Circulating Tumor DNA Is a Variant of Liquid Biopsy with Predictive and Prognostic Clinical Value in Breast Cancer Patients.

This paper introduces the reader to the field of liquid biopsies and cell-free nucleic acids, focusing on circulating tumor DNA (ctDNA) in breast cancer (BC). BC is the most common type of cancer in women, and progress with regard to treatment has been made in recent years. Despite this, there remain a number of unresolved issues in the treatment of BC; in particular, early detection and diagnosis, reliable markers of response to treatment and for the prediction of recurrence and metastasis, especially for unfavorable subtypes, are needed. It is also important to identify biomarkers for the assessment of drug resistance and for disease monitoring. Our work is devoted to ctDNA, which may be such a marker. Here, we describe its main characteristics and potential applications in clinical oncology. This review considers the results of studies devoted to the analysis of the prognostic and predictive roles of various methods for the determination of ctDNA in BC patients. Currently known epigenetic changes in ctDNA with clinical significance are reviewed. The possibility of using ctDNA as a predictive and prognostic marker for monitoring BC and predicting the recurrence and metastasis of cancer is also discussed, which may become an important part of a precision approach to the treatment of BC.

Various LncRNA Mechanisms in Gene Regulation Involving miRNAs or RNA-Binding Proteins in Non-Small-Cell Lung Cancer: Main Signaling Pathways and Networks.

Long non-coding RNAs (lncRNAs) are crucial players in the pathogenesis of non-small-cell lung cancer (NSCLC). A competing binding of lncRNAs and mRNAs with microRNAs (miRNAs) is one of the most common mechanisms of gene regulation by lncRNAs in NSCLC, which has been extensively researched in the last two decades. However, alternative mechanisms that do not depend on miRNAs have also been reported. Among them, the most intriguing mechanism is mediated by RNA-binding proteins (RBPs) such as IGF2BP1/2/3, YTHDF1, HuR, and FBL, which increase the stability of target mRNAs. IGF2BP2 and YTHDF1 may also be involved in m6A modification of lncRNAs or target mRNAs. Some lncRNAs, such as DLGAP1-AS2, MALAT1, MNX1-AS1, and SNHG12, are involved in several mechanisms depending on the target: lncRNA/miRNA/mRNA interactome and through RBP. The target protein sets selected here were then analyzed using the DAVID database to identify the pathways overrepresented by KEGG, Wikipathways, and the Reactome pathway. Using the STRING website, we assessed interactions between the target proteins and built networks. Our analysis revealed that the JAK-STAT and Hippo signaling pathways, cytokine pathways, the VEGFA-VEGFR2 pathway, mechanisms of cell cycle regulation, and neovascularization are the most relevant to the effect of lncRNA on NSCLC.

Regulation of the Key Epithelial Cancer Suppressor miR-124 Function by Competing Endogenous RNAs.

A decrease in the miR-124 expression was observed in various epithelial cancers. Like a classical suppressor, miR-124 can inhibit the translation of multiple oncogenic proteins. Epigenetic mechanisms play a significant role in the regulation of miR-124 expression and involve hypermethylation of the MIR-124-1/-2/-3 genes and the effects of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) according to the model of competing endogenous RNAs (ceRNAs). More than 40 interactomes (lncRNA/miR-124/mRNA) based on competition between lncRNAs and mRNAs for miR-124 binding have been identified in various epithelial cancers. LncRNAs MALAT1, NEAT1, HOXA11-AS, and XIST are the most represented in these axes. Fourteen axes (e.g., SND1-IT1/miR-124/COL4A1) are involved in EMT and/or metastasis. Moreover, eight axes (e.g., OIP5-AS1/miR-124-5p/IDH2) are involved in key pathways, such as Wnt/b-catenin, E2F1, TGF-ß, SMAD, ERK/MAPK, HIF-1α, Notch, PI3K/Akt signaling, and cancer cell stemness. Additionally, 15 axes impaired patient survival and three axes reduced chemo- or radiosensitivity. To date, 14 cases of miR-124 regulation by circRNAs have been identified. Half of them involve circHIPK3, which belongs to the exonic ecircRNAs and stimulates cell proliferation, EMT, autophagy, angiogenesis, and multidrug resistance. Thus, miR-124 and its interacting partners may be considered promising targets for cancer therapy.

