Advances in Molecular Oncology

Malignant tumors developing in the conditions of chronic hypoxia, unlike normal tissues, quickly acquire resistance to decreased oxygen level. Hypoxia-resistant cells in malignant disease obtain various features promoting their survival: they alter metabolism to aerobic glycolysis, activate angiogenesis-stimulating programs, and rearrange signaling cascades to adapt to hypoxia. At the same time, tumor cells become resistant to chemotherapy and radiation therapy and effectively colonize metastatic niches.

This review analyzes signaling pathways which activate in the conditions of chronic hypoxia and underlie tumor adaptation to insufficient oxygen. Apart from the main pathway associated with activation of hypoxia-inducible factor 1α (HIF-1α), such cascades as STAT3, Snail cascade associated with epithelial-mesenchymal transition, NRF2 factor responsible for adaptation to reactive oxygen species, and stemness factors OCT4, SOX2 and NANOG are also considered. Effective killing of tumor cells requires simultaneous inhibition of several parts of signaling pathways because disruption of just one will lead to switching of signaling proteins to bypass the blocked one. The article lists various inhibitors of signaling pathways patricipating in tumor cell adaptation to chronic hypoxia and describes their mechanisms of action and targets. Development of successful strategies for treatment of hypoxia-resistant tumors requires identification of an effective combination of the above-mentioned and other inhibitors of the key signaling cascades participating in adaptation to hypoxia.

Currently, many scientific studies investigate the role of receptor tyrosine kinases in mechanisms of tumor cell resistance to targeted therapies and chemotherapies. Activation of FGFR and VEGR signaling pathways through fibroblast growth factors (FGF) and vascular endothelial growth factors (VEGF) is seen in many malignant neoplasms. The literature review was addressed to describe the role of FGFR and VEGFR signaling pathways in the secondary (acquired) resistance of gastrointestinal stromal tumors to the targeted drugs – tyrosine kinase receptor inhibitors (imatinib mesylate, sunitinib, regorafenib).

The review describes the molecular mechanisms of activation of FGFR and VEGR signaling pathways in tumor cells, as well as their structural and functional relationship between aforementioned pathways. Multiple studies, including our own, illustrate the activation of FGFR and VEGFR pathways in gastrointestinal stromal tumors. The review also describes the clinical effectiveness of FGFR and VEGFR inhibition for advanced and metastatic gastrointestinal stromal tumors, thereby providing a rationale to utilize activation of the aforementioned signaling pathways as the markers for gastrointestinal stromal tumors progression.

Almost the entire population of the planet is infected with the Epstein–Barr virus. Infection occurs in childhood and, as a rule, without any clinical manifestations in infected individuals. At the same time, having a transformation potential, the virus, in the presence of unfavorable factors, can become involved in the pathological process leading to the development of a tumor. The study of the properties of EBV and the conditions for the manifestation of its oncogenic potencies represents an important branch of the science of oncogenic viruses and human tumors of viral origin.

Aim. To summarize and systematize literature data, including those that have appeared recently and devoted to the study of EBV, the first human oncogenic virus.

The work was carried out based on the analysis of published works, the search for which was carried out in the databases Web of Science, Scopus, eLibrary, PubMed, Russian Index of Science Citation (RISC), etc.

The review provides a brief history of the discovery of EBV, describes its structural organization, analyzes the transmission routes and EBV circulation in the body, analyzes the types of latency and functions of latent virus genes, and also provides a spectrum of EBV-associated neoplasms of lymphoid and epithelial origin. Particular attention was paid to nasopharyngeal carcinoma, risk factors for its occurrence and the role of EBV in the carcinogenesis.

Introduction. Epithelial-mesenchymal transition (EMT) is the major driver of invasion and metastasis of cancer cells. The hallmarks of EMT are disruption of stable cell-cell adhesion and acquisition of a migratory phenotype by epithelial cells.

Aim. To study of migratory activity and intracellular distribution of cell-cell adhesion proteins, actin cytoskeleton and vimentin intermediate filaments in primary cultures of high grade serous ovarian cancer obtained from clinical samples using correlative live cell imaging/immunofluorescence microscopy.

Materials and methods. Using correlative live cell imaging/immunofluorescence microscopy, we studied cell migration and intracellular distribution of cell-cell adhesion proteins, actin cytoskeleton structures and vimentin intermediate filaments.

