DNA Methylation and Gene Expression of the Cysteinyl Leukotriene Receptors as a Prognostic and Metastatic Factor for Colorectal Cancer Patients

Abstract

Colorectal cancer (CRC), one of the leading causes of cancer-related deaths in the western world, is the third most common cancer for both men and women. As a heterogeneous disease, colon cancer (CC) is caused by both genetic and epigenetic changes. The prognosis for CRC is affected by a variety of features, including late diagnosis, lymph node and distant metastasis. The cysteinyl leukotrienes (CysLT), as leukotriene D4 and C4 (LTD4 and LTC4), are synthesized from arachidonic acid via the 5-lipoxygenase pathway, and play an important role in several types of diseases such as inflammation and cancer. Their effects are mediated via the two main G-protein-coupled receptors, CysLT1R and CysLT2R. Multiple studies from our group observed a significant increase in CysLT1R expression in the poor prognosis group, whereas CysLT2R expression was higher in the good prognosis group of CRC patients. Here, we systematically explored and established the role of the CysLTRs, cysteinyl leukotriene receptor 1(CYSLTR1) and cysteinyl leukotriene receptor 2 (CYSLTR2) gene expression and methylation in the progression and metastasis of CRC using three unique in silico cohorts and one clinical CRC cohort. Primary tumor tissues showed significant CYSLTR1 upregulation compared with matched normal tissues, whereas it was the opposite for the CYSLTR2. Univariate Cox proportional-hazards (CoxPH) analysis yielded a high expression of CYSLTR1 and accurately predicted high-risk patients in terms of overall survival (OS; hazard ratio (HR) = 1.87, p = 0.03) and disease-free survival [DFS] Hazard ratio [HR] = 1.54, p = 0.05). Hypomethylation of the CYSLTR1 gene and hypermethylation of the CYSLTR2 gene were found in CRC patients. The M values of the CpG probes for CYSLTR1 are significantly lower in primary tumor and metastasis samples than in matched normal samples, but those for CYSLTR2 are significantly higher. The differentially upregulated genes between tumor and metastatic samples were uniformly expressed in the high-CYSLTR1 group. Two epithelial–mesenchymal transition (EMT) markers, E-cadherin (CDH1) and vimentin (VIM) were significantly downregulated and upregulated in the high-CYSLTR1 group, respectively, but the result was opposite to that of CYSLTR2 expression in CRC. CDH1 expression was high in patients with less methylated CYSLTR1 but low in those with more methylated CYSLTR2. The EMT-associated observations were also validated in CC SW620 cell-derived colonospheres, which showed decreased E-cadherin expression in the LTD4 stimulated cells, but not in the CysLT1R knockdown SW620 cells. The methylation profiles of the CpG probes for CysLTRs significantly predicted lymph node (area under the curve [AUC] = 0.76, p < 0.0001) and distant (AUC = 0.83, p < 0.0001) metastasis. Intriguingly, the CpG probes cg26848126 (HR = 1.51, p = 0.03) for CYSLTR1, and cg16299590 (HR = 2.14, p = 0.03) for CYSLTR2 significantly predicted poor prognosis in terms of OS, whereas the CpG probe cg16886259 for CYSLTR2 significantly predicts a poor prognosis group in terms of DFS (HR = 2.88, p = 0.03). The CYSLTR1 and CYSLTR2 gene expression and methylation results were successfully validated in a CC patient cohort. In this study, we have demonstrated that CysLTRs’ methylation and gene expression profile are associated with the progression, prognosis, and metastasis of CRC, which might be used for the assessment of high-risk CRC patients after validating the result in a larger CRC cohort.