Department of Cell Signalling

Head

Valery V. Filonenko

Professor, Dr. Sci. (Mol. Biol.)
Phone: (380-44) 526-20-16, 522-61-00;
Fax: (380-44) 526-07-59;
E-mail: filonenko@imbg.org.ua

Education and Degrees:

1980–1985 Graduate Student, Faculty of Biology, Taras Shevchenko National University of Kyiv, Ukraine, M.Sc. (genetics)

1986–1989 Postgraduate Student, Engelhardt Institute of Molecular Biology, Moscow, Russia

1991 Ph.D. (molecular and cell biology). Thesis: “Immunochemical analysis of high-molecular weight aminoacyl-tRNA synthatase complex”

2005 Dr.Sci. (molecular biology). Thesis: “Structural and functional analysis of ribosomal protein S6 kinases (S6K1 and S6K2)”

2006 Professor (molecular biology).

Professional Employment:

1985–1991 Junior Research Scientist, Institute of Molecular Biology and Genetics (IMBG), NASU, Kyiv, Ukraine

1992–1995 Postdoctoral Fellow, University of Connecticut Health Center, Department of Biochemistry, Farmington, USA

1995–1997 Postdoctoral Associate, Miami University, Department of Biochemistry and Molecular Biology, Miami, USA

1997–2003 Senior Research Scientist, Laboratory of Cell Growth Regulation, IMBG NASU, Kyiv, Ukraine

Since 2003 Head of the Department of Cell Signaling, IMBG NASU, Kyiv, Ukraine.

Membership:

Since 1998 Member of FEBS (Federation of European Biochemical Societies)

Since 2004 Member of Ukrainian Society of Cell Biology

Since 2007 Editorial Board member of Journal “Biopolymers and Cell” (Ukraine)

Since 2011 Editorial Board member of Journal “Biotechnologia Acta” (Ukraine).

Research Area:

  • PI3K/mTOR/S6K signaling in regulation of cell metabolism, growth, proliferation and survival under normal and pathological conditions.
  • Identification and characterization of novel biomarkers for cancer treatment and diagnostics.

Current Research Activities and Recent Achievements:

mTOR/S6K (mammalian target of rapamycin/ ribosomal protein S6 kinase) pathway is a main signaling pathway that integrates input from a major intracellular and extracellular cues such as growth factors, stress, energy status, oxygen and amino acids to control major processes, including protein and lipid synthesis and autophagy. (Fig. 1).

Fig. 1. The PI3K/mTOR/S6K signaling in regulation of cell functions

Ribosomal protein S6 kinases (S6K1 and S6K2) are principal regulators of cell size, growth and methabolism. Functional peculiarities of S6Ks and their splicing isoforms in normal tissues and malignant tumours, their expression profile and subcellular localisation are under investigation. We provide evidence of existence of the mTOR splicing isoform, mTORβ, which lacks most of its protein-protein interaction modules, HEAT and FAT, but retains domains responsible for FRB, protein kinase activity, and regulation (RD and FATC) (Fig. 2).

Fig. 2. Colocalization of mTOR and intermediate filaments in human breast adenocarcinoma cells MCF7. Immunofluorescent analysis with anti-mTOR and anti-Pan mAbs

Importantly, mTORβ could form complexes in vivo with Raptor and Rictor, which are known companions of full-length mTOR (mTORα). Also, it readily phosphorylates characterized mTORβ substrates, S6K1, PKB/Akt, and 4EBP1, in vitro. In contrast to mTORα, mTORβ has the potential to shorten considerably the G1 phase of the cell cycle and to stimulate cell proliferation. Significantly, overexpression of mTORβ transforms immortal cells and is tumorigenic in nude mice. Our studies suggest that the regulation of cell proliferation via the mTOR pathway could be mediated by mTORβ, which acts as a protooncogene and therefore could be a candidate for future anticancer drug discovery.

