Blockade of CXCL12/CXCR4 signaling inhibits intrahepatic cholangiocarcinoma progression and metastasis via inactivation of canonical Wnt pathway

Shengqiang Zhao,Chengyong Qin,Jing Wang

doi:10.1186/preaccept-2045933274142844

Abstract

BackgroundIntrahepatic cholangiocarcinoma (IHCC) is the second most frequent primary malignant liver tumor following hepatocellular carcinoma. It is a highly fatal disease and has few therapeutics. The CXC chemokine ligand-12 (CXCL12)/CXC chemokine receptor type 4 (CXCR4) axis has been shown to be involved in tumorgenesis, proliferation, and angiogenesis in a variety of cancers including IHCC. However, its prognostic significance in IHCC is unclear. The purpose of this study was to examine the functional role of CXCR4 in the progression and metastasis of IHCC and explore the underlying mechanism.MethodsThe CXCR4 expression, overall survival, and the clinical characteristics including age, sex, differentiation degree, tumor size, vascular invasion, lymph node metastasis, TNM stage, and T stage were analyzed for 122 IHCC patients. Short hairpin RNA (shRNA) against CXCR4 was used to disrupt the CXCL12/CXCR4 signal transduction pathways in IHCC cell lines. In vitro assays, including CCK-8 assay, flow cytometry, and colony formation assay, and in vivo tumor formation assay were utilized to detect the cell phenotype of CXCR4 knockdown cells. Transwell and wound healing assays were used to examine the IHCC cell invasion and migration ability. The Wnt pathway was assessed by Western blot and β-Catenin/Tcf transcription reporter assay.ResultsWe demonstrated that CXCR4 expression was closely correlated with IHCC progression and metastasis characteristics. The overall survival of patients with high CXCR4 expression was significantly lower than that of patients with low CXCR4 expression. Furthermore, we showed that the abrogation of CXCR4 had significantly negative influence on the IHCC cell phenotype, including in vitro cell proliferation, cell cycle, colony formation, cell invasion, and in vivo tumorigenicity. In addition, CXCR4 knockdown downregulated Wnt target genes and mesenchymal markers such as Vimentin and Slug.ConclusionsIn conclusion, our result shows that high CXCR4 expression is associated with IHCC progression and metastasis via the canonical Wnt pathway, suggesting that CXCR4 may serve as a promising therapeutic target for IHCC.

Highlights

Intrahepatic cholangiocarcinoma (IHCC) is the second most frequent primary malignant liver tumor following hepatocellular carcinoma
Yu et al [24] found that suppressing expression of CXC chemokine receptor type 4 (CXCR4) by MicroRNA-9 could inhibit the proliferation of oral squamous cell carcinoma cells both in vitro and in vivo through the Wnt/β-catenin signaling pathway, and activation of CXCR4 expression led to the constitutive activation of β-catenin, implying the important role of Wnt/β-catenin in CXCR4 signaling, which was consistent with the previous reports in colorectal cancer [25], ovarian cancer [26], pancreatic cancer [23], and bone marrow stromal cells [27]
Association of CXCR4 expression level with IHCC cancers Immunohistochemistry staining results showed that CXCR4 expression was detected in the cytoplasm of most IHCC cells but not in the adjacent non-tumorous tissues

Summary

Introduction

Intrahepatic cholangiocarcinoma (IHCC) is the second most frequent primary malignant liver tumor following hepatocellular carcinoma. It is a highly fatal disease and has few therapeutics. The CXC chemokine ligand-12 (CXCL12)/CXC chemokine receptor type 4 (CXCR4) axis has been shown to be involved in tumorgenesis, proliferation, and angiogenesis in a variety of cancers including IHCC. Intrahepatic cholangiocarcinoma (IHCC) is a malignancy whose pathogenesis involves abnormal biliary epithelial differentiation [1] It is the most frequent primary malignant liver tumor next to hepatocellular carcinoma and is highly fatal because of its early invasion, widespread metastasis, and the lack of an effective therapy [2,3]. Yu et al [24] found that suppressing expression of CXCR4 by MicroRNA-9 could inhibit the proliferation of oral squamous cell carcinoma cells both in vitro and in vivo through the Wnt/β-catenin signaling pathway, and activation of CXCR4 expression led to the constitutive activation of β-catenin, implying the important role of Wnt/β-catenin in CXCR4 signaling, which was consistent with the previous reports in colorectal cancer [25], ovarian cancer [26], pancreatic cancer [23], and bone marrow stromal cells [27]

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