MicroRNAs are a key factor in the globalization of tumor-host relationships
Summary. A new round of molecular oncology development in the post-genomic era significantly changes the conventional understanding of the nature and origin of the malignant process. Increasingly, fundamental phenomena are emerging that change the “canonical” views of the dominant role of gene mutations that contribute to the uncontrolled proliferation and emergence of heterogeneous malignant cells. Recent numerous studies have shown that significant variability in the initiation, proliferation and control of the apoptotic program of tumor cells is due to numerous epigenetic processes, including the level of expression of microRNAs (miRNAs). This paper reviews the literature data and presents the results of own research in which miRNAs have been found to form a molecular phenotype for malignancy. Their role as a “conductor” of the functioning of a genetic-epigenetic orchestra that coordinates various aspects of malignancy has been suggested. MiRNAs have been shown to be an active participant in balancing mechanisms in the development of controversial processes in breast cancer cell lines of varying degrees of malignancy. The analysis of the literature data and the own studies of the expression profile of only a few miRNAs suggests that scientists and clinicians have received a new marker and a target that simultaneously plays the role of an active insider in both the oncogene-oncosuppressor relationship and in the globalization of this process at the tumor-host level. Further investigation of the “alarm” marker in this network will allow to reconsider the molecular genetic classification of neoplastic disease, which will provide the development of a new strategy for cancer therapy.
Submitted: July 26, 2019.
*Correspondence: E-mail: email@example.com
The problem of the relationship between tumor and host for many centuries has attracted the attention of the best representatives of ancient medicine with their amazing medical intuition; thinkers of the Romantic era with their broad erudition and boldness of scientific generalizations; numerous followers of today who are armed with virtuoso research methods.
Now that a vast amount of theoretical, experimental and clinical data have been accumulated in the field of molecular and cellular oncology about the multifaceted diversity of mechanisms and factors of carcinogenesis and anticarcinogenesis, it seems reasonable to revive an interest and revise some of the concepts of the tumor-host relationship [1, 2]. The deepening interest in this problem has grown substantially with the emergence of the possibility of identifying an “ideal” test that would integrate changes in the parameters of genetic as well as non-genetic processes, providing opportunities for monitoring carcinogenesis and anticarcinogenesis [3, 4].
Decoding of human genome has led to an understanding of the need to consider a tumor as part of the body and to study its emergence as a dynamic process that is implemented at all levels of the biological system. Hundreds of proto-oncogenes and suppressor genes have been identified that contribute to impaired cell cycle control, changes in signal transduction cascade systems, differentiation processes and activation of the apoptotic tumor cell program. However, the decipherment of numerous mechanisms of carcinogenesis and the identification of new molecular biological markers of tumor growth and targets for targeted therapy do not fully meet the needs of modern clinical oncology. Therefore, scientists are answering the questions about the possibility, in general, of identifying a key factor maintaining equilibrium in the constant counterbalance of oncogen-oncosuppressor and restraining the accumulation of abnormalities in the processes of morphogenesis; what molecular structures mediate the coordination of regulatory systems in the tumor-host relationship; do they meet the criteria of stability and accessibility when standardized as diagnostic markers and therapeutic targets?
A new round of molecular oncology development in the post-genomic era significantly changes the conventional wisdom regarding the nature and origin of the malignant process. More recently, the basic principles of genome function in malignant transformation of cell have been understood and directly related to mutations in the genome. So, in particular, the altered structure of DNA mediated by mRNA leads to the synthesis of proteins, which give new phenotypic features and antigenic properties to the cell. Recently, however, despite the proven fact of the role of genetic mutations in cancer development, fundamental phenomena that are changing the “canonical” view of the dominant role of gene mutation, the principles of regulating their activity and mRNA in reading DNA information have become increasingly available. As an example, it is worth noting that today we know about hundreds of specific translocations and corresponding gene mutations that are associated with malignant transformation of cells of different types [5–10]. At the same time, it has been proven that the presence of hereditary mutations is the cause of a small percentage of such cancers.
The modern accumulated fundamental knowledge in genetics and molecular biology shows that carcinogenesis is a multistage phenomenon in which changes in the regulation of the functioning of a large ensemble of genetic, epigenetic and metabolic processes that contribute to uncontrolled proliferation of transformed pool of heterogenous cells are simultaneously involved. At the same time, the concept has recently been put forward of “cancer cells that do not themselves exhibit the disease but rather mobilize and disrupt the function of normal body cells that switch and become part of an cumulative abnormal process” .
