Search

Background: More than 500 thousand new cases of malignant neoplasms are registered annually in the Russian Federation, of which more than 50 thousand new cases are due to hereditary forms. Improving the diagnosis of these diseases will make it possible to detect tumors at the early stages and take timely preventive and therapeutic measures. Aims: Creation of a database and development of a software for the NGS data analysis for the prevention and early diagnosis of hereditary forms of oncological diseases. Methods: The present study used 636 DNA samples obtained from cancer patients with a high hereditary risk or a burdened family history. DNA was isolated from blood lymphocytes. DNA libraries were prepared with a KAPA Target Enrichment Panel (Roche). The panel included probes for targeted enrichment of the coding region of 44 genes. NGS was performed on the MiSeq platform (Illumina). Results: We identified 65 pathogenic/ probably pathogenic nucleotide sequence variants in 96 patients in the ATM, BLM, BRCA1, BRCA2, CHEK2, EPCAM, MEN1, MLH1, MSH2, MSH3, MSH6, MUTYH, PALB2, TP53 genes. We also identified 2858 nucleotide sequence variants of unknown clinical significance. Conclusions: We have created a local database that contains both genetic variants and clinical and anamnestic data. The database contains 4763 nucleotide sequence variants at the moment, among which 2522 are unique variants identified in a single patient.

Objective The problem of drug resistance to BRAF-targeted therapy often occurs in melanoma treatment. Activation of PI3K/AKT/mTOR signaling pathway is one of the mechanisms of acquired resistance and a potential target for treatment. In the current research, we investigated that dual inhibition of mTOR and MEK synergistically reduced the viability of melanoma cells in vitro. Methods A combination of rapamycin (a macrolide immunosuppressant, mTOR inhibitor) and binimetinib (an anti-cancer small molecule, selective inhibitor of MEK) was studied using a panel of melanoma cell lines, including patient-derived cells. Results It was found, that combinatorial therapy of rapamycin (250 nM) and binimetinib (2 μM) resulted in 25% of cell viability compared to either rapamycin (85%) or binimetinib alone (50%) for A375 and vemurafenib-resistant Mel IL/R cells. The suppressed activation of mTOR and MEK by combined rapamycin and binimetinib treatment was confirmed using Western blot assay. Cell death occured via the apoptosis pathway; however, the combination treatment significantly increased the apoptosis only for Mel IL/R cells. The enhanced cytotoxic effect was also associated with enhanced cell cycle arrest in the G0/G1 phase. Conclusion In general, we provide the evidence that dual inhibition of mTOR and MEK could be promising for further preclinical investigations.

In vivo monitoring of reactive oxygen species (ROS) in tumors during treatment with anticancer therapy is important for understanding the mechanism of action and in the design of new anticancer drugs. In this work, a platinized nanoelectrode is placed into a single cell for detection of the ROS signal, and drug-induced ROS production is then recorded. The main advantages of this method are the short incubation time with the drug and its high sensitivity which allows the detection of low intracellular ROS concentrations. We have shown that our new method can measure the ROS response to chemotherapy in tumor-bearing mice in real-time. ROS levels were measured in vivo inside the tumor at different depths in response to doxorubicin. This work provides an effective new approach for the measurement of intracellular ROS by platinized nanoelectrodes.

Acral melanoma is one of the most aggressive and fast-growing forms of cutaneous melanoma and is characterized by a predominant location on the palms and feet. Primary tumors, metastases, and normal tissue samples from five acral melanoma patients were examined by massive parallel sequencing, focusing on the coding regions of 4100 genes involved in the origin and progression of hereditary and oncology diseases. Somatic mutations were found in genes related to cell division, proliferation, and apoptosis (BRAF, NRAS, VAV1, GATA1, and GCM2); cell adhesion (CTNND2 and ITGB4); angiogenesis (VEGFA); and the regulation of energy metabolism (BCS1L). Comparisons of target DNA sequences between morphologically normal and primary tumor tissues and between normal and metastatic tissues identified the candidate genes responsible for rapid metastasis in acral melanoma.

