The online version of this article (doi:10.1186/s12943-017-0629-4) contains supplementary material, which is available to authorized users.
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.
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.
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.
In conclusion, our findings support CXCL5 as a promoter of colorectal cancer metastasis and a predictor of poor clinical outcomes in colorectal cancer patients.
Additional file 1: Figure S1. X-tile analysis of survival data in CRC patients reveals a continuous distribution based on CXCL5 staining score. The plot shows the χ2 log-rank values produced when dividing the cohort with one cut-point, producing high, and low subsets. The X-axis represents all potential cut-points from low to high (left to right) that defines a low subset, whereas the Y-axis represents cut-points from high to low (top to bottom), that defines a high subset. Red coloration of cut-point indicates an inverse correlation with survival, whereas green coloration represents direct associations (A). The optimal cut-point occurs at the brightest pixel (red). The cut-point highlighted by the white circle in A is shown on a histogram of the entire cohort (B), and a Kaplan-Meier plot (C, low subset grey, high subset light green). (TIF 4341 kb)12943_2017_629_MOESM1_ESM.tif
Additional file 2: Figure S2. Increased expression of CXCL5 in CRC tissues and calibration curve. A: An immunoblot showing that CXCL5 is overexpressed in cancer tissues compared with the peritumoral normal tissues. B: Calibration plots for predicting DFS 3 years after surgery. C: Calibration plots for predicting DFS 5 years after surgery. (TIF 2163 kb)12943_2017_629_MOESM2_ESM.tif
Additional file 3: Tables and Methods. Table S1, shRNA Sequences; Table S2, antibodies for immunoblot; Table S3, antibodies for immunofluorescence; Table S4, correlations between CXCL5 expression and clinical characteristics in CRC patients; Table S5, univariate and multivariate analyses of CXCL5 expression in CRC patients; Table S6, pathological stages of patients in Fig. 2A& B; Supplementary methods. (DOC 178 kb)
Additional file 4: Figure S3. The expression of CXCL5 in CRC cell lines and CXCL5/CXCR2 promotes CRC cell migration through the induction of EMT. A: ELISAs examining CXCL5 expression levels in CRC cell lines supernatants. B: Immunofluorescence analysis verifying the effects of upregulation of CXCL5 using a CXCL5-vector and shRNA-inhibition of CXCL5 and CXCR2. Magnification, 400×. C: Morphological changes in the indicated cells. Magnification, 200×. Scale, 200 μm. (TIF 16430 kb)12943_2017_629_MOESM4_ESM.tif
Additional file 5: Figure S4. CXCL5/CXCR2 induces EMT in CRC cells via the ERK/Elk-1 pathway. A & B: Morphological changes after treatment with LY294002 or U0126 in HCT116 and SW480 cells. Scale, 200 μm. C & E: Representative images of the transwell migration assays after treatment with LY294002 or U0126 in HCT116 and SW480 cells. Scale, 200 μm. Treatment with U0126 decreases the migrated cell number. D & F: The column graph displays the migrated cell number of the indicated cells. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (TIF 12172 kb)12943_2017_629_MOESM5_ESM.tif
Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, et al. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC Cancer Base No. 11 [Internet]. Lyon: International Agency for Research on Cancer; 2013. http://globocan.iarc.fr/.
Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. 2013;63:10–29. CrossRef
Itatani Y, Kawada K, Inamoto S, Yamamoto T, Ogawa R, Taketo MM. The role of chemokines in promoting colorectal cancer invasion/metastasis. Int J Mol Sci. 2016;17:1–17. CrossRef
Ma J, Zhao J, Lu J, Wang P, Feng H, Zong Y, et al. Cadherin-12 enhances proliferation in colorectal cancer cells and increases progression by promoting EMT. Tumor Biol. 2016;37:9077–88. CrossRef
Smith JB, Rovai LE, Herschman HR. Sequence similarities of a subgroup of CXC chemokines related to murine LIX: implications for the interpretation of evolutionary relationships among chemokines. J Leukoc Biol. 1997;62(5):598–603. PubMed
- Tumor-derived CXCL5 promotes human colorectal cancer metastasis through activation of the ERK/Elk-1/Snail and AKT/GSK3β/β-catenin pathways
- BioMed Central
Neu im Fachgebiet Onkologie
Mail Icon II