Abnormal demethylation and ectopic expression of leukotriene receptors genes LTB4R/LTB4R2 in breast cancer

Background. The identification and comprehensive characterization of new molecular markers of malignant tumors remains an urgent task of oncology. Earlier, according to the results of a genome-wide screening of differential DNA methylation of normal and tumor breast tissues, abnormal demethylation of the CpG island of LTB4R/LTB4R2 genes in tumors relative to norm was revealed. Aim. The present study focuses on the molecular and clinical characterization of abnormal demethylation of this CpG island and expression of the LTB4R/LTB4R2 genes in breast cancer (BC). Methods. For exploratory analysis, we used the previously obtained results of genome wide bisulfite sequencing of breast cancer (BC) and normal mammary gland samples. From this set, 10 samples of triple-negative (TN) breast cancer with abnormal LTB4R/LTB4R2 demethylation and 6 normal breast samples were selected, for which a comparative analysis of CpG methylation levels in cancer vs norm using the Mann-Whitney test was performed. Sanger bisulfite sequencing was used to confirm abnormal demethylation in TN BC samples. The analysis of the obtained electrophoregrams was carried out using the SeqBase software developed by the authors. To validate and assess the level of predictive significance of LTB4R/LTB4R2 gene expression, level 3 data were used to measure methylation from Illumina HumanMethylation 450K arrays, RNA-seq expression, as well as clinical characteristics for 731 samples from the TCGA-BRCA project. Kaplan-Meier curves were compared using a logrank test. Results. Abnormal demethylation of LTB4R/LTB4R2 genes in breast tumors was confirmed by Sanger sequencing. Among samples from the TCGA-BRCA project, a group of TN BC samples with low methylation of LTB4R/LTB4R2 genes was identified. Overall survival was significantly reduced in the TN breast cancer group with high LTB4R expression, as well as in the group of normal-like breast tumors with low LTB4R expression, and in the LumB group overall survival was significantly reduced when tumors demonstrated low LTB4R2 expression. Conclusions. Fine mapping of abnormal demethylation in individual CpG dinucleotides of the LTB4R/LTB4R2 genes will make it possible to design an effective PCR system that may potentially be used to define patients with TN breast cancer that would benefit from leukotriene receptor therapeutic inhibition. Different expression levels of LTB4R/LTB4R2 allow their use as a prognostic marker for breast cancer, but the prognosis directly depends on the molecular subtype of a tumor. This also applies to the use of different levels of LTB4R/LTB4R2 expression as a predictive marker of the sensitivity of such tumors to leukotriene receptor inhibitors in case they enter clinical trials.

Breast cancer, genome wide analysis of DNA methylation, leukotriene receptors

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