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

Background: 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. 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. Methods: The 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 beta-Catenin/Tcf transcription reporter assay. Results: We 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. Conclusions: In 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.