Tumor-derived CXCL5 promotes human colorectal cancer metastasis through activation of the ERK/Elk-1/Snail and AKT/GSK3β/β-catenin pathways

Abstract

Background: Metastasis is a major cause of death in human colorectal cancer patients. However, the contribution of chemokines in the tumor microenvironment to tumor metastasis is not fully understood.

Methods: Herein, we examinined several chemokines in colorectal cancer patients using chemokine ELISA array. Immunohistochemistry was used to detect expression of CXCL5 in colorectal cancer patients tissues. Human HCT116 and SW480 cell lines stably transfected with CXCL5, shCXCL5 and shCXCR2 lentivirus plasmids were used in our in vitro study. Immunoblot, immunofluorescence and transwell assay were used to examine the molecular biology and morphological changes in these cells. In addition, we used nude mice to detect the influence of CXCL5 on tumor metastasis in vivo.

Results: We found that CXCL5 was overexpressed in tumor tissues and associated with advanced tumor stage as well as poor prognosis in colorectal cancer patients. We also demonstrated that CXCL5 was primarily expressed in the tumor cell cytoplasm and cell membranes, which may indicate that the CXCL5 was predominantly produced by cancer epithelial cells instead of fibroblasts in the tumor mesenchyme. Additionally, overexpression of CXCL5 enhanced the migration and invasion of colorectal cancer cells by inducing the epithelial-mesenchymal transition (EMT) through activation of the ERK/Elk-1/Snail pathway and the AKT/GSK3β/β-catenin pathway in a CXCR2-dependent manner. The silencing of Snail and β-catenin attenuated CXCL5/CXCR2-enhanced cell migration and invasion in vitro. The elevated expression of CXCL5 can also potentiate the metastasis of colorectal cancer cells to the liver in vivo in nude mice intrasplenic injection model.

Conclusion: In conclusion, our findings support CXCL5 as a promoter of colorectal cancer metastasis and a predictor of poor clinical outcomes in colorectal cancer patients.

Keywords: CXCL5; Colorectal cancer; EMT; Snail; β-catenin.

Fig. 1

Expression profiles of chemokines in four CRC tissues. a A heat map displays the hierarchical clustering of the chemokine expression profiles. b An overlapping pie chart displays the distribution of chemokines in the four samples. c The protein microarray shows the expression of CXCL5, which was the primary focus of this study. The representative image is from sample 3. These four patient samples are selected according to their pathological stages which rages from Dukes I to Dukes IV. Pathological stages of the four tissues tested are, respectively: sample 1, Dukes I; sample 2, Dukes III; sample 3, Dukes II; sample 4, Dukes IV

Fig. 2

Increased expression of CXCL5 in CRC tissues, Kaplan-Meier analysis and nomogram plots. a & b RT-PCR results showing high expression of CXCL5 in CRC tissues. Data are showed by 2^-ΔΔCt (a) and 2^-ΔCt (b). c & d Representative immunohistochemistry images showing that CXCL5 is highly expressed in 61.5% of the cancer tissues in tissue microarray (tissues used are different from Figs. 1 and 2a & b). Scale: 200 μm. ***P<0.001. e & f Kaplan-Meier analysis of OS and DFS. The CXCL5^high group has a worse prognosis in OS and DFS compared with the CXCL5^low group. g Nomogram to predict DFS 3 and 5 years after surgery. CXCL5 line: 1 represents positive staining; 0 represents negative staining. Stage line: 1, 2, 3 and 4 represent Dukes stage I, II, III and IV. Calculation: find their points on the “point line” according to the CXCL5 staining and Dukes stage. Total points are equal to CXCL5 points plus Dukes stage points. The 3- or 5-year survival rates are indicated on the “3-year survival” line or “5-year survival” line according to their total points. For example, for a patient with CXCL5 positive staining and Dukes II. CXCL5 point is 0, Stage point is 5.2, total point is 5.2, 3-year survival prediction is 78%, 5-year survival prediction is 52%

Fig. 3

CXCL5 localization in CRC tissues, the expression of CXCL5 in CRC cell lines and CXCL5/CXCR2 promotes CRC cell migration through the induction of EMT. a & b Immunohistochemistry and immunofluorescence images showing the location of CXCL5 and αSMA (marker of fibroblasts) in CRC specimens. CXCL5 is mainly expressed in tumor tissue instead of mesenchymal tissue. c Immunoblots examining CXCL5 expression levels in CRC cell lines and normal colon epithelial cell. d Representative data from the immunoblot analysis verifying the changes of CXCL5 or CXCR2 using a CXCL5-vector or shRNA-inhibition of CXCL5 and CXCR2. e & f Representative images from the transwell migration assay in indicated CRC cells. Magnification, 200×. Scale, 200 μm. Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. g Immunoblot analysis of EMT marker expression in indicated cells. h Immunofluorescence images showing the changes in E-cadherin and Vimentin after different treatments with CXCL5-vector or shRNA-inhibition of CXCL5 and CXCR2. Scale, 50 μm

