MUC13 promotes intrahepatic cholangiocarcinoma progression via EGFR/PI3K/AKT pathways

doi:10.1016/j.jhep.2019.11.021

Journal of Hepatology

Volume 72, Issue 4, April 2020, Pages 761-773

https://doi.org/10.1016/j.jhep.2019.11.021 Get rights and content

Highlights

•
Mucin 13 is overexpressed and predicts poor survival among patients with intrahepatic cholangiocarcinoma.
•
Mucin 13 promotes intrahepatic cholangiocarcinoma progression by activating the EGFR/PI3K/AKT signaling pathway.
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Mucin 13 is upregulated by hypermethylation-induced miR-212-3p downregulation in intrahepatic cholangiocarcinoma.

Background & Aims

Mucin 13 (MUC13) is reportedly overexpressed in human malignancies. However, the clinicopathological and biological significance of MUC13 in human intrahepatic cholangiocarcinoma (iCCA) remain unclear. The aim of this study was to define the role of MUC13 in the progression of iCCA.

Methods

Expression levels of MUC13 in human iCCA samples were evaluated by immunohistochemistry, western blot, and real-time PCR. In vitro and in vivo experiments were used to assess the effect of MUC13 on iCCA cell growth and metastasis. Crosstalk between MUC13 and EGFR/PI3K/AKT signaling was analyzed by molecular methods. The upstream regulatory effects of MUC13 were evaluated by Luciferase and DNA methylation assays.

Results

MUC13 was overexpressed in human iCCA specimens and iCCA cells. MUC13 overexpression positively correlated with clinicopathological characteristics of iCCA, such as vascular invasion and lymph node metastasis, and was independently associated with poor survival. Results from loss-of-function and gain-of-function experiments suggested that knockdown of MUC13 attenuated, while overexpression of MUC13 enhanced, the proliferation, motility, and invasiveness of iCCA cells in vitro and in vivo. Mechanistically, we found that the phosphatidylinositol 3-kinase-AKT signal pathway and its downstream effectors, such as tissue inhibitor of metalloproteinases 1 and matrix metallopeptidase 9, were required for MUC13-mediated tumor metastasis of iCCA. MUC13 interacted with epidermal growth factor receptor (EGFR) and subsequently activated the EGFR/PI3K/AKT signaling pathway by promoting EGFR dimerization and preventing EGFR internalization. We also found that MUC13 was directly regulated by miR-212-3p, whose downregulation was related to aberrant CpG hypermethylation in the promoter area.

Conclusions

These findings suggest that aberrant hypermethylation-induced downregulation of miR-212-3p results in overexpression of MUC13 in iCCA, leading to metastasis via activation of the EGFR/PI3K/AKT signaling pathway.

Lay summary

Mucin 13 overexpression has been implicated in the development of malignancies, although its role in intrahepatic cholangiocarcinoma has not been studied. Herein, we show that mucin 13 plays a critical role in intrahepatic cholangiocarcinoma. Mucin 13 could have therapeutic value both as a prognostic marker and as a treatment target.

Graphical abstract

Introduction

Intrahepatic cholangiocarcinoma (iCCA) is the second most common liver cancer after hepatocellular carcinoma (HCC). It accounts for 5–10% of all primary liver malignancies.¹ Epidemiological data indicate marked increases in iCCA incidence and mortality during the last decade.² Conventional chemotherapy and radiation therapy have not been effective at improving long-term survival. Therefore, curative treatment for iCCA is limited to surgical resection.³ However, iCCA is always diagnosed at an advanced stage. Consequently, the efficacy of curative surgery is very limited. For this reason, the molecular mechanisms underlying iCCA must be explored in order to develop novel therapeutic strategies.

The gene encoding the recently identified transmembrane protein mucin 13 (MUC13) is localized on chromosome band 3q13.3.⁴ MUC13 has a tandem repeat domain characteristic of all mucins which provides a scaffold for building oligosaccharide structures. MUC13 also contains 3 epidermal growth factor (EGF)-like domains and a cytoplasmic domain which may participate in cell signaling.⁴ MUC13 is aberrantly expressed in ovarian, pancreatic, gastric, renal, and colon cancers.[5], [6], [7], [8], [9] Its overexpression promotes malignant growth and metastasis by upregulating/activating the key oncogenes HER2, PAK1, ERK, AKT, and S100A4. It downregulates the P53 tumor suppressor in ovarian and pancreatic cancers.⁶^,⁷ McGuckin et al. reported that MUC13 activates the NF-κB signaling pathway and its downstream targets in colorectal and renal cancer cells.¹⁰^,¹¹ However, the expression profile of MUC13 and its role in the progression of iCCA are unknown. Therefore, in this study, we sought to determine the expression and clinical significance, functional effects, and modes of action of MUC13 in iCCA.

