Mutual Regulation of ncRNAs and Chromatin Remodeling Complexes in Normal and Pathological Conditions.

Chromatin remodeling is the one of the main epigenetic mechanisms of gene expression regulation both in normal cells and in pathological conditions. In recent years, a growing number of investigations have confirmed that epigenetic regulators are tightly connected and form a comprehensive network of regulatory pathways and feedback loops. Genes encoding protein subunits of chromatin remodeling complexes are often mutated and change their expression in diseases, as well as non-coding RNAs (ncRNAs). Moreover, different mechanisms of their mutual regulation have already been described. Further understanding of these processes may help apply their clinical potential for establishment of the diagnosis, prognosis, and treatment of the diseases. The therapeutic targeting of the chromatin structure has many limitations because of the complexity of its regulation, with the involvement of a large number of genes, proteins, non-coding transcripts, and other intermediary molecules. However, several successful strategies have been proposed to target subunits of chromatin remodeling complexes and genes encoding them, as well as the ncRNAs that regulate the operation of these complexes and direct them to the target gene regions. In our review, we focus on chromatin remodeling complexes and ncRNAs, their mutual regulation, role in cellular processes and potential clinical application.

Chromosomal Instability in Gastric Cancer: Role in Tumor Development, Progression, and Therapy.

According to the Cancer Genome Atlas (TCGA), gastric cancers are classified into four molecular subtypes: Epstein-Barr virus-positive (EBV+), tumors with microsatellite instability (MSI), tumors with chromosomal instability (CIN), and genomically stable (GS) tumors. However, the gastric cancer (GC) with chromosomal instability remains insufficiently described and does not have effective markers for molecular and histological verification and diagnosis. The CIN subtype of GC is characterized by chromosomal instability, which is manifested by an increased frequency of aneuploidies and/or structural chromosomal rearrangements in tumor cells. Structural rearrangements in the CIN subtype of GC are not accidental and are commonly detected in chromosomal loci, being abnormal because of specific structural organization. The causes of CIN are still being discussed; however, according to recent data, aberrations in the TP53 gene may cause CIN development or worsen its phenotype. Clinically, patients with the CIN subtype of GC demonstrate poor survival, but receive the maximum benefit from adjuvant chemotherapy. In the review, we consider the molecular mechanisms and possible causes of chromosomal instability in GC, the common rearrangements of chromosomal loci and their impact on the development and clinical course of the disease, as well as the driver genes, their functions, and perspectives on their targeting in the CIN subtype of GC.

MiRNA-21a, miRNA-145, and miRNA-221 Expression and Their Correlations with WNT Proteins in Patients with Obstructive and Non-Obstructive Coronary Artery Disease.

MicroRNAs and the WNT signaling cascade regulate the pathogenetic mechanisms of atherosclerotic coronary artery disease (CAD) development. OBJECTIVE: To evaluate the expression of microRNAs (miR-21a, miR-145, and miR-221) and the role of the WNT signaling cascade (WNT1, WNT3a, WNT4, and WNT5a) in obstructive CAD and ischemia with no obstructive coronary arteries (INOCA). METHOD: The cross-sectional observational study comprised 94 subjects. The expression of miR-21a, miR-145, miR-221 (RT-PCR) and the protein levels of WNT1, WNT3a, WNT4, WNT5a, LRP6, and SIRT1 (ELISA) were estimated in the plasma of 20 patients with INOCA (66.5 [62.8; 71.2] years; 25% men), 44 patients with obstructive CAD (64.0 [56.5; 71,0] years; 63.6% men), and 30 healthy volunteers without risk factors for cardiovascular diseases (CVD). RESULTS: Higher levels of WNT1 (0.189 [0.184; 0.193] ng/mL vs. 0.15 [0.15-0.16] ng/mL, p < 0.001) and WNT3a (0.227 [0.181; 0.252] vs. 0.115 [0.07; 0.16] p < 0.001) were found in plasma samples from patients with obstructive CAD, whereas the INOCA group was characterized by higher concentrations of WNT4 (0.345 [0.278; 0.492] ng/mL vs. 0.203 [0.112; 0.378] ng/mL, p = 0.025) and WNT5a (0.17 [0.16; 0.17] ng/mL vs. 0.01 [0.007; 0.018] ng/mL, p < 0.001). MiR-221 expression level was higher in all CAD groups compared to the control group (p < 0.001), whereas miR-21a was more highly expressed in the control group than in the obstructive (p = 0.012) and INOCA (p = 0.003) groups. Correlation analysis revealed associations of miR-21a expression with WNT1 (r = -0.32; p = 0.028) and SIRT1 (r = 0.399; p = 0.005) protein levels in all CAD groups. A positive correlation between miR-145 expression and the WNT4 protein level was observed in patients with obstructive CAD (r = 0.436; p = 0.016). Based on multivariate regression analysis, a mathematical model was constructed that predicts the type of coronary lesion. WNT3a and LRP6 were the independent predictors of INOCA (p < 0.001 and p = 0.002, respectively). CONCLUSIONS: Activation of the canonical cascade of WNT-ß-catenin prevailed in patients with obstructive CAD, whereas in the INOCA and control groups, the activity of the non-canonical pathway was higher. It can be assumed that miR-21a has a negative effect on the formation of atherosclerotic CAD. Alternatively, miR-145 could be involved in the development of coronary artery obstruction, presumably through the regulation of the WNT4 protein. A mathematical model with WNT3a and LRP6 as predictors allows for the prediction of the type of coronary artery lesion.