The question about relationship between cancer and stroma cells requires additional comprehensive analysis. Earlier it was shown that fibroblasts are capable to inhibit the growth and proliferation of tumor cells in the early stages of oncogenesis, whereas in the later stages activated tumorassociated fibroblasts, demonstrate the ability to stimulate proliferation, invasion and angiogenesis. Three-dimensional culture is promising way of modeling the different stages of carcinogenesis in vitro . This approach allows to reproduce some morphological and molecular characteristics of initial tissue more precisely than traditional monolayer culture. For investigation of tumor-stroma interaction in context of PI3K/mTOR/S6K signaling 3D culture model of human dermal fibroblasts and malignant cells was developed.

Identification of novel protein-partners of S6Ks, PTEN phosphatase and TSC1/TSC2 (tuberous sclerosis complex) complex by yeast-two hybrid technique revealed novel regulatory mechanisms and functional links within PI3K/mTOR/S6Kdependent signaling.

Recently we have identified PP5 as new binding partner for TSC2, a component of TSC1/2 complex and demonstrated that PP5 dephosphorylates specifically TSC2 at sites, associated with its activation via AMP kinase (AMPK) pathway. Taken together, these results suggest that PP5 exerts negative regulation on TSC1/2 function through dephosphorylation of AMPK-mediated sites and in turn positively regulates mTOR activity.

Molecular cloning and characterization of CoA synthase (befunctional enzime that is responsible for the last two steps in CoA biosynthesis) interaction with S6K1 uncovered a potential link between mTOR/S6K signaling pathway and energy metabolism that requires CoA and its thioester derivatives, but its physiological relevance should to be further elucidated.

RCD8 (EDC4), Fyn and Csk kinases, phospholipase Cγ, NADPH oxidaseactivator 1 – p67phox, and structural membrane skeleton protein spectrin have been identified as CoAsy binding partners.

Analysis of regulatory mechanisms in CoA biosynthesis disclosed Shp2 protein tyrosine phosphatase as a positive regulator of CoA synthase. At the same time the main protein of Processing bodies (PB) RCD8, identified by massspec analysis as CoA synthase protein-partners, negatively regulates CoAsy activity.

Monoclonal antibodies specific to the components of signaling pathways (S6K1, S6K2, mTOR, Rictor, Raptor, mutant form of FGFR3) and oncomarkers (Ki-67, Napi2B, PRAME, NYBR1) have been generated. (Fig.3)

Fig. 3. A – Immunofluorescent detection of Ki-67 in cultured MCF-7 cells at different stages of cell cycle: a, interphase; b, prophase; c, metaphase; d, anaphase; e, telophase. Arrows indicate positive reaction for Ki-67 antigen in cells in interphase and at different phases of mitosis correspondently (Oc10x, Ob 40x). B – Immunohistochemical determination of Ki-67 content in proliferating human melanoma cells on paraffin sections. Arrows indicate positive reaction for Ki-67 antigen in melanocytes (hematoxylin, (Oc10x, Ob 100x)

Application of SEREX methodology (SErological analysis of Recombinant cDNA EXpression libraries) allowed to identify more than 100 autoantigens of colon, thyroid, melanoma and medullary breast carcinoma tumors. Among them Ki-67, catenin beta like1, α-catenin, HSP 105, ovarian cancer biomarker - sodium-dependent phosphate transporter NaPi2b, LGALS3BP, RAD50, FAM50A, RBPJ, PABPC4, LRRFIP1. Characterization of their gene expression profile in different type of malignancy, subcellular localization and immunogenicity in sera of cancer patients led to identification of potential molecular markers of human malignancy which can be used for both diagnosis and therapy of oncological diseases, and for the understanding of the molecular mechanisms of malignant transformation of cell, tumor progression and anti-tumor immune response (Fig.4).

Fig.4. The prospects of tumor-associated antigens application

National Grants:

Projects of National Academy of Sciences of Ukraine:

  • 2012–2016 N 2.2.4.21 Project: “Genotype-phenotype correlation in malignant tumors”
  • 2010–2014 N 29/10 Project: “Generation and characterization of monoclonal antibodies specific to oncogenic, mutant forms of fibroblast growth factor FGFR3 for diagnostics and tumor therapy”
  • 2006–2008 Bilateral cooperation program between of NASU and Siberian Branch of Russian Academy of Sciences Project: “Innovation technologies in cancer treatment”. Partners – Institute of Chemical Biology and Fundamental Medicine, Institute of Cytology and Genetics, Novosibirsk, Institute of Organic Chemistry.