Herein, we describe the design, synthesis, and biological evaluation of novel betulin and N-acetyl-d-galactosamine (GalNAc) glycoconjugates and suggest them as targeted agents against hepatocellular carcinoma. We prepared six conjugates derived via the C-3 and C-28 positions of betulin with one or two saccharide ligands. These molecules demonstrate high affinity to the asialoglycoprotein receptor (ASGPR) of hepatocytes assessed by in silico modeling and surface plasmon resonance tests. Cytotoxicity studies in vitro revealed a bivalent conjugate with moderate activity, selectivity of action, and cytostatic properties against hepatocellular carcinoma cells HepG2. An additional investigation confirmed the specific engagement with HepG2 cells by the enhanced generation of reactive oxygen species. Stability tests demonstrated its lability to acidic media and to intracellular enzymes. Therefore, the selected bivalent conjugate represents a new potential agent targeted against hepatocellular carcinoma. Further extensive studies of the cellular uptake in vitro and the real-time microdistribution in the murine liver in vivo for fluorescent dye-labeled analogue showed its selective internalization into hepatocytes due to the presence of GalNAc ligand in comparison with reference compounds. The betulin and GalNAc glycoconjugates can therefore be considered as a new strategy for developing therapeutic agents based on natural triterpenoids.

Background: Circulating tumor DNA (ctDNA) holds great potential for cancer therapy and can provide diagnostic and prognostic information before and during treatment. Methods: Plasma DNA samples from 97 melanoma patients, 20 healthy donors and 3 patients with benign skin tumors were analyzed by microarray analysis and droplet digital PCR (ddPCR). Results: A microarray for simultaneous detection of six BRAF V600 mutations in ctDNA has been developed. The method allows the detection of 0.05% mutated DNA from WT DNA background. For paired samples (pre-surgery plasma and tumor tissue) isolated from 74 patients, the concordance of genotypes between tumor DNA and ctDNA was 65% (48/74). BRAF mutations in ctDNA were detected in 27/50 patients with BRAF-positive tumors and in 3/24 patients with BRAF wild-type tumors. The presence of ctDNA BRAF mutations in 23 plasma samples from melanoma patients undergoing therapy correlated significantly with tumor progression (P=0.005). The increase in cell-free DNA levels measured by ddPCR also correlated with disease progression (P=0.02). The concordance of results obtained by microarray identification of BRAF mutations and those obtained by ddPCR was 91%. Conclusion: The novel microarray-based approach can be a useful non-invasive tool for accurate identification of ctDNA BRAF mutations to monitor disease progression.

Flow cytometry using fluorescent antibodies (FC) is the method of choice for the quantitation of proteins expressed at the surface or inside the cell, but, however, does not allow to selectively measure nuclear expression. We therefore sought to develop a method for the extraction of intact cell nuclei, which can be used for their subsequent immunofluorescent analysis by FC. The studied protein was vascular endothelial growth factor-receptor-type 1 (VEGFR-1) which is important in tumor survival and metastasis. Two human cell lines, A431 (epidermoid carcinoma of skin with low invasive and metastatic potential) and BRO (highly aggressive amelanotic melanoma), were used as examples for tumor cells, and normal human fibroblasts PHF served as a control line. The quality of the extracted nuclei was assessed by their intactness and purity from cytoplasm. The high content of the nuclear markers (PCNA = proliferating cell nuclear antigen, lamin A/C) in the extracted nuclei with almost complete absence of the cytoplasmic β-tubulin demonstrated that the protocol can be used to obtain a pure suspension of single intact cell nuclei. The measurement of the nuclear VEGFR-1 content revealed that it was present only in tumor cell nuclei and that in more malignant BRO cells the receptor content was 1.75 times higher than in A431 (p = 0.014). Thus, the developed method of extraction of cell nuclei for subsequent FC analysis is suitable for the quantitative evaluation of protein content in the native nucleus.