The CFTR Gene Germline Heterozygous Pathogenic Variants in Russian Patients with Malignant Neoplasms and Healthy Carriers: 11,800 WGS Results.

More than 275 million people in the world are carriers of a heterozygous mutation of the CFTR gene, associated with cystic fibrosis, the most common autosomal recessive disease among Caucasians. Some recent studies assessed the association between carriers of CFTR variants and some pathologies, including cancer risk. The aim of this study is to analyze the landscape of germline pathogenic heterozygous CFTR variants in patients with diagnosed malignant neoplasms. For the first time in Russia, we evaluated the frequency of CFTR pathogenic variants by whole-genome sequencing in 1800 patients with cancer and compared this with frequencies of CFTR variants in the control group (1825 people) adjusted for age and 10,000 healthy individuals. In the issue, 47 out of 1800 patients (2.6%) were carriers of CFTR pathogenic genetic variants: 0.028 (42/1525) (2.8%) among breast cancer patients, 0.017 (3/181) (1.7%) among colorectal cancer patients and 0.021 (2/94) (2.1%) among ovarian cancer patients. Pathogenic CFTR variants were found in 52/1825 cases (2.85%) in the control group and 221 (2.21%) in 10,000 healthy individuals. Based on the results of the comparison, there was no significant difference in the frequency and distribution of pathogenic variants of the CFTR gene, which is probably due to the study limitations. Obviously, additional studies are needed to assess the clinical significance of the heterozygous carriage of CFTR pathogenic variants in the development of various pathologies in the future, particularly cancer.

Epigenomic Profiling Advises Therapeutic Potential of Leukotriene Receptor Inhibitors for a Subset of Triple-Negative Breast Tumors.

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.

Histone Modifications and Non-Coding RNAs: Mutual Epigenetic Regulation and Role in Pathogenesis.

In the last few years, more and more scientists have suggested and confirmed that epigenetic regulators are tightly connected and form a comprehensive network of regulatory pathways and feedback loops. This is particularly interesting for a better understanding of processes that occur in the development and progression of various diseases. Appearing on the preclinical stages of diseases, epigenetic aberrations may be prominent biomarkers. Being dynamic and reversible, epigenetic modifications could become targets for a novel option for therapy. Therefore, in this review, we are focusing on histone modifications and ncRNAs, their mutual regulation, role in cellular processes and potential clinical application.

Methylation and Noncoding RNAs in Gastric Cancer: Everything Is Connected.

Despite recent progress, gastric cancer remains one of the most common cancers and has a high mortality rate worldwide. Aberrant DNA methylation pattern and deregulation of noncoding RNA expression appear in the early stages of gastric cancer. Numerous investigations have confirmed their significant role in gastric cancer tumorigenesis and their high potential as diagnostic and prognostic biomarkers. Currently, it is clear that these epigenetic regulators do not work alone but interact with each other, generating a complex network. The aim of our review was to summarize the current knowledge of this interaction in gastric cancer and estimate its clinical potential for the diagnosis, prognosis, and treatment of the disease.

Towards Unravelling the Role of ERα-Targeting miRNAs in the Exosome-Mediated Transferring of the Hormone Resistance.