Results. In primary cultures of high grade serous ovarian cancer many traits of incomplete EMT were observed, namely: dramatic remodeling of the actin cytoskeleton, reorganization of adherens junctions with retention of E-cadherin expression in the majority of samples, expression of mesenchymal markers such as vimentin and N-cadherin, and enhanced migratory activity. Different cell populations exhibited heterogeneous levels of EMT markers, for example, a prominent vimentin intermediate filament network was detected in actively migrating cancer cells.

Conclusion. High grade serous ovarian cancer cells with enhanced migration potential undergo incomplete EMT which is the optimal choice for promoting invasion and metastasis.

Introduction. Regulatory T cells (Treg) have pro-oncogenic activity, inhibit the immune response and therefore can be a significant target for immunotherapeutic methods of treating colorectal cancer (CRC). Currently, the role of regulatory T cells in the mechanisms of carcinogenesis, as well as the participation of co-inhibitory and co-stimulating proteins of the immune system in the formation of tumor immunosuppression in CRC, is insufficiently studied.

Aim. To determine the content of Treg subtypes in the primary tumor focus in patients with CRC and to evaluate the expression level of co-stimulating and co-inhibitory proteins on their membrane.

Materials and methods. The main group consisted of 105 patients with stage III colon adenocarcinoma; the control group – 75 patients with non-neoplastic diseases of the colon. The object of the study was tumor-infiltrating T lymphocytes, which were isolated by an enzymatic method. Determination of Treg subtypes, as well as expression of programmed cell death 1 (PD-1) and inducible T-cell costimulator (ICOS) proteins, was performed by flow cytometry. Treg was identified as CD19⁻CD3⁺CD4⁺CD25^highCD127⁻. Based on the expression of CD45RA and CD197, Treg subtypes were determined as naive (CD45RA⁺CD197⁺), central memory (CD45RA^–CD197⁺), effector memory (CD45RA^–CD197^–), and terminally differentiated (CD45RA⁺CD197^–). The significance of differences in the parameters of the main and control groups was assessed using the nonparametric Mann–Whitney U test.

Results. In the primary focus of tumor growth in CRC, the content of Treg increases by 4.4 times. At the same time, the ratio of Treg subtypes in the tumor microenvironment changes, which is expressed in a 1.4-fold decrease in the number of naive cells, as well as a 2.8-fold increase in the number of effector memory Treg and a 3.1-fold increase in terminally differentiated lymphocytes. Tumor-infiltrating Treg expressed the ICOS 2 times more strongly compared to the control. A 20 % increase in the number of Treg PD-1⁺ICOS⁺ was established. The number of Treg PD-1⁻ICOS⁻ cells in the primary tumor focus decreased by 1.4 times compared to the control group.

Conclusion. In patients with CRC, the number of naive Treg decreases in the primary tumor focus and the relative content of differentiated effector cells increases. In the tumor microenvironment in CRC, the number of Tregs increases, expressing on their surface both the costimulating molecule ICOS and the co-inhibitory receptor PD-1.

Introduction. Absence of objective morphological signs of malignancy complicates the problem of preoperative differential diagnosis of nodular thyroid lesions with follicular structure. In ambiguous cases, selection of surgical treatment is justified but results of histological examination of operative material often show benign nature of the nodules. A possible solution for this problem is development of molecular diagnostic methods for this pathology. For example, the miR-THYROID test system is based on the differences in expression patterns of regulatory microRNAs (miRNAs) in follicular adenoma cells and follicular thyroid cancer cells.

Aim. To develop and validate a set of reagents (miR-THYROID) for differential diagnosis of follicular adenoma and follicular thyroid cancer using reverse transcription with subsequent polymerase chain reaction (RT-PCR).

Materials and methods. Selection of potential marker molecules (n = 53) is based on the results of previous study of histologically verified samples of follicular adenoma and follicular thyroid cancer using high-throughput sequencing. In this study, a test system based on RT reaction with two-tailed primer and quantitative PCR with TaqMan probe was developed. To evaluate analytical characteristics of this technology, synthetic analogues of miRNA molecules were used; for analysis of diagnostic characteristics of the method, cytology samples of follicular adenoma (n = 20) and follicular thyroid cancer (n = 20) were used.

