Gastrointestinal
Functional Expression of Mucin1 in Human Duodenal Adenocarcinoma

https://doi.org/10.1016/j.jss.2019.01.006Get rights and content

Abstract

Objective

Mucin1 (MUC1), a member of the mucin family, is a glycoprotein which is often expressed in malignant cells. However, the expression and function of MUC1 in human duodenal adenocarcinoma (DAC) has not yet been characterized because of its low frequency. Here, we examined the functional roles of core protein (MUC1-C) in DAC.

Materials and methods

Using a human duodenal cancer cell line, HuTu80, proliferation, migration, invasion, ALDH activity was assessed by cell counting kit–8, scratch wound healing, matrigel invasion, and ALDEFUOR assays, respectively. The function of MUC1 protein was evaluated with knockdown using specific siRNA as well as anti-MUC1-C peptide, GO203. MUC1 expression in human DAC was evaluated immunohistochemically in surgically resected tumors.

Results

The positive expression of MUC1 in HuTu80 was confirmed by RT-PCR and flow cytometry. In vitro cell growth was inhibited by the addition of 50-100 μM GO203 as well as treatment with siRNA for MUC1-C. Silencing of MUC1-C also significantly reduced migration, invasion, ALDH activity. Local injection of GO-203 (14 mg/kg) significantly suppressed the growth of subcutaneous HuTu80 tumors in nude mice. Immunohistochemically, MUC1 was strongly detected in seven DAC cases, but not in 11 others. The outcome of patients with high MUC1 expression was significantly worse than those without MUC1 expression.

Conclusions

These results suggest that MUC1 is functionally associated with the malignant potential of DAC and could be a novel therapeutic target for this rare tumor.

Introduction

Duodenal adenocarcinoma (DAC) is a rare malignancy that constitutes 0.4% of all gastrointestinal malignancies.1 Despite technical advances in the diagnosis and surgical resection with decreased perioperative mortality and morbidity, the 5-y survival is still 45%-55%.1, 2, 3, 4 Although pancreatoduodenectomy with concomitant en bloc lymphadenectomy remains the mainstay of the treatment for the patients with DAC, the resectability rate has been reported to be 45% to 87%, probably because the diagnosis is often established relatively late in the course of the disease as well as the technical difficulties associated with curative resection.3, 5, 6, 7, 8, 9 The role of adjuvant radiotherapy and/or chemotherapy in the treatment of DAC is not yet well defined.10 More importantly, due to the rarity of the disease, only limited information is available regarding prognostic factors associated with DAC, although nodal and margin status after surgical resection have been reported to be associated with patient outcomes.4, 7, 11

Mucins are broadly defined as proteins containing from 50% to 90% of their molecular mass as O-linked oligosaccharides. Mucin1 (MUC1) is a transmembrane mucin with a large, heavily glycosylated extracellular domain and a highly conserved cytoplasmic tail, which is normally expressed on the apical surface of various secretory epithelia and several hematopoietic cell lineages.12 MUC1 consists of two subunits assembled by a noncovalent link, MUC1 N-terminal (MUC1-N) and MUC1 C-terminal (MUC1-C).13 The MUC1-N subunit (>250k Da) consists of variable numbers of 20 amino-acid tandem repeats (TRs) on which are linked hundreds of O-glycans. The MUC1-C subunit is a transmembrane domain and a 72 amino-acid cytoplasmic tail containing a CQC motif, which can interact with a variety of proteins involved in cell proliferation and adhesions such as EGFR family, c-Src, PKC-δ, and β-catenin.14, 15, 16, 17

Previous studies have demonstrated that MUC1 is critically related to the malignant features of tumor cells, such as EMT18, 19, 20, 21, 22 and stemness characteristics.23, 24 High expression of MUC1 correlates with metastatic potential and poor prognosis in gastric,25 colorectal,26 breast,27 lung,27 and pancreatic28 cancers. However, the biological and therapeutic relevance of MUC1 to DAC is unknown. In this study, we characterized the functional expression of MUC1 in human DAC both in vitro and immunohistochemically and asked whether MUC1 could be used as a therapeutic target.

Section snippets

Cell lines, reagents, and antibodies

The human duodenal cancer cell line HuTu80, pancreatic cancer cell line Panc1, cervix adenocarcinoma cell line HeLa and colon cancer cell line Caco2 were obtained from the American Tissue Culture Collection (Manassas, VA) and cultured in complete DMEM medium (Sigma-Aldrich, St. Louis, MO, USA) with 10% FBS (ThermoFisher Scientific, Massachusetts), 1% Pen-Strep-Glut (ThermoFisher Scientific). Cells were maintained at 37°C and 5% CO2. The cells were passaged at >80% confluence using 0.25% (w/v)

MUC1 was expressed on the human duodenal cancer cell line, HuTu80, and was successfully suppressed by siRNA

First, we examined MUC1 expression in various cancer cells for both mRNA and protein levels. As shown in Figure 1A, MUC1 mRNA was strongly detected in HuTu80 as well as HeLa and Panc1 as compared with Caco2. Consistent with these results, flow cytometry analysis confirmed positive expression of MUC1 protein in these three tumor cell lines but only weak expression on Caco2 (Fig. 1B). However, after treatment with siRNA specific for MUC1-C for 72 hr, the downregulation of MUC1 gene and protein

Discussion

MUC1 is a highly glycosylated heterodimeric transmembrane protein which is aberrantly overexpressed in various malignancies.25, 26, 27, 28 Previous studies have shown that MUC1 core protein (MUC1-C) plays a vital role in the induction of malignant property in tumor cells.18, 19, 20, 21, 22 In the present study, we confirmed that suppression of MUC1-C with MUC1-C siRNA strongly reduces growth of HuTu80 cells by the induction of cell cycle arrest at the GO/G1 phase. This is consistent with other

Acknowledgment

Authors' contributions: S.S., A.M., H.O., T.T., and J.K. conceived and designed the experiments. S.S., H.O., and T.T. performed the experiments. S.S., A.M., Y.S., J.K., and N.S. analyzed the data. A.M., H.O., T.T., Y.S., J.K., and N.S. contributed reagents/materials/analysis tools. S.S., A.M., A.K.L., J.K., and N.S. contributed to the writing of the manuscript. This work was supported by a Japan Society for the Promotion of Science (17H04286). The management of LSRFortessa at Jichi Medical

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