Hormone therapy is one of the most effective breast cancer treatments, however, its application is limited by the progression of hormonal resistance, both primary or acquired. The development of hormonal resistance is caused either by an irreversible block of hormonal signalling (suppression of the activity or synthesis of hormone receptors), or by activation of oestrogen-independent signalling pathways. Recently the effect of exosome-mediated intercellular transfer of hormonal resistance was revealed, however, the molecular mechanism of this effect is still unknown. Here, the role of exosomal miRNAs (microRNAs) in the transferring of hormonal resistance in breast cancer cells has been studied. The methods used in the work include extraction, purification and RNAseq of miRNAs, transfection of miRNA mimetics, immunoblotting, reporter analysis and the MTT test. Using MCF7 breast cancer cells and MCF7/T tamoxifen-resistant sub-line, we have found that some miRNAs, suppressors of oestrogen receptor signalling, are overexpressed in the exosomes of the resistant breast cancer cells. The multiple (but not single) transfection of one of the identified miRNA, miR-181a-2, into oestrogen-dependent MCF7 cells induced the irreversible tamoxifen resistance associated with the continuous block of the oestrogen receptor signalling and the activation of PI3K/Akt pathway. We suppose that the miRNAs-ERα suppressors may act as trigger agents inducing the block of oestrogen receptor signalling and breast cancer cell transition to an aggressive oestrogen-independent state.

Secretion of Mutant DNA and mRNA by the Exosomes of Breast Cancer Cells.

Exosomes are the small vesicles that are secreted by different types of normal and tumour cells and can incorporate and transfer their cargo to the recipient cells. The main goal of the present work was to study the tumour exosomes' ability to accumulate the parent mutant DNA or RNA transcripts with their following transfer to the surrounding cells. The experiments were performed on the MCF7 breast cancer cells that are characterized by the unique coding mutation in the PIK3CA gene. Using two independent methods, Sanger sequencing and allele-specific real-time PCR, we revealed the presence of the fragments of the mutant DNA and RNA transcripts in the exosomes secreted by the MCF7 cells. Furthermore, we demonstrated the MCF7 exosomes' ability to incorporate into the heterologous MDA-MB-231 breast cancer cells supporting the possible transferring of the exosomal cargo into the recipient cells. Sanger sequencing of the DNA from MDA-MB-231 cells (originally bearing a wild type of PIK3CA) treated with MCF7 exosomes showed no detectable amount of mutant DNA or RNA; however, using allele-specific real-time PCR, we revealed a minor signal from amplification of a mutant allele, showing a slight increase of mutant DNA in the exosome-treated MDA-MB-231 cells. The results demonstrate the exosome-mediated secretion of the fragments of mutant DNA and mRNA by the cancer cells and the exosomes' ability to transfer their cargo into the heterologous cells.

Genetic Polymorphisms Associated with Rheumatoid Arthritis Development and Antirheumatic Therapy Response.

Rheumatoid arthritis (RA) is the most common inflammatory arthropathy worldwide. Possible manifestations of RA can be represented by a wide variability of symptoms, clinical forms, and course options. This multifactorial disease is triggered by a genetic predisposition and environmental factors. Both clinical and genealogical studies have demonstrated disease case accumulation in families. Revealing the impact of candidate gene missense variants on the disease course elucidates understanding of RA molecular pathogenesis. A multivariate genomewide association study (GWAS) based analysis identified the genes and signalling pathways involved in the pathogenesis of the disease. However, these identified RA candidate gene variants only explain 30% of familial disease cases. The genetic causes for a significant proportion of familial RA have not been determined until now. Therefore, it is important to identify RA risk groups in different populations, as well as the possible prognostic value of some genetic variants for disease development, progression, and treatment. Our review has two purposes. First, to summarise the data on RA candidate genes and the increased disease risk associated with these alleles in various populations. Second, to describe how the genetic variants can be used in the selection of drugs for the treatment of RA.

Roles of E-cadherin and Noncoding RNAs in the Epithelial-mesenchymal Transition and Progression in Gastric Cancer.

The epithelial-mesenchymal transition (EMT) is thought to be at the root of invasive and metastatic cancer cell spreading. E-cadherin is an important player in this process, which forms the structures that establish and maintain cell-cell interactions. A partial or complete loss of E-cadherin expression in the EMT is presumably mediated by mechanisms that block the expression of E-cadherin regulators and involve the E-cadherin-associated transcription factors. The protein is involved in several oncogenic signaling pathways, such as the Wnt/ß-catenin, Rho GTPase, and EGF/EGFR, whereby it plays a role in many tumors, including gastric cancer. Such noncoding transcripts as microRNAs and long noncoding RNAs-critical components of epigenetic control of gene expression in carcinogenesis-contribute to regulation of the E-cadherin function by acting directly or through numerous factors controlling transcription of its gene, and thus affecting not only cancer cell proliferation and metastasis, but also the EMT. This review focuses on the role of E-cadherin and the non-coding RNAs-mediated mechanisms of its expressional control in the EMT during stomach carcinogenesis.