Oncolytic viruses, including live attenuated measles virus (MV) vaccine strains, have recently been shown as promising therapeutic agents against human malignancies. In this study, the oncolytic potential of the attenuated vaccine strain Leningrad-16 (L-16) of MV was evaluated in a panel of human metastatic melanoma cell lines. The L-16 measles virus was shown to replicate within melanoma cells mediating direct cell killing of tumor cells, although all melanoma cell lines varied in regard to their ability to respond to L-16 MV infection, as revealed by the different pattern of the Interferon Stimulated Gene expression, cytokine release and mechanisms of cell death. Furthermore, the statistically significant L-16 measles virus related tumor growth inhibition was demonstrated in a melanoma xenograft model. Therefore, L-16 MV represents an appealing oncolytic platform for target delivery of therapeutic genes along with other attenuated measles virus strains.

The oncolytic potential of the attenuated mumps virus (MV) vaccine strain Leningrad-3 (L-3) was evaluated in a panel of four human metastatic melanoma cell lines. The lines were shown to be susceptible and permissive to MV infection. Efficient MV replication led to death of melanoma cells, but the effect differed among the cell lines. Possible mechanisms mediating the selectivity of MV L-3 towards the cell lines were explored. Replicative and oncolytic activity of MV was found to depend on the expression pattern of type I interferon genes. None of the melanoma cell lines showed induction of expression of the total spectrum of genes required to inhibit virus replication. Based on the results, MV L-3 was assumed to be a promising oncolytic agent for human melanoma cells.

The discovery of B-RAF activating mutations in malignant melanoma cells has led to the development of a number of targeted drugs, which block exclusively the mutant B-RAF protein. Tumor cells often acquire resistance to B-RAF inhibitors via activation of alternative signaling pathways. One of the resistance mechanisms is activation of PDGF, VEGF, c-KIT, and certain other tyrosine kinases. The possibility of overcoming the resistance to the B-RAF inhibitor Vemurafenib by inactivating receptor tyrosine kinases (RTKs) was studied in metastatic melanoma cell lines differing in B-RAF mutations and RTK activity. It was found that RTK inactivation may help to overcome resistance to B-RAF inhibitors via inhibition of tyrosine kinase phosphorylation and a subsequent blocking of the PI3K-AKT-mTOR and MEK-ERK1/2 downstream signaling pathways. The changes eventually mitigated the cell growth and enhanced the Vemurafenib-dependent cell cycle arrest.

// Marina Emelyanova 1 , Lilit Ghukasyan 1 , Ivan Abramov 1, 2 , Oxana Ryabaya 2 , Evgenia Stepanova 2 , Anna Kudryavtseva 1, 3 , Asiya Sadritdinova 1, 3 , Cholpon Dzhumakova 2 , Tatiana Belysheva 2 , Sergey Surzhikov 1 , Lyudmila Lyubchenko 2 , Alexander Zasedatelev 1, 2 and Tatiana Nasedkina 1, 2 1 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russian Federation 2 Blokhin Cancer Research Center, Ministry of Health of the Russian Federation, Moscow, Russian Federation 3 P. Hertsen Moscow Oncology Research Institute, Moscow, Russian Federation Correspondence to: Marina Emelyanova, email: emel_marina@mail.ru Keywords: biochip, somatic mutations, melanoma, diagnostic tool, targeted therapy Received: December 30, 2016 Accepted: March 30, 2017 Published: April 10, 2017 ABSTRACT Target inhibitors are used for melanoma treatment, and their effectiveness depends on the tumor genotype. We developed a diagnostic biochip for the detection of 39 clinically relevant somatic mutations in the BRAF , NRAS , KIT , GNAQ , GNA11 , MAP2K1 and MAP2K2 genes. We used multiplex locked nucleic acid (LNA) PCR clamp for the preferable amplification of mutated over wild type DNA. The amplified fragments were labeled via the incorporation of fluorescently labeled dUTP during PCR and were hybridized with specific oligonucleotides immobilized on a biochip. This approach could detect 0.5% of mutated DNA in the sample analyzed. The method was validated on 253 clinical samples and six melanoma cell lines. Among 253 melanomas, 129 (51.0%) BRAF , 45 (17.8%) NRAS , 6 (2.4%) KIT , 4 (1.6%) GNAQ , 2 (0.8%) GNA11 , 2 (0.8%) MAP2K1 and no MAP2K2 gene mutations were detected by the biochip assay. The results were compared with Sanger sequencing, next generation sequencing and ARMS/Scorpion real-time PCR. The specimens with discordant results were subjected to LNA PCR clamp followed by sequencing. The results of this analysis were predominantly identical to the results obtained by the biochip assay. Infrequently, we identified rare somatic mutations. In the present study we demonstrate that the biochip-based assay can effectively detect somatic mutations in approximately 70% of melanoma patients, who may require specific targeted therapy.

