M2-like macrophages induce colon cancer cell invasion via matrix metalloproteinases.

The inflammatory milieu plays an important role in colon cancer development and progression. Previously, we have shown that tumor-associated macrophages (TAMs), an important component of the tumor microenvironment, are enriched in tumors compared with normal tissue and confer a poorer prognosis. In the present study, we found that matrix metallopeptidase-9 (MMP-9), which degrades extracellular matrix proteins, was increased in biopsies from colon cancer patients and in mouse xenografts with SW480 cell-derived tumors. SW480 colon cancer cells exposed to M2-like macrophage-conditioned medium (M2-medium) exhibited increased MMP-9 mRNA, protein expression and gelatinase activity. A similar effect was obtained by the addition of tumor necrosis factor-α (TNFα) and leukotriene D4 (LTD4 ). MMP-9 expression and activity were reduced by a TNFα blocking antibody adalimumab and a cysteinyl leukotriene receptor 1 (CysLTR1, the receptor for LTD4 ) antagonist montelukast. M2-medium also induced changes in the epithelial-mesenchymal transition (EMT) markers E-cadherin, ß-catenin, vimentin, and snail in SW480 cells. We also found that both M2-medium and TNFα and LTD4 induced stabilization/nuclear translocation of ß-catenin. Furthermore, we also observed an elongated phenotype that may indicate increased invasiveness, as confirmed in a collagen I invasion assay. M2-medium increased the invasive ability, and a similar effect was also obtained by the addition of TNFα and LTD4 . The specific MMP inhibitor I or adalimumab and montelukast reduced the number of invasive cells. In conclusion, our findings show that M2-medium enriched in TNFα and LTD4 promote colon cancer cell invasion via MMP-9 expression and activation and the induction of EMT.

The inflammatory mediator leukotriene D4 induces subcellular ß-catenin translocation and migration of colon cancer cells.

The abnormal activation of the Wnt/ß-catenin pathway frequently occurs in colorectal cancer. The nuclear translocation of ß-catenin activates the transcription of target genes that promote cell proliferation, survival, and invasion. The pro-inflammatory mediator leukotriene D4 (LTD4) exerts its effects through the CysLT1 receptor. We previously reported an upregulation of CysLT1R in patients with colon cancer, suggesting the importance of leukotrienes in colon cancer. The aim of this study was to investigate the impact of LTD4 on Wnt/ß-catenin signaling and its effects on proliferation and migration of colon cancer cells. LTD4 stimulation led to an increase in ß-catenin expression, ß-catenin nuclear translocation and the subsequent transcription of MYC and CCND1. Furthermore, LTD4 significantly reduced the expression of E-cadherin and ß-catenin at the plasma membrane and increased the migration and proliferation of HCT116 colon cancer cells. The effects of LTD4 can be blocked by the inhibition of CysLT1R. Furthermore, LTD4 induced the inhibition of glycogen synthase kinase 3 (GSK)-3ß activity, indicating a crosstalk between the G-protein-coupled receptor CysLT1 and the Wnt/ß-catenin pathway. In conclusion, LTD4, which can be secreted from macrophages and leukocytes in the tumor microenvironment, induces ß-catenin translocation and the activation of ß-catenin target genes, resulting in the increased proliferation and migration of colon cancer cells.

The cysteinyl leukotriene 2 receptor contributes to all-trans retinoic acid-induced differentiation of colon cancer cells.

BACKGROUND: Cysteinyl leukotrienes (CysLTs) are potent pro-inflammatory mediators that are increased in samples from patients with inflammatory bowel diseases (IBDs). Individuals with IBDs have enhanced susceptibility to colon carcinogenesis. In colorectal cancer, the balance between the pro-mitogenic cysteinyl leukotriene 1 receptor (CysLT(1)R) and the differentiation-promoting cysteinyl leukotriene 2 receptor (CysLT(2)R) is lost. Further, our previous data indicate that patients with high CysLT(1)R and low CysLT(2)R expression have a poor prognosis. In this study, we examined whether the balance between CysLT(1)R and CysLT(2)R could be restored by treatment with the cancer chemopreventive agent all-trans retinoic acid (ATRA). METHODS: To determine the effect of ATRA on CysLT(2)R promoter activation, mRNA level, and protein level, we performed luciferase gene reporter assays, real-time polymerase chain reactions, and Western blots in colon cancer cell lines under various conditions. RESULTS: ATRA treatment induces CysLT(2)R mRNA and protein expression without affecting CysLT(1)R levels. Experiments using siRNA and mutant cell lines indicate that the up-regulation is retinoic acid receptor (RAR) dependent. Interestingly, ATRA also up-regulates mRNA expression of leukotriene C4 synthase, the enzyme responsible for the production of the ligand for CysLT(2)R. Importantly, ATRA-induced differentiation of colorectal cancer cells as shown by increased expression of MUC-2 and production of alkaline phosphatase, both of which could be reduced by a CysLT(2)R-specific inhibitor. CONCLUSIONS: This study identifies a novel mechanism of action for ATRA in colorectal cancer cell differentiation and demonstrates that retinoids can have anti-tumorigenic effects through their action on the cysteinyl leukotriene pathway.

Nuclear expression of glycogen synthase kinase-3ß and lack of membranous ß-catenin is correlated with poor survival in colon cancer.

Dysregulation of Wnt/ß-catenin signaling is a hallmark of colon cancer. Glycogen synthase kinase-3ß (GSK-3ß) can be a positive regulator of survival and proliferation of cultured colon cancer cell but its role in clinical colon cancer is unknown. Our objectives were to evaluate the role of GSK-3ß in colon cancer. A tumor tissue microarray of primary colon cancers and metastases was used to evaluate expression and subcellular localization of GSK-3ß and ß-catenin. In total, 85 primary colon cancer samples were evaluated by immunohistochemistry. Immunoreactivity was correlated to known markers of adverse prognosis. Overall survival was the primary end-point. We found nuclear accumulation of GSK-3ß in 39% (33/85) of evaluated tumors. Nuclear GSK-3ß was significantly associated with shorter overall survival (p = 0.008), larger tumor size (p = 0.015), distant metastasis (p = 0.029) and loss of membranous ß-catenin (p = 0.007). Loss of membranous ß-catenin occurred in 37% (30/82) of the tumors and was associated with poor survival (p = 0.016). The combination of nuclear GSK-3ß and lack of membrane ß-catenin occurred in a total of 26% of the studied tumors (21/61) and was significantly and independently associated with poor prognosis. Our results suggest that nuclear expression of GSK-3ß and loss of membrane ß-catenin identify a subset of colon carcinomas with worse prognosis.