promoter was determined by luciferase reporter assay. The activation of Erk, Akt and Sp1 was detected by western blot and the binding of Sp1 to the MUC1 promoter was determined by ChIP.ResultsThe co-expression of CCR7 and MUC1 was detected in 153 ESCC samples by IHC, and both were correlated with lymph node metastasis, regional lymphatic recurrence and poor prognosis. Correspondingly, increasing levels of MUC1 mRNA and protein were detected in the ESCC cell lines KYSE410 and Eca9706 after treatment with CCL21 in a time- and dose-dependent manner. Furthermore, silencing MUC1 could remarkably suppress the invasion and migration of ESCC cells induced by CCL21. Moreover, heterologous CCR7 promoted the invasion and migration of KYSE150 and up-regulated MUC1 expression. Increasing levels of activated ERK1/2 and Akt were detected in KYSE410 after treating the cells with CCL21, and inhibiting the activation of ERK1/2 but not Akt caused the increased transcription of MUC1. Finally, the phosphorylation of Sp1 induced by ERK1/2 and subsequent increases in the binding of Sp1 to the muc1 promoter at −99/−90 were confirmed to cause the up-regulation of MUC1 induced by CCL21-CCR7.ConclusionsOur findings suggested that MUC1 plays an important role in CCL21-CCR7-induced lymphatic metastasis and may serve as a therapeutic target in ESCC.

Targeting hypoxia-mediated mucin 2 production as a therapeutic strategy for mucinous tumors.

Excessive accumulation of mucin 2 (MUC2; a gel-forming secreted mucin) protein in the peritoneal cavity is the major cause of morbidity and mortality in pseudomyxoma peritonei (PMP). Hypoxia (hypoxia-inducible factor-1α; HIF-1α) has been shown to regulate the expression of similar mucins (eg, MUC5AC). We hypothesized that hypoxia (HIF-1α) drives MUC2 expression in PMP and is therefore a novel target to reduce mucinous tumor growth. The regulation of MUC2 by 2% hypoxia (HIF-1α) was evaluated in MUC2-secreting LS174T cells. The effect of BAY 87-2243, an inhibitor of HIF-1α, on MUC2 expression and mucinous tumor growth was evaluated in LS174T cells, PMP explant tissue, and in a unique intraperitoneal murine xenograft model of PMP. Invitro exposure of LS174T cells to hypoxia increased MUC2 messenger RNA (mRNA) and protein expression and increased HIF-1α binding to the MUC2 promoter. Hypoxia-mediated MUC2 protein overexpression was downregulated by transfected HIF-1α small interfering RNA (siRNA) compared with scrambled siRNA in LS174T cells. BAY 87-2243 inhibited hypoxia-induced MUC2 mRNA and protein expression in LS174T cells and PMP explant tissue. In a murine xenograft model of PMP, chronic oral therapy with BAY 87-2243 inhibited mucinous tumor growth and MUC2, HIF-1α expression in the tumor tissue. Our data suggest that hypoxia (HIF-1α) induces MUC2 promoter activity to increase MUC2 expression. HIF-1α inhibition decreases MUC2 production and mucinous tumor growth, providing a preclinical rationale for the use of HIF-1α inhibitors to treat patients with PMP.

The canonical Wnt pathway regulates the metastasis-promoting mucin MUC4 in pancreatic ductal adenocarcinoma

Aberrant Wnt signaling frequently occurs in pancreatic cancer (PC) and contributes to disease progression/metastases. Likewise, the transmembrane-mucin MUC4 is expressed de novo in early pancreatic intraepithelial neoplasia (PanINs) and incrementally increases with PC progression, contributing to metastasis. To determine the mechanism of MUC4 upregulation in PC, we examined factors deregulated in early PC progression, such as Wnt/β-catenin signaling. MUC4 promoter analysis revealed the presence of three putative TCF/LEF-binding sites, leading us to hypothesize that MUC4 can be regulated by β-catenin. Immunohistochemical (IHC) analysis of rapid autopsy PC tissues showed a correlation between MUC4 and cytosolic/nuclear β-catenin expression. Knock down (KD) of β-catenin in CD18/HPAF and T3M4 cell lines resulted in decreased MUC4 transcript and protein. Three MUC4 promoter luciferase constructs, p3778, p3000, and p2700, were generated. The construct p3778, encompassing the entire MUC4 promoter, elicited increased luciferase activity in the presence of stabilized β-catenin. Mutation of the TCF/LEF site closest to the transcription start site (i.e., −2629/−2612) and furthest from the start site (i.e., −3425/−3408) reduced MUC4 promoter luciferase activity. Transfection with dominant negative TCF4 decreased MUC4 transcript and protein levels. Chromatin immunoprecipitation confirmed enrichment of β-catenin on −2629/−2612 and −3425/−3408 of the MUC4 promoter in CD18/HPAF. Functionally, CD18/HPAF and T3M4 β-catenin KD cells showed decreased migration and decreased Vimentin, N-cadherin, and pERK1/2 expression. Tumorigenicity studies in athymic nude mice showed CD18/HPAF β-catenin KD cells significantly reduced primary tumor sizes and metastases compared to scrambled control cells. We show for the first time that β-catenin directly governs MUC4 in PC.