CCR9 initiates epithelial-mesenchymal transition by activating Wnt/β-catenin pathways to promote osteosarcoma metastasis

Abstract

Background: Osteosarcoma (OS) patients with lung metastasis have poor prognoses, and effective therapeutic strategies for delaying or inhibiting the spread of lung metastasis from the primary OS site are lacking. Hence, it is critical to elucidate the underlying mechanisms of OS metastasis and to identify additional new effective treatment strategies for patients.

Methods: Differential expression and functional analyses were performed to identify key genes and relevant signaling pathways associated with OS lung metastasis. The expression of CCR9 in OS cell lines and tissues was measured by RT-qPCR, western blotting and immunohistochemistry. Cell migration and invasion were assessed by wound healing and Transwell Matrigel invasion assays, respectively. The regulatory relationship between CCR9 and the Wnt/β-catenin signaling pathway was further evaluated by rescue experiments.

Results: The expression of CCR9 was elevated in OS cell lines and patients with lung metastasis. CCR9 promoted MG63 and HOS cell migration and invasion by activating the Wnt/β-catenin signaling pathway. Furthermore, knockdown of CCR9 repressed epithelial-mesenchymal transition (EMT) by downregulating mesenchymal markers (N-cadherin and Vimentin) and EMT-associated transcription factors (twist and snail) and upregulating an epithelial marker (E-cadherin).

Conclusions: Our findings suggest that CCR9 promotes EMT by activating Wnt/β-catenin pathways to promote OS metastasis. CCR9 may be a promising therapeutic target to inhibit lung metastasis and serve as a novel prognostic marker for OS.

Keywords: CCR9; Epithelial–mesenchymal transition; Lung metastasis; Osteosarcoma; Wnt/β-catenin.

Fig. 1

a Volcano plot of DEGs, including 1355 upregulated genes and 1484 downregulated genes. The threshold for the X axis was Log2fc ≥ 1 and Log2fc ≤ -1, and the threshold for the Y axis was P-value < 0.05. b GO enrichment analysis of DEGs revealed enriched biological process terms. c GO enrichment analysis of DEGs revealed enriched cellular component terms. d GO enrichment analysis of DEGs revealed enriched molecular function terms. e Top 5 enriched terms in the BP, MF and CC categories. f The top ten KEGG signaling pathways (KEGG analysis) enriched with the DEGs

Fig. 2

a PPI network of DEGs and a core module of the PPI network. CCR9 was the highest-ranked PPI hub gene. b Immunohistochemistry staining for CCR9 in OS tissues with or without lung metastasis. c CCR9 immunohistochemistry intensity score in OS tissues (21 without lung metastasis and 10 with lung metastasis), and the score was higher in OS tissues with lung metastasis. d Relative CCR9 mRNA expression levels in HD-MSCs, U2OS, HOS, MG63 and SAOS2 cell lines. The expression of CCR9 was higher in OS cell lines. e Protein expression levels of CCR9 in HD-MSCs, U2OS, HOS, MG63 and SAOS2 cell lines. f Quantitative western blot data for CCR9 protein expression. The expression of CCR9 was higher in OS cell lines. Mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3

a MG63 and HOS cells were transfected with blank control, negative control siRNA (NC) or CCR9 siRNA (si-1, si-2 and si-3). The relative CCR9 mRNA expression levels were obviously lower in si-CCR9 group. b Protein expression levels of CCR9 in MG63 and HOS cells treated with either blank control, si-NC or si-CCR9, as determined by western blot analysis. The protein expression levels were apparently lower in si-CCR9 group. c CCK8 assay results showed no significant difference in two groups. d Colony formation capacity of MG63 and HOS cells, as assessed by a colony formation assay after 10 days. e Quantification of the cell colony number, and the results showed no significant difference in two groups. f The apoptosis rates of MG63 and HOS cells were analyzed by Annexin V-FITC/PI staining. The percentage of apoptotic cells was calculated as the sum of the early apoptotic (Q3) and late apoptotic cells (Q2) percentages. g Quantitative apoptosis rates of MG63 and HOS cells, and no significant difference in two groups. Mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4

a The migration of MG63 and HOS cells was evaluated by a wound healing assay, and the si-CCR9 group showed slower wound healing. b Quantification of the percentage area of wound healing. c Transwell migration and invasion assays of MG63 and HOS cells, and knockdown of CCR9 significantly inhibited cell invasion. d Quantitative results of Transwell migration and invasion assays. Mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 5

a The GSEA results showed significant enrichment of the gene signature associated with EMT and cell adhesion molecules. b The protein expression level of E-cadherin was higher in OS tissues with lung metastasis, and the expression of N-cadherin and Vimentin was downregulated. c The expression N-cadherin, vimentin, twist, snail and MMP-1, was obviously downregulated, and E-cadherin expression was significantly upregulated in si-CCR9 group. d The protein expression levels of EMT-related markers in MG63 and HOS cells. e Quantitative data for the protein expression levels of EMT-related markers. Mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 6

a The protein expression levels of active β-catenin and total β-catenin were downregulated in si-CCR9 group. b The protein expression levels and quantitative data of active β-catenin and total β-catenin in MG63 and HOS cells were shown. Overexpression of CCR9 activated the Wnt/β-catenin pathway, and the Wnt signaling inhibitor XAV-939 counteracted the activation. c Cell migration assay of MG63 and HOS cells. Overexpression of CCR9 increased the wound healing rate and the inhibitor of XAV-939 inhibited this effect. d Quantification of the area percentage of wound healing. e Transwell invasion assay of MG63 and HOS cells. Overexpression of CCR9 promoted the invasion ability and the inhibitor of XAV-939 inhibited this effect. f Quantitative results of the Transwell migration and invasion assays. Mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 7

a Hypothesis diagram of the mechanism by which CCR9 works