Fig. 4

CXCL5/CXCR2 induces EMT in CRC cells via the ERK/Elk-1 pathway. a & b CXCL5 induces the phosphorylation of ERK and Akt in CRC cells in a CXCR2-dependent manner. No differences are observed in STAT3 and JNK pathway. c & d Representative immunofluorescence images showing that inhibition of the ERK pathway using U0126 upregulates E-cadherin expression and downregulates Vimentin expression in HCT116^CXCL5 and SW480^shRNA cells. Inhibition of the AKT pathway using LY294002 is not able to change expression of E-cadherin or Vimentin in HCT116^CXCL5 and SW480^shRNA cells. Scale, 50 μm. e & f Immunoblots showing that inhibition of the ERK pathway using U0126 upregulates E-cadherin expression and downregulates Vimentin expression in HCT116^CXCL5 and SW480^shRNA cells. Inhibition of the AKT pathway using LY294002 is not able to change the expression of E-cadherin or Vimentin in HCT116^CXCL5 and SW480^shRNA cells. Changes in Elk-1 are consistent with changes in ERK1/2

Fig. 5

Snail is critical to CXCL5/CXCR2-mediated CRC migration. a Immunoblot analysis of Snail, Slug and ZEB1 levels in the indicated cells. CXCL5 is able to induce Snail expression. b Inhibition of the ERK pathway (U0126) instead of the AKT pathway (LY294002) downregulates Snail expression in the indicated cells. c Downregulation of Snail inhibits CXCL5-induced downregulation of E-cadherin and upregulation of Vimentin and N-cadherin in HCT116. Downregulation of Snail upregulates E-cadherin expression and downregulates Vimentin and N-cadherin expression in SW480. d Inhibition of Snail altered the morphology of cells in both the HCT116^CXCL5 and SW480 groups. e & f Inhibition of Snail decreased the number of migrating cells in the indicated cell lines. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. g & h Immunofluorescence images show that downregulation of Snail inhibits CXCL5-induced downregulation of E-cadherin and upregulation of Vimentin and N-cadherin in HCT116. Downregulation of Snail upregulates E-cadherin expression and downregulates Vimentin and N-cadherin expression in SW480. Scale, 50 μm

Fig. 6

The CXCL5/CXCR2 axis regulates CRC cell invasion through the AKT/GSK3β/β-catenin/MMP7 pathway. a & b CXCL5 promotes CRC cell invasion in a CXCR2-dependent manner in HCT116. Inhibition of CXCL5 reverses this process in a CXCR2-dependent manner in SW480. Scale, 200 μm. Data are presented as the mean ± SD. c Immunofluorescence images show that CXCL5 promotes translocation of β-catenin in a CXCR2-dependent manner in HCT116. Inhibition of CXCL5 reverses this process in a CXCR2-dependent manner in SW480. Scale, 50 μm. d The inhibitory effects of β-catenin- shRNA on β-catenin in HCT116^CXCL5 and SW480 cells. Scale, 50 μm. e & f Inhibition of β-catenin decreases the invasion of CRC cells. Scale, 200 μm. Data are presented as the mean ± SD. g & h CXCL5/CXCR2 enhances the expression of MMP7 through activation of the AKT/GSK3β/β-catenin pathway. i & j Inhibition of the AKT pathway (LY294002) instead of the ERK pathway (U0126) reverses the activation of GSK3β/β-catenin and upregulates the expression of MMP7. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

CXCL5/CXCR2 promotes CRC liver metastasis in vivo and schematic diagram. a & c Representative images of liver metastasis in the indicated HCT116 and SW480 cells in vivo. Scale, 100 μm. Six mice are included in each group. b & d The number of metastatic nodules in each indicated group. Data are presented as the mean ± SD. *P < 0.05, ***P < 0.001. e Schematic diagram shows that tumor-derived CXCL5 promotes CRC cell migration by inducing EMT through the activation of ERK/Elk-1/Snail and enhances invasion through the activation of AKT/GSK3β signaling, which inhibits the degradation of β-catenin. Accumulated β-catenin in the cytoplasm translocates to the nucleus to promote the expression of MMP7, which facilitates cell invasion