Patients with metastatic melanoma are difficult to treat and have a very poor prognosis because of high resistance to therapy. Recent evidence indicates that tumors could overcome death through autophagy, a survival mechanism, which cancer cells use under lack of energy and nutrient deprivation. Melanoma cells have different sensitivity to temozolomide (TMZ) treatment. In this study, we showed that the combination of autophagy inhibitors chloroquine or LY294002 and TMZ induced enhanced cytotoxicity of alkylating agents on human melanoma cell lines. All assays were performed on patient-derived melanoma cell lines. The effectiveness of the combined treatment of TMZ and autophagy inhibitors was determined using an MTT assay. Next, we analyzed the expression mRNA level of Beclin 1, LC3B, and p62/STSQM1 and the relative expression of LC3B protein under combined treatment. Autophagic flux was determined by analysis of colocalization of Lysotracker Red and LC3B puncta. Apoptosis was measured by Annexin V/PI staining. Cell cycle analyses were carried out by flow cytometry. We showed that autophagy inhibition could enhance melanoma cell death combined with TMZ therapy. Chloroquine synergistically enhanced the TMZ-induced growth arrest and increased the G0/G1 population in Mel Z and Mel IL cell lines, but not Mel MTP. The expression analysis showed that autophagy involvement in TMZ enhanced cytotoxicity. Furthermore, LY294002, an early-stage autophagy, and PI3K inhibitor were found to exert similar effects. Both chloroquine and LY294002 improved the cytotoxic effect of TMZ treatment, making this combination applicable as a potent antitumor treatment for metastatic melanoma.

Alofanib (RPT835) is a novel selective allosteric inhibitor of fibroblast growth factor receptor 2 (FGFR2). We showed previously that alofanib could bind to the extracellular domain of FGFR2 and has an inhibitory effect on FGF2-induced phoshphorylation of FRS2α. In the present study, we further showed that alofanib inhibited phosphorylation of FRS2α with the half maximal inhibitory concentration (IC50) values of 7 and 9 nmol/l in cancer cells expressing different FGFR2 isoforms. In a panel of four cell lines representing several tumour types (triple-negative breast cancer, melanoma, and ovarian cancer), alofanib inhibited FGF-mediated proliferation with 50% growth inhibition (GI50) values of 16-370 nmol/l. Alofanib dose dependently inhibited the proliferation and migration of human and mouse endothelial cells (GI50 11-58 nmol/l) compared with brivanib and bevacizumab. Treatment with alofanib ablated experimental FGF-induced angiogenesis in vivo. In a FGFR-driven human tumour xenograft model, oral administration of alofanib was well tolerated and resulted in potent antitumour activity. Importantly, alofanib was effective in FGFR2-expressing models. These results show that alofanib is a potent FGFR2 inhibitor and provide strong rationale for its evaluation in patients with FGFR2-driven cancers.