Circulating MicroRNAs as a New Class of Biomarkers of Physiological Reactions of the Organism to the Intake of Dietary Supplements and Drugs.

BACKGROUND: The analysis of individual microRNAs (miRNAs) as a diagnostic and prognostic tool for the effective treatment of various diseases has aroused particular interest in the scientific community. The determination of circulating miRNAs makes it possible to assess biological changes associated with nutritional processes, the intake of dietary supplements and drugs, etc. The profile of circulating miRNAs reflects the individual adaptation of the organism to the effect of specific environmental conditions. OBJECTIVE: The objective of this study is to systematize the data and show the importance of circulating miRNAs as new potential biomarkers of the organism's response to the intake of various dietary supplements, drugs, and consider the possibility of their use in doping control. METHODS: A systematic analysis of scientific publications (ncbi.nlm.nih.gov) on the miRNA expression profile in response to the intake of dietary supplements and drugs most often used by athletes, and supposed their role as potential markers in modern doping control was carried out. RESULTS: The profile of circulating miRNAs is highly dependent on the intake of a particular drug, and, therefore, may be used as a marker of the effects of biologically active supplements and drugs including the substances from the Prohibited List of the World Anti-Doping Agency (WADA). CONCLUSION: Monitoring of circulating miRNAs can serve as a high-precision marker for detecting doping abuse in elite sports. However, it is necessary to conduct additional studies on the effect of complex drugs on the profile of circulating miRNAs and individual circulating miRNAs on a particular biological process.

Dysregulation of lncRNA-miRNA-mRNA Interactome as a Marker of Metastatic Process in Ovarian Cancer.

Ovarian cancer (OC) is one of the most common types of cancer among malignancies of the female reproductive system. This pathology is asymptomatic until advanced stages and has a poor prognosis. Our study aimed to search for lncRNA-miRNA-mRNA competing triplets that promote ovarian tumorigenesis. For this purpose, we analyzed tumor samples from the TCGA database and verified the results experimentally in a set of 46 paired samples of tumor and matched histologically unchanged ovarian tissues from OC patients. The list of RNAs selected in silico for experimental studies included 13 mRNAs, 10 lncRNAs, and 5 miRNAs related to epithelial-mesenchymal transition and angiogenesis. We evaluated the expression of these RNAs by qRT-PCR and assessed the correlation between levels of miRNAs, mRNAs, and lncRNAs. Sixteen significant triplets were revealed, in some of which, e.g., OIP5-AS1-miR-203a-c-MET and OIP5-AS1-miR-203a-ZEB2, both lncRNA and mRNA had sites for miR-203a direct binding. Transfection of the OVCAR-3 and SKOV-3 cell lines with the miR-203a mimic was used to confirm the novel links of miR-203a with ZEB2 and c-MET in OC. These connections suggest that the interactomes have the potential for diagnostics of metastasis at early onset.

Aberrant Methylation of 20 miRNA Genes Specifically Involved in Various Steps of Ovarian Carcinoma Spread: From Primary Tumors to Peritoneal Macroscopic Metastases.

Our work aimed to differentiate 20 aberrantly methylated miRNA genes that participate at different stages of development and metastasis of ovarian carcinoma (OvCa) using methylation-specific qPCR in a representative set of clinical samples: 102 primary tumors without and with metastases (to lymph nodes, peritoneum, or distant organs) and 30 peritoneal macroscopic metastases (PMM). Thirteen miRNA genes (MIR107, MIR124-2, MIR124-3, MIR125B-1, MIR127, MIR129-2, MIR130B, MIR132, MIR193A, MIR339, MIR34B/C, MIR9-1, and MIR9-3) were hypermethylated already at the early stages of OvCa, while hypermethylation of MIR1258, MIR137, MIR203A, and MIR375 was pronounced in metastatic tumors, and MIR148A showed high methylation levels specifically in PMM. We confirmed the significant relationship between methylation and expression levels for 11 out of 12 miRNAs analyzed by qRT-PCR. Moreover, expression levels of six miRNAs were significantly decreased in metastatic tumors in comparison with nonmetastatic ones, and downregulation of miR-203a-3p was the most significant. We revealed an inverse relationship between expression levels of miR-203a-3p and those of ZEB1 and ZEB2 genes, which are EMT drivers. We also identified three miRNA genes (MIR148A, MIR9-1, and MIR193A) that likely regulate EMT-MET reversion in the colonization of PMM. According to the Kaplan-Meier analysis, hypermethylation of several examined miRNA genes was associated with poorer overall survival of OvCa patients, and high methylation levels of MIR130B and MIR9-1 were related to the greatest relative risk of death.