Implication of Gastric Cancer Molecular Genetic Markers in Surgical Practice.

INTRODUCTION: We have investigated aberrant methylation of genes CDH1, RASSF1A, MLH1, N33, DAPK, expression of genes hTERT, MMP7, MMP9, BIRC5 (survivin), PTGS2, and activity of telomerase of 106 gastric tumor samples obtained intra-operatively and 53 gastric tumor samples from the same group of patients obtained endoscopically before surgery. Biopsy specimens obtained from 50 patients with chronic calculous cholecystitis were used as a control group. Together with tissue samples obtained from different sites remote to tumors, a total of 727 samples have been studied. The selected parameters comprise a system of molecular markers that can be used in both diagnostics of gastric cancer and in dynamic monitoring of patients after surgery. Special attention was paid to the use of molecular markers for the diagnostics of malignant process in the material obtained endoscopically since the efficacy of morphological diagnostics in biopsies is compromised by intratumoral heterogeneity, which may prevent reliable identification of tumor cells in the sampling. Our data indicated that certain molecular genetic events provided more sensitive yet specific markers of the tumor. CONCLUSION: We demonstrated that molecular profiles detected in preoperative biopsies were confirmed by the material obtained intra-operatively. The use of endoscopic material facilitates gastric tumors pre-operative diagnostics, improving early detection of gastric cancer and potential effective treatment strategies.

CDKN2A Gene Mutations: Implications for Hereditary Cancer Syndromes.

Malignant neoplasms, including pancreatic cancer and melanoma, are major global health challenges. This study investigates melanoma pancreatic syndrome, a rare hereditary tumor syndrome associated with CDKN2A gene mutations. CDKN2A mutations contribute to a lifetime risk of melanoma ranging from 28% to 67%. This study reports the clinical features of six individuals with CDKN2A mutations and identifies recurrent alterations such as c.307_308del, c.159G>C and c.71G>C. It highlights the need for CDKN2A mutation testing in suspected cases of familial atypical multiple mole melanoma. Clinically significant variants show associations with melanoma and pancreatic cancer. The challenges of treating individuals with CDKN2A mutations are discussed, and the lack of specific targeted therapies is highlighted. Preclinical studies suggest a potential benefit of CDK4/6 inhibitors, although clinical trials show mixed results. This study underscores the importance of continued research into improved diagnostic and therapeutic strategies to address the complexities of hereditary cancer syndromes.

DNA Methylation and Prospects for Predicting the Therapeutic Effect of Neoadjuvant Chemotherapy for Triple-Negative and Luminal B Breast Cancer.

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.

Genetic and Clinical Factors Associated with Olokizumab Treatment in Russian Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease and its treatment is an urgent problem of rheumatology. Olokizumab (OKZ) is a new humanized monoclonal antibody targeting IL-6 and is one of the few promising drugs for RA therapy. One-hundred-and-twenty-five DNA samples from Russian patients with RA, treated with olokizumab, were genotyped with an NGS panel containing 60 single nucleotide polymorphisms (SNPs) and the whole coding sequences of IL6, IL6R, TNFRSF1A, CTLA4, IL10, IL23R, and PADI4; and by RT-PCR for HLA-DRB1 and HLA-B. Associations of polymorphic variants with olokizumab efficacy according to the scores ACR20, ACR50, and DAS28-CRP were determined. We analyzed the obtained data by using logistic regression, ROC curves, and multivariate ANOVA. A high predictive value of the response to olokizumab therapy at 24 weeks was found for the combination of HLA-DRB1*04 and HLA-B*27 alleles with SNPs located in non-HLA genes (IL1B, IL17A, PADI4, DHODH, GLCCI1, IL23R, and TNFAIP3), and clinical characteristics (age, RA duration, and intensity) according to ACR20. Thus, the comprehensive assessment of polymorphic variants of HLA and non-HLA genes considering population characteristics in combination with clinical parameters allows for the elaboration of an RA prognostic panel.

Comprehensive Molecular Genetic Diagnostics of Birt-Hogg-Dube Syndrome in a Russian Patient with Renal Cancer and Lung Cysts: A Case Report.