The rapid progress in the study of the regulation of gene expression and the high plasticity of malignant cells has revealed epigenetic heterogeneity as a key factor in the selection of the aggressive tumor phenotype. Potentially inverse epigenetic processes do not affect the structure and function of genes, but substantially modify their expression by altering chromatin structure and DNA methylation level. Chromatin remodeling enzymes and histone modifiers are the most studied and characterized to date [12, 13]. Interaction of processes of acetylation, methylation, phosphorylation, ADP-ribosylation, etc. and other reactions composes a histone code, which is implemented in the form of corresponding events in the nucleus. Posttranslational histone modification involves more than 50 known sites of epigenetic regulation of genes. In this process, DNA-organizing methylation proteins are involved providing support for the condensed state of chromatin [14, 15]. Hypomethylation in tumor cells can contribute to chromatin decondensation and, consequently, to chromosome reorganization and instability and to the formation of a drug-resistant pool of malignant cells [16–18].
According to existing concepts, DNA methylation is the most common epigenomic modification in the mammalian genome, which is inherited, but is reversible [19, 20]. Genes have been shown to be expressed if chromatin is decondensed, cytosine is demethylated, and histones are acetylated, whereas in the case of condensed chromatin, methylated cytosine, and deacetylated histones, transcription is blocked, and therefore genes are not expressed. However, it is known that tumor cells are characterized by a paradoxical change in the state of methylation, in particular, against the background of total DNA hypomethylation, local hypermethylation of individual genes is observed as one of the key factors for repression of transcription of promoter regions of genes, including tumor suppressor genes. Each of these disorders may contribute to the emergence of different variations in the heterogeneous tumor cell pool and have profound implications for tumor progression [21–23].
However, in the design of various histone modifications, there remains a great number of mysteries of this unique phenomenon of nature. In addition, numerous processes are extremely dynamic, unstable and due to a number of processes are still incomprehensible and unavailable for registration. At the same time, there is a growing demand for experimental and clinical oncology to search for stable, conservative molecules in the parameters of general biological, genetic and epigenetic events and technological processes of their identification.
As a result of the “Big Bang” of the post-genomic era, a small player of numerous processes has crystallized in biomedical research as the ideal (optimal) nominee for an integral indicator of landscape change in tumor-host relationships [24–30]. In the golden triad of DNA methylation, posttranslational histone modification and chromatin remodeling and expression of non-coding miRNA, the latter acquired the characteristics of a “conductor” of the functioning of the genetic-epigenetic orchestra, which satisfies various aspects of malignant growth, including alteration of proliferation and cell metabolism, angiogenesis, metastasis, avoiding immunological control, and insensitivity to chemotherapy and radiation therapy. The discovery of RNA directional gene silencing or RNA interference allowed, along with the canonical system of protein regulation of gene activity, to identify the existence of short non-coding RNAs as coordinators of gene functional activity at the post-translational level as negative regulators of their expression level. Simultaneous impairment of their expression levels gives them the signs of both oncogene and tumor suppressor. In recent years, the number of publications dedicated to the detection, biogenesis, identification of multifaceted mechanisms of their action and creation of a data bank for their functions has increased tenfold [31–36].
The interest of researchers and clinicians in miRNAs is due to the multifaceted aspects of their participation in balancing the fundamental processes of cellular differentiation, signal transduction, proliferation, metabolic transformations of genome stability, DNA repair and apoptosis, as well as the response of cells to the effects of different exogenous and exogenous factors [34, 37–42]. In 2006, the Nobel Prize was awarded to the authors of these studies for their interpretation of the RNA interference mechanisms underlying gene silencing . These specific molecules have been shown to be able to recognize complementary sequences in DNA and mRNA and inhibit translation of promoter regions of DNA.
Each mRNA can be regulated by a few miRNAs, and one miRNA can recognize a few targets [44, 45]. For example, it has been found that the well-known tumor suppressor p53 activates transcription of a number of miRNAs that provide cell cycle arrest and initiation of apoptosis program [46–48]. Such functionality of miRNAs allows them to form a regulatory network that provides control of numerous biological processes [49–55]. As it has been found out, each type of cancer is characterized by a specific miRNA expression profile [56–59]. It is established that a complex system of interacting miRNAs and transcription factors regulates the switching of synthesis of both protein products and the level of expression of the miRNA itself [60, 61]. The conservative nature of the different miRNA sequences that emerged in the early stages of eukaryotic evolution and their involvement in maintaining genome stability and the malignant process at different stages of its development, testify to the high hopes for finding new markers and targets of influence [36, 62–64].