LncRNAs in the Regulation of Genes and Signaling Pathways through miRNA-Mediated and Other Mechanisms in Clear Cell Renal Cell Carcinoma.

The fundamental novelty in the pathogenesis of renal cell carcinoma (RCC) was discovered as a result of the recent identification of the role of long non-coding RNAs (lncRNAs). Here, we discuss several mechanisms for the dysregulation of the expression of protein-coding genes initiated by lncRNAs in the most common and aggressive type of kidney cancer-clear cell RCC (ccRCC). A model of competitive endogenous RNA (ceRNA) is considered, in which lncRNA acts on genes through the lncRNA/miRNA/mRNA axis. For the most studied oncogenic lncRNAs, such as HOTAIR, MALAT1, and TUG1, several regulatory axes were identified in ccRCC, demonstrating a number of sites for various miRNAs. Interestingly, the LINC00973/miR-7109/Siglec-15 axis represents a novel agent that can suppress the immune response in patients with ccRCC, serving as a valuable target in addition to the PD1/PD-L1 pathway. Other mechanisms of action of lncRNAs in ccRCC, involving direct binding with proteins, mRNAs, and genes/DNA, are also considered. Our review briefly highlights methods by which various mechanisms of action of lncRNAs were verified. We pay special attention to protein targets and signaling pathways with which lncRNAs are associated in ccRCC. Thus, these new data on the different mechanisms of lncRNA functioning provide a novel basis for understanding the pathogenesis of ccRCC and the identification of new prognostic markers and targets for therapy.

Long Noncoding RNA GAS5 in Breast Cancer: Epigenetic Mechanisms and Biological Functions.

Long noncoding RNAs (lncRNAs) have been identified as contributors to the development and progression of cancer through various functions and mechanisms. LncRNA GAS5 is downregulated in multiple cancers and acts as a tumor suppressor in breast cancer. GAS5 interacts with various proteins (e.g., E2F1, EZH2, and YAP), DNA (e.g., the insulin receptor promoter), and various microRNAs (miRNAs). In breast cancer, GAS5 binds with miR-21, miR-222, miR-221-3p, miR-196a-5p, and miR-378a-5p that indicates the presence of several elements for miRNA binding (MREs) in GAS5. Mediated by the listed miRNAs, GAS5 is involved in the upregulation of a number of mRNAs of suppressor proteins such as PTEN, PDCD4, DKK2, FOXO1, and SUFU. Furthermore, the aberrant promoter methylation is involved in the regulation of GAS5 gene expression in triple-negative breast cancer and some other carcinomas. GAS5 can stimulate apoptosis in breast cancer via diverse pathways, including cell death receptors and mitochondrial signaling pathways. GAS5 is also a key player in the regulation of some crucial signal pathways in breast cancer, such as PI3K/AKT/mTOR, Wnt/ß-catenin, and NF-κB signaling. Through epigenetic and other mechanisms, GAS5 can increase sensitivity to multiple drugs and improve prognosis. GAS5 is thus a promising target in the treatment of breast cancer patients.

Novel miRNA genes deregulated by aberrant methylation in ovarian carcinoma are involved in metastasis.

