I. Development of pharmacological (in vitro and in vivo) models based on primary xenografts and development of bioinformatics and system-biological approaches to study of cancer diseases
Ilya Serebriiskii, Anna Gaponova, Alexander Deneka, Grigorii Andrianov,
Identification of genes involved in the development of estrogen independent
About 70% of newly diagnosed cases of breast cancer are characterized by a high level of expression of estrogen receptor alpha (ERα). Nevertheless, the acquisition of resistance to anti-estrogen drug de novo limits treatment of such patients, requiring the use of cytotoxic chemotherapy; many of these patients have a poor prognosis. To solve this problem, we used a biological system of approaches to identify a group of genes as new vulnerability points in estrogen independent breast cancer:
1. Using systems biology to construct ERa-networks.
2. Screening of respective siRNAs-library to identify genes that are not essential for estrogen-dependent cells MCF7, but necessary for the survival of two estrogen-independent cell lines MCF7.
3. Validation and characterization of identified candidate genes using phosphoproteomic profiling and depletion analysis.
Study of the genes involved in DNA double strand breaks in familial colon cancer
For most familial colorectal carcinomas, the genetic basis remains undefined (uFCRC). The prevalence of DNA structural lesions in CRCs suggested that relevant pathways might be defective in uFCRC.
- Germline DNA from 25 uFCRC patients attending a high-risk cancer tertiary care clinic will be subjected to exome sequencing and compared to 1508 controls.
- Primary T-cells from 25 patients and matched controls were cultured and assayed for DNA double strand breaks (DSBs).
- Representative variant genes will be studied by knockdown assays in HCT 116 CRC cells, enzymatic assay of recombinant proteins, and/or transfection.
The role of RNA-binding protein MSI2 in the development of non-small cell lung cancer (NSCLC)
Two murine cell lines have been received: metastatic (344SQ) and nonmetastatic (393P), where p53 allele lost its function (P53R172HDG/+) and an activated allele KrasG12D (KrasLA1/+). It has been found that overexpression of MSI2 accompanies NSCLC metastases in mice and humans. The effect of MSI2 on the expression of CLDN7, FN1, ERBB3/HER3, KDR/VEGFR2, STAT5A has been shown.
Identification of rare alleles that may be predisposed to kidney cancer
There has been a search on biological databases in order to find the genes responsible for the development of kidney cancer. Next prioritization of found genes will be implemented: the construction of interaction networks, the definition of damage genes, an additional literature search. After the selection of candidate genes exome sequencing of kidney cancer patients whose relatives had also cancer will be held. The sequencing results will serve to further reference search and confirmation of candidate genes.
Anna Gaponova, MS, Junior Researcher
Preclinical trials of a new class of anticancer drugs - HSP90 (Heat Shock Protein 90) inhibitor conjugates in small cell lung cancer
Small cell lung cancer (SCLC) is characterized by a poor prognosis for patients. No mutations activating the development of SCLC have not been found, as well as there are no effective therapeutic targets. The treatment with cytotoxic chemotherapeutic agents - inhibitors of topoisomerase I (irinotecan) and II (etoposide and topotecan) and DNA-damaging agents such as carboplatin is ineffective and leads to development of resistance in patients.
STA-12-8666 is a new compound combining the ability to bind active heat shock protein 90 (HSP90) with a cleavable linker attached to SN-38 - the active metabolite of irinotecan.
Various studies have demonstrated that HSP90 inhibitors are accumulated in tumors at levels more than 30 times than in normal tissue. Cleavage of the linker in tumor tissue provides a prolonged release of active SN-38 at high local concentration and significantly limits toxicity in non-transformed tissue. The aim of this work is to evaluate the effectiveness of STA-12-8666 in the treatment of SCLC.
- Evaluation of the effectiveness of a new class of anticancer drugs in various SCLC models in vitro and in vivo.
- Determination of the biological mechanisms of drugs.
- Determination of prognosis and predictors markers in the appointment of therapy with this drug.
1) Determination of optimal drug dosage and ability of efficient combination with other drugs, and evaluation of the toxic properties and drug side effects.
2) Assessment of change in the activation of cells Keane SCLC under the influence of the drug using MIBs technology (multiplexed kinase inhibitor beads) and subsequent mass spectrometric quantification.
3) Validation of the data obtained by Western blot analysis.
4) Immunohistochemical evaluation of the expression of markers of proliferation (Ki-67), apoptosis (Casp3) and DNA damage (γH2AX) in tumors treated with the drug as compared to control.
5) Determination of the effect of this drug on the dynamics of the cell cycle by flow cytometry.
Alexander Deneka, MD, Junior Researcher
Study of interaction of Aurora-A and EGFR inhibitors and their impact on non-canonical signaling pathway of Aurora-A kinase in tumor cells
Traditional functions of Aurora-A kinase (AURKA) is the regulation of mitosis and cell kinetic activity. AURKA is overexpressed in multiple tumors leading to aneuploidy by cytokinetyc defects. Aurora A inhibitors such as alisertib let achieve a positive effect in the treatment of cancers that are characterized by overexpression of AURKA, but sometimes cause severe side effects.