The discovery of a miRNA regulatory molecule is analogous to the value of the axis on which balance functionally active oncogenes and oncosuppressors, which prevent the accumulation of anomalies in cells by initiating reparative processes and early start of the apoptotic program. Today it is difficult to imagine the mechanism of balancing fundamental dynamic processes in the absence of an active regulator of the controversial phenomena of nature.
Detailed studies on the occurrence of miRNAs, increase or decrease of their concentrations indicate that more than half of miRNA genes are located on chromosome regions associated with amplification, deletion, or translocation, which may be a direct cause of altering their expression and cancer pathogenesis [65–67].
Given the direct involvement of miRNAs in the fundamental processes of regulating the expression level of molecular markers of tumor cells and their adaptation and legalization regarding the potential incorporation into diagnostic standards of markers using the real-time polymerase chain reaction method , we studied miRNA expression in human breast cancer cell lines with varying degrees of malignancy depending on their receptor status, proliferative, adhesive, invasive activity, level of their stemness and degree of initiation of apoptotic program [68, 69]. The results show that in MDA-MB-231 and MDA-MB-468 cells with the aggressive molecular phenotype, the level of miR-221 expression increased by more than 5–10 times (miR-221 is involved in the formation of receptor status and proliferative activity), and expression of oncogenic miR-10b implicated in invasive activity also increased. It should also be noted that, in contrast to the triple-negative breast cancer cell lines, inhibition of miR-10b and miR-221 expression was observed in the cell lines of low malignancy (Table).
Table. Differences in miRNA expression in human breast cancer cells of varying degrees of malignancy
Note: *p < 0.05 compared to cells with low grade of malignancy.
Numerous clinical studies have shown that normal breast epithelial cells express 7–10% ERα and 80–85% ERβ . Experimental data show that under physiological conditions miR-221, miR-10b, miR-20b activate expression of ERα, that is, they participate in the formation of a classic “loop” of negative feedback in the ERα/ERβ ratio, which may be a crucial factor in the progression of estrogen-dependent tumors [70, 71].
An inverse correlation was found for oncosuppressive miR-34a and miR-29b, in particular in receptor-negative cell lines their levels were much lower than in receptor-positive cells (Fig. 1). In the analysis of the participation of miR-221 in the mechanisms of formation of the molecular phenotype of cells, it was found that overexpression of this miRNA suppresses the expression of cell cycle regulators CDKN1B (p27) and CDKNIC (p57) . At the same time, miR-34a activates the expression of PRDM4, which is a transcription factor for the progesterone receptor . The observed harmonization of the association between estrogen stimulation and the activity of the posttranscriptional gene expression control system in the breast cancer cells with the participation of miRNAs may be a strong argument for extrapolating these data to the appropriateness of assigning the latter to the cohort of standardized markers of malignant process.
Fig. 1. Expression levels of miR-221, -34a and -29b depending on the receptor status of breast cancer cells
To confirm the cooperative association of miRNA expression with the molecular profile of the breast cancer cells of various degrees of malignancy, an analysis of the processes involved in cell proliferation involving cyclins and cyclin-dependent kinases was performed. Because cyclin D1 is a trigger factor in the G1/S transition, it binds to CDK 4/6, forming an active complex that causes the release of Rb from the E2F complex, which allows to activate the transcription of genes containing these sites in the promoters of cyclin E, cyclin A, DNA polymerase, thymidine kinase. The authors of  have shown that cyclin D1 and cyclin E are overexpressed in many types of malignant cells, and their interaction with the corresponding regulatory miRNAs influences cell division and tumor growth. In particular, it is known that the increase in miR-221 causes activation of cyclin E-CdK2 and cyclin A-CdK2 complexes, which provides G1/S phase transition and DNA synthesis and is consistent with our data on their level in cells with high and low malignancy grade.
It is important to note that in this study CD44+/CD24– markers were associated with the level of stem cell motility and the content of oncogenic miRNAs, that is consistent with the general biological processes of the aggressive breast cancer course. Our data on the levels of miR-320a and miR-34a, which inversely correlate with the degree of malignancy, testify to the balance of pathogenetic processes in breast cancer cells with high and low aggressiveness (Fig. 2). Increased expression of these miRNAs is characteristic cells of the luminal breast cancer subtypes (T47D, MCF7), and in the actively proliferating triple-negative breast cancer lines (MDA-MB-231, MDA-MB-468), their levels are much lower.