Methylation of promoter CpG islands may suppress the function of miRNAs by inhibiting their expression. Our work analyzes the role of promoter methylation in altering the expression of 12 miRNAs associated with epithelial ovarian cancer (EOC): miR-124-3p, -125b-5p, -127-5p, -129-5p, -132-3p, -137, -148a-3p, -191-5p, -193a-5p, -203a, -339-3p, and -375. The role of methylation in the deregulation of these miRNAs has not been previously assessed in a representative set of EOC samples. Using 76 paired (tumor/matched normal) ovarian samples and methylation-specific PCR, we demonstrated significant aberrations in the methylation patterns of 11 miRNA genes and identified 8 novel hypermethylated miRNA genes (MIR-124-1, -124-2, -124-3, -127, -132, -137, -193A, and -339) as well as one hypomethylated miRNA gene (MIR-191). Quantitative PCR on a subset of 29 paired EOC samples allowed us to establish a strong correlation between methylation status and alterations in expression levels for all 12 miRNAs studied. These findings demonstrate the functional role of aberrant methylation of examined miRNA genes in EOC. Moreover, we showed a significant association of hypermethylation of 10 miRNA genes (MIR-124-2, -124-3, -125B-1, -127, -129-2, -137, -193A, -203A, -339, -375) with EOC metastasis to lymph nodes, peritoneum, and distant organs. Interestingly, MIR-203A and MIR-375 were hypermethylated only in disseminated ovarian tumors, implying that non-suppressor miR-203a and miR-375 have anti-metastatic properties. Hypermethylation of 10 miRNA genes in EOC metastases was validated using an additional sample set of 13 primary tumors and matched peritoneal metastases. Together, these results show the impact of aberrant methylation on deregulation of 12 miRNAs in EOC, the involvement of 10 hypermethylated miRNA genes in metastasis (including peritoneal macro-metastases), and suggest novel potential biomarkers.

DNA methylation contributes to deregulation of 12 cancer-associated microRNAs and breast cancer progression.

The methylation of promoter CpG islands and the interaction between microRNAs (miRNAs) and messenger RNAs (mRNAs) of target genes are considered two crucial mechanisms for gene and pathway deregulation in malignant tumors. The aim of this study was to analyze the role of promoter methylation in altering the expression of 13 miRNAs that are associated with breast cancer (BC): miR-124, -125b, -127, -132, -137, -148a, -191, -193a, -203, -212, -34b, -375, -9. The role of methylation in the deregulation of these miRNAs has not been previously assessed in the representative set of BC samples. We used a set of 58 paired (tumor/normal) breast tissue samples and methylation-specific PCR to demonstrate significant aberrations in the methylation patterns of 9 miRNA genes. In particular, we observed hypermethylation of MIR-127, -132, and -193a, and hypomethylation of MIR-191 for the first time. Using quantitative PCR, we established a strong correlation between promoter methylation and expression levels for 12 miRNA genes (all except MIR-212); this finding demonstrates the functional importance of altered methylation patterns. We also performed a correlation analysis between expression levels of the 13 miRNAs and 5 cancer-associated genes, namely RASSF1(A), CHL1, APAF1, DAPK1, and BCL2, which were predicted as targets for these miRNAs, to investigate the impact of these miRNAs on these genes with key cellular functions in BC. Significant negative correlation was revealed for the following miRNA-mRNA pairs: miR-127-5p and DAPK1, miR-375 and RASSF1(A), and miR-124-3p and BCL2. Additionally, we also found a strong association between hypermethylation of MIR-127 and MIR-125b-1 and BC progression, particularly metastasis. Thus, our findings provide evidence for the significant role of methylation in the deregulation of 12 miRNA genes in BC, identify putative novel functional miRNA-mRNA pairs, and suggest MIR-127 and MIR-125b-1 hypermethylation to be potential biomarkers of BC metastasis.

Expression and DNA methylation alterations of seven cancer-associated 3p genes and their predicted regulator miRNAs (miR-129-2, miR-9-1) in breast and ovarian cancers.