Melanoma is one of the most aggressive and treatment-resistant tumors that responsible for majority of skin-cancer related deaths. Here we propose a combination of MEK inhibitor binimetinib with metformin as a promising therapy against human melanoma cells in vitro, including BRAF -mutated A375, Mel Z, and Mel IL cells, and NRAS-mutated Mel MTP and Mel Me cells. Additionally, we obtained two close to clinical practice models of melanoma progression. The first one was vemurafenib-resistant Mel IL/R melanoma cells with acquired resistance to BRAF inhibition-targeted therapy, and the second one was tumor spheroids, which are 3D in vitro model of small-size solid tumors in vivo. The cytotoxicity of binimetinib and metformin was synergistic in both 2D and 3D melanoma culture and mediated through apoptotic pathway. The combination reduced the number of melanoma-formed colonies, inhibited cell invasion and migration, and led to G0/G1 cell cycle arrest through cyclin D/CDK4/CDK6 pathway. The mechanism of metformin and binimetinib synergy in melanoma cells was associated with increased activation of p-AMPKα and decreased p-ERK, but not with alterations in p-mTOR. In summary, the combination of metformin and binimetinib resulted in stronger anti-proliferative effects on melanoma cells compared to binimetinib alone, and therefore could be promising for clinical applications.

Autophagy is an evolutionarily established process of maintaining the cell integrity by lysosomal recycling of their own cytosolic proteins, macromolecules, organelles, and protein aggregates. Autophagy is regulated by a group of autophagy-related genes (ATG) and is detected at the basal level in almost all cells of the body. Alterations in the autophagy-associated signaling pathways often occur in various pathologies, including malignant neoplasms. Numerous experimental and clinical trials demonstrated the important role of autophagy in mechanisms of chemoresistance or chemosensitivity to a wide range of antitumor agents. This review describes the molecular mechanisms of autophagy and its interaction with apoptosis. The possibilities of modulating the autophagy level in tumor cells to increase the efficacy of anticancer therapy are estimated.

Previous studies have shown that aranosa-lio initiate cell death by activation of CD95-receptor of apoptosis. The mechanism of liposomal aranosa action is still unknown. Anticancer drugs may cause tumor cell death by the activation of autophagy. The purpose of this study was to determine the influence of liposomal aranosa on autophagy. The study was assessed on human melanoma cell lines mel Mtp, mel Ibr, mel Kor, mel Z and mel Mtp clone X. The cells were treated with aranosa-lio or liposomal aranosa at concentration 450 μg/ml for 24 hours. The expression of autophagy were analyzed the expression of mRNA Beclin 1. Expression of mRNA Beclin 1 was reduced on the majority of cell lines, which might be due to the fact that the liposomal form of the drug caused cell death by intrinsic apoptosis which is suppose to suppress autophagy.

The incidence of malignant melanoma is increasing. The discovery of agents specifically targeting the mutated cascades has provided a good response for patients with oncogenic B-Raf proto-оncogene, serine/threonine kinase (BRAF). However, numerous studies continue to focus on novel methods of treatment to overcome acquired resistance to novel drugs. Recently, it has been revealed that inhibition of endoplasmic reticulum (ER) stress chaperon 78 kDa glucose-regulated protein 78 (GRP78) leads to down-regulation of autophagy and increased sensitivity to temozolomide (TMZ) treatment. Melanoma cells have a different sensitivity to TMZ treatment, which corresponds to the basal autophagy level. In the present study, we demonstrated that downregulation of GRP78 mitigated chemoresistance to TMZ in three melanoma cell lines. We found that downregulation of GRP78 led to inhibition of autophagy, cell cycle arrest in the G0/G1 phase, and activation of caspase-7-induced apoptosis, and this was affected by the initial autophagy level. Moreover, inhibition of GRP78 mitigated the combined TMZ and chloroquine effect. Our data revealed that autophagy inhibition through downregulation of ER stress response could overcome resistance to TMZ treatment in melanoma cells with a high basal level of autophagy treatment, which makes this combination a potential potent antitumor treatment for metastatic melanoma.

