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Cell transmembrane receptors and extracellular matrix components play a pivotal role in regulating cell activity and providing for the concerted integration of cells in the tissue structures. We have assessed DNA methylation in the promoter regions of eight integrin genes, two nidogen genes, and the dystroglycan gene in normal breast tissues and breast carcinomas (BC). The protein products of these genes interact with the basement membrane proteins LAMA1, LAMA2, and LAMB1; abnormal hypermethylation of the LAMA1, LAMA2, and LAMB1 promoters in BC has been described in our previous publications. In the present study, the frequencies of abnormal promoter hypermethylation in BC were 13% for ITGA1, 31% for ITGA4, 4% for ITGA7, 39% for ITGA9, 38% for NID1, and 41% for NID2. ITGA2, ITGA3, ITGA6, ITGB1, and DAG1 promoters were nonmethylated in normal and BC samples. ITGA4, ITGA9, and NID1 promoter hypermethylation was associated with the HER2 positive tumors, and promoter hypermethylation of ITGA1, ITGA9, NID1 and NID2 was associated with a genome-wide CpG island hypermethylated BC subtype. Given that ITGA4 is not expressed in normal breast, one might suggest that its abnormal promoter hypermethylation in cancer is non-functional and is thus merely a passenger epimutation. Yet, this assumption is not supported by our finding that it is not associated with a hypermethylated BC subtype. ITGA4 acquires expression in a subset of breast carcinomas, and methylation of its promoter may be preventive against expression in some tumors. Strong association of abnormal ITGA4 hypermethylation with the HER2 positive tumors (p = 0.0025) suggests that simultaneous presence of both HER2 and integrin α4 receptors is not beneficial for tumor cells. This may imply HER2 and integrin α4 signaling pathways interactions that are yet to be discovered. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype, with a poor survival rate compared to others subtypes. For a long time, chemotherapy was the only systemic treatment for TNBC, and the identification of actionable molecular targets might ultimately improve the prognosis for TNBC patients. We performed a genome-wide analysis of DNA methylation at CpG islands on a collection of one hundred ten breast carcinoma samples and six normal breast tissue samples using reduced representation bisulfite sequencing with the XmaI restriction enzyme (XmaI-RRBS) and identified a subset of TNBC samples with significant hypomethylation at the LTB4R/LTB4R2 genes' CpG islands, including CpG dinucleotides covered with cg12853742 and cg21886367 HumanMethylation 450K microarray probes. Abnormal DNA hypomethylation of this region in TNBC compared to normal samples was confirmed by bisulfite Sanger sequencing. Gene expression generally anticorrelates with promoter methylation, and thus, the promoter hypomethylation detected and confirmed in our study might be revealed as an indirect marker of high LTB4R/LTB4R2 expression using a simple methylation-sensitive PCR test. Analysis of RNA-seq expression and DNA methylation data from the TCGA dataset demonstrates that the expression of the LTB4R and LTB4R2 genes significantly negatively correlates with DNA methylation at both CpG sites cg12853742 (R = −0.4, p = 2.6 × 10−6; R = −0.21, p = 0.015) and cg21886367 (R = −0.45, p = 7.3 × 10−8; R = −0.24, p = 0.005), suggesting the upregulation of these genes in tumors with abnormal hypomethylation of their CpG island. Kaplan–Meier analysis using the TCGA-BRCA gene expression and clinical data revealed poorer overall survival for TNBC patients with an upregulated LTB4R. To this day, only the leukotriene inhibitor LY255283 has been tested on an MCF-7/DOX cell line, which is a luminal A breast cancer molecular subtype. Other studies compare the effects of Montelukast and Zafirlukast (inhibitors of the cysteinyl leukotriene receptor, which is different from LTB4R/LTB4R2) on the MDA-MB-231 (TNBC) cell line, with high methylation and low expression levels of LTB4R. In our study, we assess the therapeutic effects of various drugs (including leukotriene receptor inhibitors) with the DepMap gene effect and drug sensitivity data for TNBC cell lines with hypomethylated and upregulated LTB4R/LTB4R2 genes. LY255283, Minocycline, Silibinin, Piceatannol, Mitiglinide, 1-Azakenpaullone, Carbetocin, and Pim-1-inhibitor-2 can be considered as candidates for the additional treatment of TNBC patients with tumors demonstrating LTB4R/LTB4R2 hypomethylation/upregulation. Finally, our results suggest that the epigenetic status of leukotriene B4 receptors is a novel, potential, predictive, and prognostic biomarker for TNBC. These findings might improve individualized therapy for TNBC patients by introducing new therapeutic adjuncts as anticancer agents. [ABSTRACT FROM AUTHOR]

