The Functional And Mechanistic Studies Of IL-13 On Epithelial-mesenchymal Transition In Colorectal Cancer Cells

Metastasis is one of the main characteristics of malignant tumor and the major cause of death in cancer patients. Tumor metastasis is a complex process with multiple stages and steps. Increasing studies suggest that epithelial-mesenchymal transition (EMT) plays a critical and complex role in tumor invasion and metastasis. EMT is a biological program characterized by losing the epithelial phenotypes and gaining mesenchymal properties. The EMT allows epithelial cells to lose apical-basal polarity and connection with basement membrane, and then acquire mesenchymal-like morphological characteristics and biological functions such as enhanced migratory capacity, invasiveness and elevated stem-like traits. The underlying molecular mechanisms of EMT are very complex and remain to be intangible.In order to screen and identify the novel regulators in colorectal cancer (CRC), our lab performed cDNA microarray (Affymetrix, GeneChip(?) Human Genome U133 Plus 2.0) to detect the gene expression of cancer cells and normal intestinal mucosa epithelial cells, and successfully screened 381 genes of differential expression. Among them, the expression level of interleukin-13 (IL-13) in tumor cells was 2.5 times higher than normal cells.The cytokine IL-13 is mainly secreted by T helper 2 (Th2, CD4+) cells and extensively involoved in organ fibrosis, inflammation and tumor development, but its role and mechanism in CRC progression is still not clear. Therefore, our study is to investigate the significance of IL-13 in human CRC and explore relevant moleculer mechanisms. We simulated the paracrine pathway in vitro and stimulated CRC cell lines HT29 and SW480 with recombinant human IL-13 protein. We observed the IL-13 induced cells changed from cobblestone-like phenotypes to a more fibroblast-like, spindle-shaped morphology and this morphological transformation indicated that cells induced by IL-13 might undergo EMT-related changes. The immunofluroecence assay showed that E-cadherin located on the cell membrane was significantly inhibited and Vimentin located in the cytoplasm was obviously induced by IL-13 in HT29 and SW480 cell lines. Further immunoblotting and qRT-PCR assays showed that IL-13 treatment of HT29 and SW480 cells markedly decreased epithelial markers E-cadherin and ZO-1 expression while at the same time increased the expression of mesenchymal markers Vimentin and MMP9. Moreover, the expression of ZEB1, ZEB2 and Snail were up-regulated in IL-13-induced cells when compared with untreated cells, and the change of ZEB1 was the most obvious. IL-13 is also involved in regulating cell motility in CRC cells. Collectively, these observations suggested that IL-13 promotes EMT and aggressiveness in CRC cells. Our signaling experiments showed that IL-13 activated two different signaling pathways:STAT6 and PI3K/AKT, but not STAT3 and MAPK signaling. We treated cells with JAK inhibitor JAKi 1 or PI3K inhibitor before IL-13 treatment and observed that the blockade of the STAT6 activation by the pharmacological inhibitor JAKi 1 significantly reversed the IL-13-induced changes of EMT markers. To further confirm the relationship between STAT6 and EMT, we sought to selectively suppress STAT6 expression in HT-29 and SW480 cells using STAT6 shRNA. The cells treated with sh-STAT6 demonstrated a more epithelial phenotype in the presence of IL-13, which was similar to that displayed by cells in the absence of IL-13. More importantly, knocking down of STAT6 significantly reversed IL-13-mediated EMT changes and enhanced cell migration and invasion in HT29 and SW480 cells, In addition, IL-13 could not activate STAT6 phosphorylation, nor induce an EMT phenotype in the STAT6-defective CRC cell line Caco2. We then overexpressed STAT6 in Caco2 cells and IL-13 could promote EMT in STAT6-tansfected Caco2 cells. These observations further demonstrated STAT6 is required for IL-13-induced EMT phenotypes and invasiveness in CRC cells. Besides, numerous findings reveal that EMT endows cells with stem-like properties and enables them to migrate from the primary tumor and then colonize distant sites. In our study, we found that the expression of stem cell markers were significantly up-regulated in IL-13-treated cells and reversed by inhibitor JAki 1. Likewise, silencing STAT6 in HT29 and SW480 cells reversed IL-13-induced elevation of stem cell markers. These results indicated that IL-13 induces cancer stem cell (CSC) through STAT6 pathway. Then, we detected the mRNA expression levels of IL-13 receptors IL-13Rα1 and IL-13Rα2. The results showed that IL-13Rα2 was almost not expressed in CRC cell lines except RKO cells, but IL-13Rα1 is highly expressed in CRC cell lines. Similarily, we identified that IL-13Rα1 mRNA expression level but not IL-13Rα2 was higher in CRC tissues when compared with adjacent non-tumor tissues from 33 patients. Meanwhile, we found the HT29 and SW480 cells used in this study only expressed IL-13Rα1 which mediated signal transduction through the canonical JAK/STAT6 pathway. Thus, IL-13/IL-13Rα1/STAT6 pathway was shown to play a central role in regulating EMT process and promoting cells aggressiveness. Besides, IL-13Rα1 mRNA expression but not IL-13Rα1 was found to elevate in EMT model induced by TGF-β and TNF-α. This implied that IL-13 might play a synergistic role with other EMT inducers in promoting EMT in CRC cells and the underlying mechanism needed further verification.Collectively, this study uncovered the contribution of IL-13/IL-13Rα1/STAT6 pathway to EMT and aggressiveness in CRC and suggested novel biomarkers and therapeutic opportunities for the diagnosis and treatment of malignant diseases.