CXCL16 Promotes Gastric Cancer Tumorigenesis via ADAM10-Dependent CXCL16/CXCR6 Axis and Activates Akt and MAPK Signaling Pathways

Abstract

Abnormal expression of CXC motif chemokine ligand 16 (CXCL16) has been demonstrated to be associated with tumor progression and metastasis, served as a prognostic factor in many cancers, with higher relative expression behaving as a marker of tumor progression. However, its role and mechanisms underlying progression and metastasis of gastric cancer (GC) are yet to be elucidated. In our investigation, public datasets and human GC tissue samples were used to determine the CXCL16 expression levels. Our results revealed that CXCL16 was upregulated in GC. The high expression CXCL16 in GC was significantly associated with histologic poor differentiation and pTNM staging. And high CXCL16 was positively correlated with the poor survival of GC patients. Gain-and loss-of-function experiments were employed to investigate the biological role of CXCL16 in proliferation and migration both in vitro and in vivo. Mechanically, Gene set enrichment analysis (GSEA) revealed that the epithelial‑mesenchymal transition (EMT), Akt and MAPK signal pathway related genes were significantly enriched in the high CXCL16 group, which was confirmed by western blot. Moreover, overexpression CXCL16 promoted the disintegrin and metalloproteases (ADAM10) and the CXC motif chemokine receptor 6 (CXCR6) expression, which mediated the CXCL16/CXCR6 positive feedback loop in GC, with activating Akt and MAPK signaling pathways. Knocking down ADAM10 would interrupted the CXCL16/CXCR6 axis in the carcinogenesis and progression of GC. In conclusion, our findings offered insights into that CXCL16 promoted GC tumorigenesis by enhancing ADAM10-dependent CXCL16/CXCR6 axis activation.

Keywords: ADAM10; CXCL16; gastric cancer; tumorigenesis.

Figure 1

CXCL16 up-regulated in human gastric cancer tissues and cell lines. (A) CXCL16 expression was examined by RT-qPCR in GC and adj-normal (n=149). (B and C) Kaplan-Meier survival analysis of DFS and OS according to CXCL16 levels in GC patients. (D) The expression level of CXCL16 in human GC compared with normal tissues in TCGA database. (E) Kaplan-Meier survival analysis of DFS according to CXCL16 mining of public microarray datasets (GSE22377 and GSE15459). (F and G) Compared to the cell lines derived from normal gastric mucosa GES-1, the relative expression level of CXCL16 were determined by RT-qPCR or western blot in primary GC cell lines (MKN45, MKN28, MGC803, BGC823, SGC7901, N87 and AGS). Data are presented as the mean ± SD from three independent experiments. *P<0.05, ***P <0.001.

Figure 2

CXCL16 promotes GC cell proliferation and migration. (A) The efficiency of CXCL16 overexpression was determined by western blot in MGC803 and SGC7901 cells. (B-E) Proliferation assays, colony formation assays, migration assays and invasion assays were preformed to exam the biological function in CXCL16 overexpressed MGC803 and SGC7901 cells. (F) The knockdown efficiency of CXCL16 was determined by western blot in BGC823 cell. (G-J) Proliferation assays, colony formation assays, migration assays and invasion assays were preformed to exam the knockdown CXCL16 in BGC823 cell. Data are presented as the mean ± SD from three independent experiments. (K) The representative images and the quantification of xenograft upon NT or shCXCL16 in BGC823 injected nude mice, respectively (n=6 for each group). Tumor volumes were calculated after injection every 3 days for 21 days. Tumor weights are represented as mean ± SD. (L) The representative images and the quantification of lung metastatic colonization of nude mice treated with tail vein injection of BGC823 cells stably transfected with NT or shCXCL16 (n=5 for each group). *P<0.01, **P<0.005, ***P<0.001 vs. the control group.

Figure 3

CXCL16 promotes EMT via Akt and MAPK signaling pathways. (A) GSEA analysis results of TCGA STAD dataset revealed that HALLMARK EPITHELIAL MESENCHYMAL TRANSITION, HALLMARK APICAL JUNCTION, HALLMARK PI3K-AKT-MTOR SIGNALING and KEGG MAPM SIGNALING PATHWAY were significantly enriched based on CXCL16 high expression. NES, normalized enrichment score; the P value indicates the significance of the enrichment score. (B) Pearson correlation analysis using TCGA STAD database revealed that CXCL16 expression were significantly correlated with CDH2, CDH1, CTNNB1, SNAIL1, and JPT1 in GC. (C) Representative images of immunofluorescence (IF) staining of E-cadherin (red) on CXCL16 overexpression or knockdown GC cell lines. (D) The effect of CXCL16 overexpression or knockdown cell lines on the protein levels of EMT markers: N-cadherin, E-cadherin, ZO-1, β-catenin, snail and slug in GC cells. (E) The effect of CXCL16 overexpression or knockdown cell lines on Akt and MAPK signaling pathway in GC cells.

Figure 4

High CXCL16 was positively correlated with increased CXCR6 and ADAM10 protein expression level in GC. (A) Verification of CXCR6 and ADAM10 protein expression in CXCL16 overexpressed MGC803 and SGC7901 cells, and CXCL16 knockdown BGC823 cell. (B) Pearson correlation analysis using TCGA STAD database revealed that CXCL16 expression were significantly correlated with ADAM10 and CXCR6 in GC. (C) Immunohistochemistry, using DAB (brown), showed the protein expression level of CXCR6 in adj-normal tissues and GC tissues: a, negative staining of CXCR6 in adj-normal tissues; negative, weak and high (-, + and ++) staining of CXCR6 in cancer tissues. Magnification, x200 and x400. (D) Kaplan‑Meier overall survival curves of patients with GC stratified by negative (n=15) and positive (n=40) expression levels of CXCR6 (P=0.025). Patients with positive CXCR6 expression had the poor outcome. (E) Pearson correlation analysis confirmed ADAM10 positively relate to the expression of CXCL16 in our previous RNA array dataset (n=198) and GEO public dataset GSE62254 (n=300) respectively. (F)The expression level of ADAM10 significantly increased in human gastric cancer compared with normal tissues in TCGA database.

Figure 5

Knocking down ADAM10 abrogated CXCL16 function in GC cells. (A) The knockdown efficiency of ADAM10 was determined by western blot in MGC803 and SGC7901 cells, respectively. (B-D) Proliferation assays, colony formation assays and migration assay of ADAM10 knockdown cells with or without CXCL16 overexpression in BGC823 and SGC7901 cells respectively. (E and F) Western blot was performed to detect the protein levels of EMT markers, Akt and MAPK signaling pathway in ADAM10 knockdown with or without CXCL16 overexpression in SGC7901 cells. *P<0.01, ***P<0.001.

Figure 6

Schematic diagram representation of the mechanism of CXCL16 promoting tumorigenesis in GC. Cleaved by ADAM10, upregulation of CXCL16 resulted into accumulation of CXCL16 outside the tumor cells. CXCL16 binding to its orphan receptor, CXCR6, activated the CXCL16/CXCR6 axis which led to the tumorigenesis in GC. And during this process Akt and MAPK signaling pathway were activated.