We report a case of Birt-Hogg-Dube syndrome (BHDS), a rare hereditary syndrome, the main visible sign of which is the development of multiple skin fibrofolliculomas. In our case, there was a manifestation of BHDS consisting in the absence of fibrofolliculomas and presence of other characteristic features of this syndrome: lung cysts and renal cancer. The 26-year-old woman was admitted to a clinic for diagnosis and treatment of a neoplasm of the left kidney and had a history of renal cell cancer (RCC) of the right kidney and spontaneous pneumothorax. Multiple tumors of the left kidney and lung cysts were observed upon clinical and laboratory testing. Tumors of the left kidney were resected and diagnosed by a pathologist as chromophobe RCC. Sequencing of FLCN exons 4-14 from blood DNA revealed the heterozygous germline nonsense mutation c.1429C>T (p.R477*), confirming the diagnosis of BHDS. Several somatic variants were detected by tumor DNA sequencing using the Comprehensive Cancer Panel and Ion S5 platform. Medical-genetic counseling was conducted, and follow-up management was outlined. To our knowledge, this case report is the first comprehensive clinical and genetic examination of a patient with BHDS in Russia. The p.R477* mutation has been described by other authors in patients with fibrofolliculomas and lung cysts, but not in those with RCC, while RCC was the first manifestation of BHDS in our case. The case report may help geneticists, oncologists, and other specialists to better understand the clinical and genetic heterogeneity of BHDS in various populations.

Genetic Factors of Predisposition and Clinical Characteristics of Rheumatoid Arthritis in Russian Patients.

Rheumatoid arthritis (RA) is a multifactorial disease caused by a genetic predisposition and environmental factors. Predisposing alleles of various genes have a relatively small influence on the disease risk when they appear separately, but in combination, they predispose an individual to RA development. We genotyped 125 patients with RA including 60 SNPs and sequenced coding part of six genes by next-generation sequencing (NGS) technology on a target panel (IAD177464_185). According to our data, the alleles HLA-DRB1*04, HLA-DRB1*01, HLA-B*27, PTPN22 (rs2476601), TNF (rs1800629), TPMT (rs2842934), and IL4 (rs2243250), and genotypes HLA-DRB1*04:04, HLA-DRB1*01:16, PTPN22 (rs2476601), TPMT (rs2842934), were significantly associated with the RA development. Associations with clinical criteria (DAS28-CRP, HAQ-DI, and CDAI) and biochemical factors were investigated. We have shown that the PADI4 genotypes (rs11203367, rs2240340, rs11203366, and rs874881) are significantly associated with the baseline levels of DAS28-CRP, HAQ-DI, and CDAI; genotypes IL23R (rs7530511) and TNFRSF1A (rs748004, rs2228144) with the level of anti citrullinated peptide antibodies (ACPA); the genotypes DHODH (rs3213422) and MTHFR (rs180113) with the concentration of C-reactive protein (CRP); and the genotypes IL2RA (rs2104286), IRAK3 (rs11541076), and IL4R (rs1801275) with the level of rheumatoid factor (RF). Application of targeted NGS panel contributes to expanded genotyping to identify risk groups among the RA patients.

Mutations in Epigenetic Regulation Genes in Gastric Cancer.

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.

Long Non-Coding PROX1-AS1 Expression Correlates with Renal Cell Carcinoma Metastasis and Aggressiveness.

Long non-coding RNAs (lncRNAs) can be specifically expressed in different tissues and cancers. By controlling the gene expression at the transcriptional and translational levels, lncRNAs have been reported to be involved in tumor growth and metastasis. Recent data demonstrated that multiple lncRNAs have a crucial role in renal cell carcinoma (RCC) progression-the most common malignant urogenital tumor. In the present study, we found a trend towards increased PROX1 antisense RNA 1 (PROX1-AS1) expression in RCC specimens compared to non-tumoral margins. Next, we found a positive correlation between PROX1-AS1 expression and the occurrence of distant and lymph node metastasis, higher tumor stage (pT1 vs. pT2 vs. pT3-T4) and high-grade (G1/G2 vs. G3/G4) clear RCC. Furthermore, global demethylation in RCC-derived cell lines (769-P and A498) and human embryonic kidney 293 (HEK293) cells induced a significant increase of PROX1-AS1 expression level, with the most remarkable change in HEK293 cells. In line with this evidence, bisulfite sequencing analysis confirmed the specific demethylation of bioinformatically selected CpG islands on the PROX1-AS1 promoter sequence in the HEK293 cell line but not in the tumor cells. Additionally, the human specimen analysis showed the hemimethylated state of CG dinucleotides in non-tumor kidney tissues, whereas the tumor samples presented the complete, partial, or no demethylation of CpG-islands. In conclusion, our study indicated that PROX1-AS1 could be associated with RCC progression, and further investigations may define its role as a new diagnostic marker and therapeutic target.

Abnormal promoter DNA hypermethylation of the integrin, nidogen, and dystroglycan genes in breast cancer.

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.