Crosstalk between CCL7 and CCR3 promotes metastasis of colon cancer cells via ERK-JNK signaling pathways - PubMed (original) (raw)
Crosstalk between CCL7 and CCR3 promotes metastasis of colon cancer cells via ERK-JNK signaling pathways
Yeo Song Lee et al. Oncotarget. 2016.
Abstract
Chemokine ligand 7 (CCL7) enhances cancer progression and metastasis via epithelial-mesenchymal transition (EMT). However, little is known about the molecular mechanism of CCL7-induced EMT signaling cascade in colon cancer. Thus, the objective of this study was to investigate CCL7-induced EMT signaling pathway and its role in the progression and metastasis of colon cancer. To demonstrate the effect of CCL7 on EMT induction, HCT116 and HT29 cells overexpressing CCL7 were generated. CCL7-induced EMT and its downstream signaling pathway were evaluated by both in vitro and in vivo experiments. In in vitro studies, CCL7 was found to interplay with CC chemokine receptor 3 (CCR3), resulting in enhanced cellular proliferation, invasion, and migration via ERK and JNK signaling pathway. To validate these findings, we established ectopic and orthotopic mouse models injected with CCL7-overexpressed cells. In ectopic mouse models, we observed that CCL7-overexpressed cells grew significantly faster than control cells. In orthotopic mouse models, we found that liver and lung metastasis developed only in mice injected with CCL7-overexpressed cells. This study is the first one focusing on the EMT cascade via CCL7-CCR3-ERK-JNK signaling axis in colon cancer. Our novel findings will improve our understanding on the mechanism of metastatic process and provide potential therapeutic strategies for preventing metastasis in colon cancer.
Keywords: CCL7; CCR3; ERK-JNK signaling; colon cancer; metastasis.
Conflict of interest statement
The authors have no conflicts of interest to disclose.
Figures
Figure 1. CCL7 induces cell proliferation in HCT116 cells
Cell proliferation of HCT116 cells was evaluated by A. WST-1 indirect assay or B. Cell counting (direct method) using a hemocytometer and trypan blue staining at 24, 48, and 72 hours with or without recombinant CCL7 (200 ng/ml). C-D. The same experiment was carried out in HCT116 cells overexpressing CCL7 or GFP (control). Both experiments were performed in parallels in triplicates. Results shown are mean value ± SE. *P < 0.05; **P < 0.01.
Figure 2. CCL7 increases expression of chemokine receptor CCR3
A. CCL7 overexpression induces morphological changes in HCT116 cells. Representative images of cells taken at 400× magnification are shown. B. Total cell lysates were subjected to western blot analysis to confirm CCL7 overexpression. Actin was used as a loading control. C. Transcriptional levels of CCL7 were measured using real-time PCR. β-actin expression was used as an internal control to obtain the relative quantification of gene expression. D. CCL7 secretion was measured by multiplex magnetic immunoassay of HCT116 cell lysates and supernatants. Expression patterns of CCR1, -2, -3, and -5 protein were monitored with E. Western blot and F, G. FACS analysis in CCL7 overexpressing (E, F) or CCL7 recombinant protein treated HCT116 cells (G). Columns: means ± SEs. **P < 0.01; ***P < 0.001.
Figure 3. CCL7 induces migration and invasion of colon cancer cells via CCR3
A. Quantitation of E-cadherin expression on the surface of HCT116 cells treated with or without recombinant CCL7 (200 ng/ml) using FACS analysis. B. Expression of E-cadherin, N-cadherin, and vimentin in HCT116 cells stably transfected with GFP or CCL7 were measured by western blotting (left panel). Expression of CCL7 and EMT markers in negative siRNA control-treated or CCL7 specific siRNAs-treated HCT116 cell extracts (right panel). Actin was used as a loading control. C. A wound healing assay was performed by creating a wound on a confluent monolayer of stable GFP/CCL7 overexpressing cells using l-Dish 35-mm high culture inserts. D. Transwell matrigel invasion assays of HCT116 cells after transfection with 100 nM of negative siRNA control or CCL7 specific siRNAs. E. Cell migration and F. Invasion was measured using a trans-well migration chamber (left panels). Mean fluorescence intensity (MFI) of invaded area is presented in bar graphs (right panels). G. HCT116 cells were treated with 20 nM CCR3 inhibitor SB 328437. Representative images of invaded cells are shown (left panels). MFI values are presented in bar graphs (right panels). H. Expression of EMT markers in HCT116 cells stably transfected with GFP or CCL7 with or without treatment with 20 nM SB 328437 (CCR3 inhibitor) was measured by western blotting. Actin was used as a loading control. Columns: means ± SEs. ** P < 0.01; *** P < 0.001.
Figure 4. Involvement of CCR3 in CCL7-induced ERK and JNK activation
A. Protein phosphorylation array in HCT116 cells stably transfected with GFP/CCL7 with or without treatment with 20 nM SB 328437 (CCR3 inhibitor). B. Expression of phosphorylated and total ERK/JNK in negative siRNA control-treated or CCL7 specific siRNA-treated HCT116 cell. C. Western blot analysis of phosphorylated and total ERK/JNK in HCT116 cells stably transfected with GFP/CCL7 with or without treatment with 20 nM SB 328437. D. Expression of phosphorylated and total ERK/JNK and EMT markers in HCT116 cells stably transfected with GFP or CCL7 with or without treatment with 20 uM U0126 (ERK/JNK inhibitor) was measured by western blotting. Actin was used as a loading control.
Figure 5. CCL7 overexpression provokes tumorigenicity and metastasis in HCT116 cells in vivo
A. Tumor images and B. tumor volumes at 3 weeks after transplantation of HCT116 cells overexpressing GFP (control) or CCL7 into nude mice (n = 5). Tumor size was measured once a week with a caliper. Tumor volume was calculated using the following formula: (short length × long length × width)/2. C. Haematoxylin and eosin (H&E) stain of stable GFP/CCL7 overexpressing HCT116 primary tumors at 22 days after subcutaneous injection. Scale bar, 100 μm (upper panel). CCL7 (middle panel) and Ki-67 (lower panel)-stained sections of primary tumors formed by stable GFP/CCL7 overexpressing HCT116 cells. Scale bar, 100 μm. D. Western blot analysis for CCL7, CCR3, CCR1, CCR2, CCR5 (left panel), and EMT markers (right panel) in primary tumors formed by stable GFP/CCL7 overexpressing HCT116 cells. E. Magnetic resonance imaging (MRI) detection of liver metastasis (yellow arrows) in orthotopic colorectal cancer mouse models. F. H&E stain of primary cecal tumor (upper left), liver metastatic tumor (middle left), lung metastatic tumor (lower left), E-cadherin stain of primary cecal tumor (upper right), liver metastatic tumor (middle right), and lung metastatic tumor (lower right) isolated from mice that received orthotopic cecum injection of stable GFP/CCL7 overexpressing HCT116 cells. Scale bar, 100 μm; T, tumor; G. Representative RT-PCR analysis showing CCL7, CCR3 and EMT-related gene, E-cadherin, and vimentin expression in primary cecal tumor and liver metastatic tumor.
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