Fig. 2. Expression levels of miR-221, -320a, -34a, 133a, -122 and -29b depending on the proliferative activity of breast cancer cells
In the study of the molecular profile of cell lines of varying degrees of malignancy, we found a clear dependence of the expression level of miR-221, -10b, -29b, -320a, 34a and 200b on the invasive and metastatic markers of the studied cells (Fig. 3). It is known that these indicators of aggressiveness of malignant cells are regulated by a number of mediated signaling pathways, such as Notch1, EGF, Slug, c-Met and others. In particular, Notch signaling is involved in the regulation of differentiation, proliferation, adhesion, migration, angiogenesis, and epithelial-mesenchymal cell transition, which is inhibited by miR-34a through the inhibition of Notch-2 expression and the Jagged-1 protein, which is a Notch-1 receptor. Another target of this miRNA is c-Met, which, through activation of cyclin-dependent kinases, is associated with the level of metastatic activity of cells. We have shown that in cells with high malignancy (MDA-MB-231), this index was 20 times lower than in T47D cells with low malignancy grade.
Fig. 3. Expression levels of miR-221, -10b, 29b, 320a, -34a and -200b depending on the invasiveness of breast cancer cells
A characteristic feature of triple-negative breast cancer cell lines is a high degree of epithelial-mesenchymal transition due to decreased expression of E-cadherins. This is caused by the activation of the Slug signaling pathway, which is one of the key markers of mesenchymal cells and an activator of expression of oncogenic miR-221 . We have shown that the level of activation of this miRNA is much higher in the cell line MDA-MB-231 compared to the cell line of the luminal type T47D RMZ. Correlation of expression level of miR-10b with the most malignant phenotype of the MDA-MB-231 line was also confirmed. Its expression is 8.5 times higher than in the T47D and MCF-7 cell lines.
Our interest in the level of expression of miR-29b in breast cancer cell lines of various degrees of malignancy is caused by the existing data on the change of miRNA levels in the development of neoplasms, its participation in a wide network of intracellular events, the formation of tumor microenvironment and its effect on the interaction of a tumor with the host [76–79]. Existing sources of literature data provide contradictory information. However, a comprehensive study by Jonathan et al. showed that micro-RNA-29b regulates a network of pro-metastatic regulators involved in angiogenesis, including vascular endothelial growth factor, collagen reconstruction and proteolysis processes involving metalloproteinase-9, and integrins that constrain the metastatic potential of malignant cells. The analysis of the results of our own studies clearly indicates a significant inhibition of miR-29b in the cell lines of the aggressive molecular phenotype compared to the breast cancer cell lines of the luminal phenotype. The results obtained may indicate that decreased expression of the specified miRNA may be an important biomarker of cancer progression.
The analysis of the literature data and the results of our own studies of the expression profile of only a few miRNAs suggests that scientists and clinicians have received a new marker and a target that simultaneously plays the role of an active insider in the oncogene-oncosuppressor relationship. Deregulation of oncogenes and tumor suppressors induced by genetic and epigenetic factors caused by disruption of miRNA expression reveals new links in the pathogenesis of the malignant process. The first rays of such hopes are the data on the key role of miRNAs in the interaction between the tumor microenvironment of stem tumor cells and macrophages, in the formation and disruption of processes of intracellular homeostasis, autophagy, metabolism, etc. [80–83]. However, particular interest in miRNAs is associated not only with the important role of this regulatory molecule in the balance of gene expression, but also as a potential factor in systemic communication of physiological and pathobiological processes, which plays a key role in the globalization of tumor-host relationships [84–86]. Functional triad of oncogene-miRNA-oncosuppressor, as a factor of light at the end of the tunnel, will open a new era in understanding the fundamental problems of carcinogenesis, find optimal solutions for early diagnosis, monitoring of the course, evaluating the effectiveness of therapy, determining prognostic and predictive processes with a perspective for nomination as “a gold marker”. The basis for such a definition of a “young” biomarker is its role and place in vital processes, ranging from the regulation of stem cell function, cell cycle, proliferation, apoptosis. It is important that their involvement in numerous target processes has a tissue-dependent character and shows all the hallmarks of an “alarm” cancer signal.
Further study of the functional network of the oncogene-miRNA-oncosuppressor will allow to review the molecular-genetic classification of the tumor disease, which will provide the development of a new strategy for cancer therapy.
The study was supported by scientific grant of the NAS of Ukraine 2015–2019 “Molecular and Cell Biotechnologies for the needs of medicine, industry and agriculture” “Development and introduction of a panel of predictive miRNAs for the personalized design of neoadjuvant therapy for breast cancer patients” (220.127.116.115, 2019, 0119U101242).
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