The methylation of promoter CpG islands and interactions between microRNAs (miRNAs) and messenger RNAs (mRNAs) of target genes are considered two crucial epigenetic mechanisms for inducing gene and pathway deregulation in tumors. Here, the expression levels of seven cancer-associated 3p genes (RASSF1(isoform A), RARB(isoform 2), SEMA3B, RHOA, GPX1, NKIRAS1, and CHL1) and their predicted regulator miRNAs (miR-129-2, miR-9-1) were analyzed in breast (BC, 40 samples) and ovarian (OC, 14 samples) cancers using RT-PCR and qPCR. We first revealed a negative correlation between the level of the miR-129-2 precursor and RASSF1(A) and GPX1 mRNA levels in BC (Spearman's correlation coefficient (rs) was − 0.26 in both cases). Similar results were observed for the miR-129-2 precursor and the RASSF1(A), GPX1, RARB(2), and CHL1 genes in OC (rs was in the range − 0.48 to − 0.54). Using methylation-specific PCR, a significant correlation was shown between promoter hypermethylation and the down-regulation of the RASSF1(A), GPX1, RARB(2), SEMA3B, MIR-129-2, and MIR-9-1 genes in BC (rs = 0.41 to 0.75) and of the RASSF1(A) gene in OC (rs = 0.67). We first demonstrated a high hypermethylation frequency of MIR-129-2 and SEMA3B (up to 45 to 48%) in both BC (69 samples) and OC (41 samples). Moreover, we observed a positive correlation between the hypermethylation of MIR-129-2 and the up-regulation of the RASSF1(A) and GPX1 genes in BC (rs = 0.38 and 0.42, respectively). QPCR analysis of the expression of RASSF1(A) and mature miR-129-2 in additional BC sample set (24 samples) revealed a negative correlation between them (rs = − 0.41) that strengthened the results obtained during the analysis of miR-129-2 precursor level. In summary, the obtained data indicate the involvement of methylation in the down-regulation of the studied coding and miRNA genes and suggest the involvement of miR-129-2 in the deregulation of RASSF1(A) via a direct interaction or/and mediators in common pathways (according to KEGG, Gene Ontology (FDR < 0.01), and GeneCards data) in the examined gynecological tumors.

Tumor Suppressor Function of the SEMA3B Gene in Human Lung and Renal Cancers.

The SEMA3B gene is located in the 3p21.3 LUCA region, which is frequently affected in different types of cancer. The objective of our study was to expand our knowledge of the SEMA3B gene as a tumor suppressor and the mechanisms of its inactivation. In this study, several experimental approaches were used: tumor growth analyses and apoptosis assays in vitro and in SCID mice, expression and methylation assays and other. With the use of the small cell lung cancer cell line U2020 we confirmed the function of SEMA3B as a tumor suppressor, and showed that the suppression can be realized through the induction of apoptosis and, possibly, associated with the inhibition of angiogenesis. In addition, for the first time, high methylation frequencies have been observed in both intronic (32-39%) and promoter (44-52%) CpG-islands in 38 non-small cell lung carcinomas, including 16 squamous cell carcinomas (SCC) and 22 adenocarcinomas (ADC), and in 83 clear cell renal cell carcinomas (ccRCC). Correlations between the methylation frequencies of the promoter and the intronic CpG-islands of SEMA3B with tumor stage and grade have been revealed for SCC, ADC and ccRCC. The association between the decrease of the SEMA3B mRNA level and hypermethylation of the promoter and the intronic CpG-islands has been estimated in renal primary tumors (P < 0.01). Using qPCR, we observed on the average 10- and 14-fold decrease of the SEMA3B mRNA level in SCC and ADC, respectively, and a 4-fold decrease in ccRCC. The frequency of this effect was high in both lung (92-95%) and renal (84%) tumor samples. Moreover, we showed a clear difference (P < 0.05) of the SEMA3B relative mRNA levels in ADC with and without lymph node metastases. We conclude that aberrant expression and methylation of SEMA3B could be suggested as markers of lung and renal cancer progression.

Genetic and epigenetic analysis of non-small cell lung cancer with NotI-microarrays.

This study aimed to clarify genetic and epigenetic alterations that occur during lung carcinogenesis and to design perspective sets of newly identified biomarkers. The original method includes chromosome 3 specific NotI-microarrays containing 180 NotI clones associated with genes for hybridization with 40 paired normal/tumor DNA samples of primary lung tumors: 28 squamous cell carcinomas (SCC) and 12 adenocarcinomas (ADC). The NotI-microarray data were confirmed by qPCR and bisulfite sequencing analyses. Forty-four genes showed methylation and/or deletions in more than 15% of non-small cell lung cancer (NSCLC) samples. In general, SCC samples were more frequently methylated/deleted than ADC. Moreover, the SCC alterations were observed already at stage I of tumor development, whereas in ADC many genes showed tumor progression specific methylation/deletions. Among genes frequently methylated/deleted in NSCLC, only a few were already known tumor suppressor genes: RBSP3 (CTDSPL), VHL and THRB. The RPL32, LOC285205, FGD5 and other genes were previously not shown to be involved in lung carcinogenesis. Ten methylated genes, i.e., IQSEC1, RBSP3, ITGA 9, FOXP1, LRRN1, GNAI2, VHL, FGD5, ALDH1L1 and BCL6 were tested for expression by qPCR and were found downregulated in the majority of cases. Three genes (RBSP3, FBLN2 and ITGA9) demonstrated strong cell growth inhibition activity. A comprehensive statistical analysis suggested the set of 19 gene markers, ANKRD28, BHLHE40, CGGBP1, RBSP3, EPHB1, FGD5, FOXP1, GORASP1/TTC21, IQSEC1, ITGA9, LOC285375, LRRC3B, LRRN1, MITF, NKIRAS1/RPL15, TRH, UBE2E2, VHL, WNT7A, to allow early detection, tumor progression, metastases and to discriminate between SCC and ADC with sensitivity and specificity of 80-100%.