Results. Studies of analytical characteristics of RT-PCR system and concentration of potential marker molecules in cytology samples were performed which allowed to shorten the list of marker miRNAs from 53 to 7 (hsa-miR-15a-5p, -20-5p, -24-3p, -106b-5p, -143-3p, -146b-5p, -192-5p). Algorithm of RT-PCR result analysis was developed based on calculation of molecule concentration ratios relative to oppositely directed follicular cancer-associated changes in expression, combination of these results, and calculation of the miR-T diagnostic parameter. miR-THYROID test system based in RT-PCR technology allowed to differentiate follicular adenoma from follicular cancer with sensitivity 89.47 % and specificity 90 %, positive prognostic significance 89.47 %, negative prognostic significance 90 %, and accuracy 89.74 %.

Conclusion. Test system miR-THYROID based on 7 microRNA molecules was validated on 40 samples (20 samples of follicular adenoma and 20 samples of follicular cancer) and demonstrated high diagnostic significance.

Introduction. Cell cycle dysregulation associated with errors in the CDK4 / 6-pRb signaling pathway is characteristic of many oncological diseases. The MM-D37K drug is an innovative chimeric peptide imitating endogenous inhibitor p16INK4a. Unlike traditional ATP-competitive (adenosine triphosphate) inhibitors, MM-D37K is highly selective. It consists of p16INK4a cytotoxic fragment and pANTP transport peptide promoting effective intracellular delivery. Results of preclinical studies showed high cytotoxic effect of MM-D37K in a wide variety of tumor cells and synergy with chemotherapeutic drugs.

Aim. To evaluate the effect of MM-D37K drug on biochemical characteristics of blood and to determine dose-limiting toxicity and maximal tolerable dose in patients with advanced solid tumors.

Materials and methods. The study was performed in accordance with the 3 + 3 escalation dose scheme. Starting MM-D37K dose was 7.5 mg / m², further dose escalation for MM-D37K drug in the clinical trial was two-fold at each dosing level (7.5; 15; 30; 60; 120 and 240 mg / m²). The drug was administered intravenously 3 times a week for 2 weeks. Dose-limiting toxicity was evaluated according to the hematological and non-hematological toxicity criteria per the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v. 4.0 classification.

Results. Intravenous administration of MM-D37K in the dose range between 7.5 and 240 mg / m² did not show signs of dose-limiting toxicity. There were no statistically significant differences in the analyzed laboratory, instrumental and diagnostic parameters during intravenous administration of the drug which allowed to conclude that the maximal tested dose of the MM-D37K drug 240 mg / m² is safe and has satisfactory tolerability profile.

Conclusion. First clinical use of the new antitumor drug – targeted inhibitor of cyclin-dependent kinases 4 / 6 – MM-D37K in humans confirmed its safety and possibility of further clinical trials with the selected dose 240 mg / m².

Introduction. Loss of membrane expression of the epithelial adhesion glycoprotein EpCAM, the primary marker of circulating tumor cells (CTCs), is often associated with epithelial-mesenchymal transition, which facilitates tumor dissemination. Distinguishing phenotypic variants of tumor cells lacking membrane EpCAM expression in both blood and disseminated tumor cells (DTC) in bone marrow will enable the identification of cells capable of intra- and extravasation, i. e. the identification of populations with metastatic potential.

Aim. To determine the presence and ratio of tumor cells with membrane (mEpCAM) and intracellular (icEpCAM) localization and the degree of EpCAM expression (low and high) among CTCs and DTCs in breast cancer.

Materials and methods. The study involved CTCs and DTCs from breast cancer patients. Phenotyping was performed by flow cytometry using antibodies to CD45, EpCAM and pan-cytokeratin. EpCAM expression was analyzed based on localization (membrane/intracellular) and expression level (low/high).

Results. Cells lacking membrane EpCAM expression predominate among CTCs, whereas they were virtually undetectable in bone marrow. Not all CTC phenotypes are present in DTCs, which may be explained by cell selection during extravasation or cellular plasticity. Notably, unlike in primary tumors, CTCs and DTCs completely lack cells co-expressing membrane and intracellular EpCAM.

Conclusion. This study suggests the potential for intra- and extravasation of CTCs with different EpCAM expression patterns. However, their ability to form clinically detectable metastases requires further study.