CXCR4 promotes the growth and metastasis of esophageal squamous cell carcinoma as a critical downstream mediator of HIF-1α

Abstract

C-X-C motif chemokine receptor 4 (CXCR4) belongs to the CXC chemokine receptor family, which mediates the metastasis of tumor cells and promotes the malignant development of cancers. However, its biological role and regulatory mechanism in esophageal squamous cell carcinoma (ESCC) remain unclear. Here, we found that CXCR4 expression was associated with lymph node metastasis and a poor prognosis. In vitro and in vivo studies demonstrated that CXCR4 overexpression promoted ESCC cell proliferation, migration, invasion, and survival, whereas silencing CXCR4 induced the opposite effects. Mechanically, HIF-1α transcriptionally regulates CXCR4 expression by binding to a hypoxia response element in its promoter. HIF-1α-induced ESCC cell migration and invasion were reversed by CXCR4 knockdown or treatment with MSX-122, a CXCR4 antagonist. Collectively, these data revealed that the HIF-1α/CXCR4 axis plays key roles in ESCC growth and metastasis and indicated CXCR4 as a potential target for ESCC treatment.

Keywords: CXC chemokine receptor 4; esophageal squamous cell carcinoma; growth; hypoxia inducible factor-1α; metastasis.

FIGURE 1

Expression of CXCR4 in ESCC and its association with the clinicopathologic features of ESCC. A, Assessment of CXCR4 mRNA expression in ESCC specimens from the GEO database (GSE23400, GSE75241). B, qRT‐PCR analysis of CXCR4 mRNA expression in 30 pairs of ESCC and adjacent normal tissues. C, Western blotting analysis of CXCR4 protein expression in tumor (T) and paired normal (N) tissue specimens obtained from ESCC patients. D, Representative images of CXCR4 expression in ESCC and esophageal normal mucosa tissues. E, High expression of CXCR4 was associated with a poor prognosis. F, Western blotting analysis of CXCR4 expression in 7 ESCC cell lines and Het‐1A cells

FIGURE 2

Effect of altered CXCR4 expression on ESCC proliferation and growth in vitro and in vivo. A, B, Verification of the efficiency of stable CXCR4 knockdown or overexpression in ESCC cell lines. KYSE30 and KYSE150 cells were infected with CXCR4 knockdown (shCXCR4#1, shCXCR4#2, shCXCR4#3) or control (shCtrl) lentiviral particles; KYSE450 and KYSE510 cells were infected with CXCR4 overexpression (oeCXCR4) or control (oeCtrl) lentiviral particles. C, D, Assessment of ESCC cell proliferation in vitro by CCK‐8 (C) and colony formation (D) assays. E, Assessment growth potential of ESCC cells in vivo. tumor growth curves are shown. Data with error bars are presented as the mean ± SD. *P < .05; **P < .01

FIGURE 3

Influence of CXCR4 expression on ESCC cell migration and invasion in vitro and metastasis in vivo. A, Detection of the migration and invasion of CXCR4 knockdown KYSE30 and KYSE150 cells using a transwell assay. B, Detection of the migration and invasion of KYSE450 and KYSE510 cells overexpressing CXCR4 using a transwell assay. C, In vivo imaging of the lung metastasis of KYSE30‐fLuc cells with or without CXCR4 knockdown. The luminescence signal with the signal intensity is indicated by the scale (right panel). D, H&E staining of the lung tissues to detect the lung metastasis loci of KYSE30‐fLuc cells with or without CXCR4 knockdown. E, In vivo imaging of the lung metastasis in KYSE30‐fLuc cells with or without MSX‐122 treatment (100 nM). The luminescence signal with the signal intensity is indicated by the scale (right panel). F, H&E staining of the lung tissues to detect the lung metastasis loci of KYSE30‐fLuc cells with or without MSX‐122 treatment. Data with error bars are presented as the mean ± SD. *P < .05; **P < .01

FIGURE 4

Hypoxia‐induced CXCR4 expression in ESCC cells. A, qRT‐PCR analysis of CXCR4 mRNA expression in ESCC cells after hypoxia exposure (1% O₂) for the indicated times. B, Western blotting analysis of CXCR4 expression in ESCC cells under hypoxic conditions (1% O₂). C, Western blotting analysis of CXCR4 expression in ESCC cells after treatment with 0, 100, 150, 200, 250, or 300 μmol/L CoCl₂ for 18 h. D, Transwell assays demonstrated that the increased migration and invasion ability of KYSE30 and KYSE150 cells induced by hypoxia could be weakened by CXCR4 interference or treatment with MSX‐122 (100 nM). Data with error bars are presented as the mean ± SD. **P < .01; ***P < .001

FIGURE 5

Close relationship between HIF‐1α and CXCR4 expression in ESCC cells. A, Gene ontology analyses of differentially expressed mRNAs (DEmRNAs) in the KYSE30‐shNC and KYSE30‐shHIF‐1α groups by RNA‐seq analysis. B, Heatmap analysis of DEmRNAs in response to hypoxia in the KYSE30‐shNC and KYSE30‐shHIF‐1α groups. C, qRT‐PCR analysis of CXCR4 mRNA in KYSE30 and KYSE150 cells after HIF‐1α knockdown under normoxia or hypoxia. D, Western blotting analysis of CXCR4 expression in KYSE30 and KYSE150 cells after HIF‐1α knockdown under normoxic or hypoxic conditions. E, Western blotting analysis of CXCR4 expression in KYSE450 and KYSE510 cells after HIF‐1α overexpression. F, ELISA analysis of the CXCL12 protein level in the culture medium of KYSE30, KYSE150, KYSE450, and KYSE510 cells with HIF‐1α overexpression or knockdown under normoxic or hypoxic conditions. G, Representative images of HIF‐1α and CXCR4 expression in ESCC tissues and esophageal normal mucosa tissues. H, Assessment of the correlation between HIF‐1α and CXCR4 expression in ESCC specimens using Spearman correlation coefficient analysis (R = .54, P < .001). I, Assessment of the correlation between HIF‐1α and CXCR4 expression in ESCC specimens from the GEO database (GSE23400, GSE75241). Data with error bars are presented as the mean ± SD. **P < .01; ns, not statistically significant

FIGURE 6

Influence of HIF‐1α expression on ESCC cell migration, invasion, and chemotaxis. A, The migration and invasion of KYSE450 and KYSE510 cells with HIF‐1α overexpression transfected with siCXCR4 or after treatment with the CXCR4 antagonist MSX‐122 (100 nM). B, The chemotaxis ability of KYSE450 and KYSE510 cells with HIF‐1α overexpression and CXCR4 knockdown or after treatment with MSX‐122 (100 nM). Data with error bars are presented as the mean ± SD. ***P < .001

FIGURE 7

Direct binding of HIF‐1α to the CXCR4 promoter. A, Seven HREs located at different sites in the CXCR4 promoter sequence. B, Results of the ChIP‐PCR assay conducted using chromatin isolated from KYSE30 cells. KYSE30 cells were exposed to hypoxia for 18 h. A specific anti‐HIF‐1α antibody was used, and normal IgG was used as a control. C, CXCR4 promoter reporter plasmid (GV238‐1970), truncated CXCR4 promoter reporter plasmids (GV238‐1155, GV238‐296) and mutant reporter plasmid GV238‐mut were constructed. The transcriptional activity of reporter plasmids in 293T cells induced by HIF‐1α overexpression or hypoxia. Data with error bars are presented as the mean ± SD. * P < .05