The causes of these side effects are still unclear, and may include newly identified non-canonical functions of AURKA such as control of primary cilia and influence on Ca2 + traffic.
Their inhibition has important physiological significance and explains the deterioration of the phenotype in our mouse model of autosomal dominant polycystic kidney disease (ADPKD) - the most common inherited condition where an important role is violations signaling pathways proteins localized to the primary cilium. Combination therapy with alisertib and erlotinib (inhibitor of EGFR) gives promising results in clinical trials. We explore the non-canonical function of Aurora A inhibitors and their signaling pathways, in particular the combination of erlotinib and alisertib, in a mouse model of ADPKD. Results of in vivo experiments have shown that erlotinib decreases cyst formation and improves the phenotype observed in the monotherapy with alisertib. At the stage of in vitro we clarify the mechanism of this interaction, exploring the target proteins and signaling pathways AURKA and EGFR. We also investigate the effect of combination therapy on the morphology of primary cilia and regulation of Ca2 + traffic.
II. The identification of drug targets for neoplastic diseases
Igor Astsaturov, Linara Gabitova, Rashid Gabbasov, Vera Skripova, Anastasiya Vetkina
Linara Gabitova, Junior Researcher
Study of the effect of metabolites of cholesterol biosynthetic pathway to a nuclear receptor LXR as an important mechanism for the treatment of EGFR-positive cancers of the head and neck
Transformed cancer cells need to keep an increased level of cholesterol in order to support a high rate of their growth and reproduction. The role of cholesterol in the formation of lipid rafts - microdomains of the plasma membrane lipid bilayer, enriched with glycosphingolipids and sterols and playing an important role in cell signaling is well studied (George et al, 2012), but there is a little information about biological activity of intermediate metabolites of cholesterol biosynthesis.
Deletion of NSDHL prevents the development of Kras-mediated carcinogenesis
Vera Skripova, Junior Researcher
The development of new non-covalent inhibitors of ERCC3 for pancreatic cancer therapy based on triptolide
Triptolide is insoluble substance isolated from the Chinese herb Tripterygium wilfordii, which has a number of therapeutic effects (anti-inflammatory, immunosuppressive, anti-proliferative, and antitumor). The effect of triptolide on one of the most dangerous cancers – pancreatic cancer has been shown.
Triptolide effects caused by its covalent binding to ERCC3 - subunit of transcription factor TFIIH, inhibiting its ATPase activity that leads to the collapse of a transcriptional complex of RNA polymerase II and stop of transcription. This also explains its toxic effects even at low concentrations.
Search for compounds working on a similar mechanism with triptolide but possessing greater water solubility and lower toxicity to non-covalently binding to the target can help to develop drugs for the clinical treatment of cancer.
The aim of the project is to develop a non-covalent inhibitors of ERCC3 drugs based on the structure triptolide.
Anastasiya Vetkina, Junior Researcher
The protein NSDHL as a new target of antitumor therapy
NSDHL (Sterol-4-alpha-carboxylate 3-dehydrogenase) is an enzyme that is encoded by the NSDHL gene in humans. This enzyme is localized in the endoplasmic reticulum, consists of 373 amino acids and has one transmembrane domain. It is involved in cholesterol biosynthesis and during cholesterol synthesis, the NSDHL enzyme participates in one of several steps that convert a molecule called lanosterol to cholesterol. Specifically, the NSDHL enzyme removes a carbon atom and three hydrogen atoms (a methyl group) in the conversion of lanosterol to cholesterol. This enzyme has 3-beta-hydroxy-delta5-steroid dehydrogenase activity and sterol-4-alpha-carboxylate 3-dehydrogenase activity.
It is known that persistent activity of epidermal growth factor receptor (EGFR) is a common cause for cancer growth and drug resistance. It has been shown that blocking the activity of NSDHL or direct addition of meiosis activating sterols (MAS), a substrate for these enzymes, markedly sensitizes cancer cells to EGFR inhibitors (Sukhanova, 2013). It has been also found that arrest of the sterol pathway at the level NSDHL activates the liver X receptor (LXR), which induces the expression of cholesterol efflux proteins, depletes cellular cholesterol, and reduces expression of LDL lipoprotein receptors (LDLR) (Astsaturov, 2014) but it is well known that cholesterol can cause a number of types of cancer. The main idea is that MAS sterol metabolites accumulate because of NSDHL deficiency and negatively regulate cell growth by activating LXR, disrupting cholesterol uptake and biosynthesis in cells.
I addressed this idea by performing the following specific aims:
1. Select the conditions for expression of NSDHL and its fragments in soluble fraction in bacteria.
2. Purify NSDHL, determine its enzymatic activity after purification and crystallize the protein.
3. Study the structure of NSDHL, determine its functional domains (find out which amino acids contact with substrate, cause mutations in the protein domains in order to clarify the role of each domain in the functional activity of the protein)
4. Select inhibitors of NSDHL by screening.