Section snippets

Patient and tissue samples

A retrospective cohort of 60 patients with iCCA who had undergone routine surgical procedures at the First Affiliated Hospital of Harbin Medical University, Harbin, China, was included in the present study. Ethical approval was obtained from the First Affiliated Hospital of Harbin Medical University Research Ethics Committee, and informed consent was obtained from each patient. Inclusion criteria were: primary diagnosis of iCCA between 2005 and 2014 (at least 5 years of potential follow-up); no

MUC13 is upregulated in human iCCA and correlated with worse clinical outcomes

Results from reverse transcription PCR (RT-PCR) showed that MUC13 was significantly upregulated in tumor tissues relative to levels in the corresponding adjacent non-tumor tissues (Fig. 1A). Similar results were obtained by western blot for 16 paired human iCCA samples (Fig. 1B). Results from immunohistochemistry (IHC) showed that the staining score of the MUC13 protein in the iCCA tissues was higher than that in the adjacent non-tumor tissues (Fig. 1C). We then investigated 8 paired

Discussion

Secreted and transmembrane mucins are high-molecular-weight glycoproteins. They are often heavily O-glycosylated in the cytoplasm or the membrane. MUC2, MUC4, MUC6, and MUC7 are overexpressed in iCCA.¹⁷^,¹⁸ MUC1 and MUC4 may serve as biomarkers for early iCCA detection and prognosis.¹⁹^,²⁰ Knockdown of MUC1 inhibits proliferation of iCCA by downregulating hypoxia inducible factor-1 (HIF-1).²¹ However, the function of MUC13 in iCCA is unclear. In the present study, we explored the biological and

Financial support

This study was supported by National Natural Science Foundation of China (Grant No. 81602050, No. 81602058 and No. 81772588), Changjiang Scholars and Innovative Research Team in University (Grant No. IRT1122), Foundation of the First Affiliated Hospital of Harbin Medical University (Grant No. 201713008), China Postdoctoral Science Foundation (Grant No. 2018M631947), Hei Long Jiang Postdoctoral Foundation (LBH-Z18125), Heilongjiang Province General Undergraduate Colleges and Universities Young

Conflict of interest

The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details.

Authors' contributions

P.T.M., M.F.Z. and X.P. performed all experiments. H.J.H. and S.R.P. provided human specimens, clinical information and data analysis. L.Y.L., W.Y., X.J.L., L.Q.F., H.Z.F., L.J., G.Z.H., L.G.X., S.B.S., and Z.M. provided support with experimental materials and techniques. M.Q.H, L.D.S. and L.L.X. designed the research and wrote the manuscript.

Acknowledgements

The authors would like to thank Tohoku University for providing the HuCCT1 cell line.

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: Author names in bold designate shared co-first authorship

^†: These authors contributed equally to this work.

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Research ArticleMUC13 promotes intrahepatic cholangiocarcinoma progression via EGFR/PI3K/AKT pathways

Highlights

Background & Aims

Methods

Results

Conclusions

Lay summary

Graphical abstract

Introduction

Section snippets

Patient and tissue samples

MUC13 is upregulated in human iCCA and correlated with worse clinical outcomes

Discussion

Financial support

Conflict of interest

Authors' contributions

Acknowledgements

J Hepatol

J Biol Chem

J Biol Chem

Gynecol Oncol

Adv Genet

Biochem Biophys Res Commun

Changing pattern of epidemiology in intrahepatic cholangiocarcinoma

Hepatology

Factors affecting survival after resection of intrahepatic cholangiocarcinoma

Surg Today

Increased expression and aberrant localization of mucin 13 in metastatic colon cancer

J Histochem Cytochem

MUC13 mucin augments pancreatic tumorigenesis

Mol Cancer Ther