Despite the advantages of neoadjuvant chemotherapy (NACT), associated toxicity is a serious complication that renders monitoring of the patients' response to NACT highly important. Thus, prediction of tumor response to treatment is imperative to avoid exposure of potential non-responders to deleterious complications. We have performed genome-wide analysis of DNA methylation by XmaI-RRBS and selected CpG dinucleotides differential methylation of which discriminates luminal B breast cancer samples with different sensitivity to NACT. With this data, we have developed multiplex methylation sensitive restriction enzyme PCR (MSRE-PCR) protocol for determining the methylation status of 10 genes (SLC9A3, C1QL2, DPYS, IRF4, ADCY8, KCNQ2, TERT, SYNDIG1, SKOR2 and GRIK1) that distinguish BC samples with different NACT response. Analysis of these 10 markers by MSRE-PCR in biopsy samples allowed us to reveal three top informative combinations of markers, (1) IRF4 and C1QL2; (2) IRF4, C1QL2, and ADCY8; (3) IRF4, C1QL2, and DPYS, with the areas under ROC curves (AUCs) of 0.75, 0.78 and 0.74, respectively. A classifier based on IRF4 and C1QL2 better meets the diagnostic panel simplicity requirements, as it consists of only two markers. Diagnostic accuracy of the panel of these two markers is 0.75, with the sensitivity of 75% and specificity of 75%. [ABSTRACT FROM AUTHOR]

Simple Summary: Breast cancer (BC) is a group of diseases heterogeneous in morphology, progression, survival, and response to therapy. Although BC is among the most exhaustively studied cancers, there is still a lack of molecular markers to predict its response to neoadjuvant chemotherapy (NACT). Tumor development is determined by alterations not only of its genome, but of its epigenome as well. In order to identify epigenomic markers of BC NACT effectiveness, we have applied genome-wide DNA methylation screening of tumors in cohorts of NACT responders and nonresponders. Combining several of the most informative DNA methylation markers, we suggest tiny diagnostic panels that may be readily implemented in diagnostic laboratories. We also demonstrate that clinical characteristics predictive of NACT response, such as the clinical stage and lymph node status, are independently additive to the epigenetic classifiers and in combination improve prediction. Despite advances in the diagnosis and treatment of breast cancer (BC), the main cause of deaths is resistance to existing therapies. An approach to improve the effectiveness of therapy in patients with aggressive BC subtypes is neoadjuvant chemotherapy (NACT). Yet, the response to NACT for aggressive subtypes is less than 65% according to large clinical trials. An obvious fact is the lack of biomarkers predicting the therapeutic effect of NACT. In a search for epigenetic markers, we performed genome-wide differential methylation screening by XmaI-RRBS in cohorts of NACT responders and nonresponders, for triple-negative (TN) and luminal B tumors. The predictive potential of the most discriminative loci was further assessed in independent cohorts by methylation-sensitive restriction enzyme quantitative PCR (MSRE-qPCR), a promising method for the implementation of DNA methylation markers in diagnostic laboratories. The selected most informative individual markers were combined into panels demonstrating cvAUC = 0.83 (TMEM132D and MYO15B markers panel) for TN tumors and cvAUC = 0.76 (TTC34, LTBR and CLEC14A) for luminal B tumors. The combination of methylation markers with clinical features that correlate with NACT effect (clinical stage for TN and lymph node status for luminal B tumors) produces better classifiers, with cvAUC = 0.87 for TN tumors and cvAUC = 0.83 for luminal B tumors. Thus, clinical characteristics predictive of NACT response are independently additive to the epigenetic classifier and in combination improve prediction. [ABSTRACT FROM AUTHOR]

Simple Summary: Epigenetic mechanisms, such as DNA methylation/demethylation, covalent modifications of histone proteins, and chromatin remodeling, create specific patterns of gene expression. Epigenetic deregulations are associated with oncogenesis, relapse of the disease and metastases, and can serve as a useful clinical marker. We assessed the clinical relevance of integrity of the genes coding for epigenetic regulator proteins by mutational profiling of 25 genes in 135 gastric cancer (GC) samples. Overall, mutations in the epigenetic regulation genes were found to be significantly associated with reduced overall survival of patients in the group with metastases and in the group with tumors with signet ring cells. We have also discovered mutual exclusivity of somatic mutations in the KMT2D, KMT2C, ARID1A, and CHD7 genes in our cohort. Our results suggest that mutations in epigenetic regulation genes may be valuable clinical markers and deserve further exploration in independent cohorts. We have performed mutational profiling of 25 genes involved in epigenetic processes on 135 gastric cancer (GC) samples. In total, we identified 79 somatic mutations in 49/135 (36%) samples. The minority (n = 8) of mutations was identified in DNA methylation/demethylation genes, while the majority (n = 41), in histone modifier genes, among which mutations were most commonly found in KMT2D and KMT2C. Somatic mutations in KMT2D, KMT2C, ARID1A and CHD7 were mutually exclusive (p = 0.038). Mutations in ARID1A were associated with distant metastases (p = 0.03). The overall survival of patients in the group with metastases and in the group with tumors with signet ring cells was significantly reduced in the presence of mutations in epigenetic regulation genes (p = 0.036 and p = 0.041, respectively). Separately, somatic mutations in chromatin remodeling genes correlate with low survival rate of patients without distant metastasis (p = 0.045) and in the presence of signet ring cells (p = 0.0014). Our results suggest that mutations in epigenetic regulation genes may be valuable clinical markers and deserve further exploration in independent cohorts. [ABSTRACT FROM AUTHOR]