LRRC3B gene is frequently epigenetically inactivated in several epithelial malignancies and inhibits cell growth and replication.

Chromosome 3 specific NotI microarrays containing 180 NotI linking clones associated with 188 genes were hybridized to NotI representation probes prepared using matched tumor/normal samples from major epithelial cancers: breast (47 pairs), lung (40 pairs) cervical (43 pairs), kidney (34 pairs of clear cell renal cell carcinoma), colon (24 pairs), ovarian (25 pairs) and prostate (18 pairs). In all tested primary tumors (compared to normal controls) methylation and/or deletions was found. For the first time we showed that the gene LRRC3B was frequently methylated and/or deleted in breast carcinoma - 32% of samples, cervical - 35%, lung - 40%, renal - 35%, ovarian - 28%, colon - 33% and prostate cancer - 44%. To check these results bisulfite sequencing using cloned PCR products with representative two breast, one cervical, two renal, two ovarian and two colon cancer samples was performed. In all cases methylation was confirmed. Expression analysis using RT-qPCR showed that LRRC3B is strongly down-regulated at the latest stages of RCC and ovarian cancers. In addition we showed that LRRC3B exhibit strong cell growth inhibiting activity (more than 95%) in colony formation experiments in vitro in KRC/Y renal cell carcinoma line. All these data suggest that LRRC3B gene could be involved in the process of carcinogenesis as a tumor suppressor gene.

Differential expression of CHL1 gene during development of major human cancers.

BACKGROUND: CHL1 gene (also known as CALL) on 3p26.3 encodes a one-pass trans-membrane cell adhesion molecule (CAM). Previously CAMs of this type, including L1, were shown to be involved in cancer growth and metastasis. METHODOLOGY/PRINCIPAL FINDINGS: We used Clontech Cancer Profiling Arrays (19 different types of cancers, 395 samples) to analyze expression of the CHL1 gene. The results were further validated by RT-qPCR for breast, renal and lung cancer. Cancer Profiling Arrays revealed differential expression of the gene: down-regulation/silencing in a majority of primary tumors and up-regulation associated with invasive/metastatic growth. Frequent down-regulation (>40% of cases) was detected in 11 types of cancer (breast, kidney, rectum, colon, thyroid, stomach, skin, small intestine, bladder, vulva and pancreatic cancer) and frequent up-regulation (>40% of cases)--in 5 types (lung, ovary, uterus, liver and trachea) of cancer. Using real-time quantitative PCR (RT-qPCR) we found that CHL1 expression was decreased in 61% of breast, 60% of lung, 87% of clear cell and 89% papillary renal cancer specimens (P<0.03 for all the cases). There was a higher frequency of CHL1 mRNA decrease in lung squamous cell carcinoma compared to adenocarcinoma (81% vs. 38%, P = 0.02) without association with tumor progression. CONCLUSIONS/SIGNIFICANCE: Our results suggested that CHL1 is involved in the development of different human cancers. Initially, during the primary tumor growth CHL1 could act as a putative tumor suppressor and is silenced to facilitate in situ tumor growth for 11 cancer types. We also suggested that re-expression of the gene on the edge of tumor mass might promote local invasive growth and enable further metastatic spread in ovary, colon and breast cancer. Our data also supported the role of CHL1 as a potentially novel specific biomarker in the early pathogenesis of two major histological types of renal cancer.