We continue analysis of the phenotype of human melanoma cell Mel Ibr subclone obtained previously by treatment of the parental cell line by chicken embryo extract. The present study is focused on detection of markers of epithelial-mesenchymal transition that determine enhanced metastatic and invasive potential of malignant tumors of various locations. Analysis of the expression of E-cadherin and vimentin genes in the subclone and parental cells detected activation of epithelial-mesenchymal transition in the subclone. Immunological markers CD90, CD271, and CD95 were present in the parental population, but were not detected on the subclone cells. In contrast to the parental line, cells of the analyzed subclone retain viability in serum-free medium and formed vessel-like structures characteristic of vasculogenic mimicry.

Alofanib is a potential allosteric inhibitor of FGFR2 used in oncology. The inhibitor blocks the extracellular part of the receptor and prevents its binding with the ligand. Alofanib suppressed proliferation of endothelial cells, their migration activity, and ability to form vessellike structures in vitro and significantly decreased the number of microvessels in Matrigel implant and in ovarian cancer (SKOV-3) xenograft in vivo. The results indicate that Alofanib can inhibit angiogenesis.

Encouraged by the clinical validation of fibroblast growth factor receptor 2 (FGFR2) as the target for the treatment of several tumors, we sought to identify pharmacological agents that could target this receptor by an allosteric mechanism. We report the efficacy of RPT835, novel highly potent inhibitor of the FGFR2 extracellular domain (RusPharmTech, LLC), in vitro and in vivo. Concordant with this activity, in human umbilical vein endothelial cells (HUVEC) and mouse endothelial cells (SVEC-4-10), RPT835 inhibited FGF-stimulated proliferation with IC50 values of 11 and 10 nmol/L, respectively. Inhibition of HUVEC cells proliferation by brivanib as positive control was significantly lower (IC50=289 nmol/L, P Collectively, the data obtained with RPT835 are consistent with potent inhibition of FGF signaling, angiogenesis, and breast cancer growth. Citation Format: Ilya Tsimafeyeu, Evgenia Stepanova, Eliso Solomko, Dmitry Kochenkov, Nina Peretolchina, Oxana Ryabaya, Mikhail Byakhov, Sergey Tjulandin. Allosteric FGFR2 inhibitor RPT835 impacts on tumor growth and neovascularization. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1737. doi:10.1158/1538-7445.AM2014-1737

e22097 Background: The fibroblast growth factor (FGF)/FGF receptors (FGFR) signaling axis plays a key role in driving tumor angiogenesis. There is little data on the targeting of FGF-induced angiogenesis. We describe here the targeting angiogenesis driven by FGF using RPT835, novel inhibitor of the FGFR2 extracellular domain (received from RusPharmTech, LLC). Methods: To assess the efficacy of RPT835 on FGF-mediated endothelial cells proliferation, the human umbilical vein endothelial FGFR-expressing cells (HUVEC) were incubated in a 96-well microculture plate and were treated with serially diluted RPT835 or Brivanib as a control. Basic FGF was added at a concentration of 25 ng/ml. Control wells were left untreated. Cell growth inhibition was determined using Promega’s Cell Titer-Glo assay. In vivo angiogenesis was measured with subcutaneously implanted Matrigel plugs containing bFGF (100 ng/ml) or bFGF (100 ng/ml) + bevacizumab (10 mg/kg) or bFGF (100 ng/ml) + RPT835 (15 mg/kg). Control group was without stimulation and treatment. Each group included 3 mice. Number of endothelial cells/vessels was calculated. Results: Basic FGF significantly increased proliferation of the HUVEC cells (P=0.001) in untreated control group. RPT835 significantly inhibited FGF-triggered endothelial cell proliferation when compared with control (P