Projects of State Fund for Fundamental Researches of Ukraine:

  • 2011–2012 Bilateral cooperation program between NASU and Russian Academy of Sciences N Ô40.4/061 Project: “Proteome analysis of autoantigenic repertoire of tumors with favorable prognosis associated with lymphocyte infiltration”. Partner – Lomonosov Moscow State University
  • 2011–2012 State Key Laboratory of Molecular and Cellular Biology N Ô46/457 Project: “Macromolecules and their complexes in realization of genetic information”.

International Grants:

  • 2011–2014 7th Framework Programme (FP7) FP7- INCO-2011-6, ERA-WIDE Project: “Strengthening cooperation in Molecular Biomedicine between EU and UKRAINE”, COMBIOM (scientific supervisor – Prof. A. Elskaya)
  • 2006–2008 INTAS (The International Association for the Promotion of Co-operation with Scientists from the New Independent States of the Former Soviet Union) Project: “Studies of mTor/S6K signaling pathway by proteome analysis and DNA microarrays”. Partners – University College London (UK), Ludwig Institute for Cancer Research (USA), Biomedical Sciences Research Center “Alexander Fleming” (Greece)
  • 2006–2007 INTAS Project: “PTEN – FAB4 interaction as a link to the regulation of lipid metabolism and adipogenesis”
  • 2005–2008 Bilateral cooperation program between Ludwig Institute for Cancer Research- IMBG NASU
  • 2004–2007 INTAS Project: “Identification and characterization human of tumour-associated antigens Partners – University College London (UK), Ludwig Institute for Cancer Research (USA)”
  • 1999–2001 INTAS N 97-30890 Project: “Identification and characterization of tumor-associated antigens in thyroid cancer”
  • NATO (North Atlantic Treaty Organization) N NUKR/982645 Project: “Generation of new biodetection tools for cancer research and diagnostics”
  • 1998–2001 WELLCOME TRUST N 055427/Z/98 Project: “Identification and functional analysis of novel PI3-kinase binding partners”.

Collaboration:

with Ukrainian organizations:

  • National Cancer Institute, Ministry of Health of Ukraine (Kyiv)
  • R. E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NASU (Kyiv)
  • O. V. Palladin Institute of Biochemistry, NASU (Kyiv)
  • Institute of Cell Biology, NASU (Lviv)
  • National University Lviv Polytechnic (Lviv)
  • State Institution “V. P. Filatov Institute of Eye Diseases and Tissue Therapy of NAMS of Ukraine” (Odessa)
  • O. O. Chuiko Institute of Surface Chemistry, NASU (Kyiv)
  • Ivan Franko National University of Lviv (Lviv)
  • National Forestry University of Ukraine (Lviv)
  • Kyiv City Bureau of Forensic Expertise (Kyiv)
  • with foreign organizations:

  • Lomonosov Moscow State University (Moscow, Russia)
  • Ludwig Institute for Cancer Research (New York, USA)
  • University College London (London, UK)
  • Heidelberg University Hospital (Heidelberg, Germany)
  • Biomedical Sciences Research Center “Alexander Fleming” (Vari, Greece)
  • Selected publications:

    1. Kosach V. R., Tykhonkova I. O., Cherednyk O. V., Filonenko V. V., Khoruzhenko A. I. Phospho-mTOR (Ser2481) colocalizes with condensed chromosomes during metaphase. Biopolym. Cell. 2016; 32(2):105-110.
    2. L.Dyachenko, K. Havrysh, A.Lytovchenko, I.Dosenko, S. Antoniuk,V. Filonenko, and R.Kiyamova. Autoantibody Response to ZRF1 and KRR1 SEREX Antigens in Patients with Breast Tumors of Different Histological Types and Grades. Disease Markers, Volume 2016 (2016), Article ID 5128720.
    3. V.Skripova., I.Serebriiskii., Z.Abramova1., I.Astsaturov., R.Kiyamova. CRISPR/Cas9 Technique for Identification of Genes Regulating Oxaliplatin Resistance of Pancreatic Cancer Cell Line. BioNanoSci. DOI 10.1007/s12668-016-0272-3.
    4. Kostianets O., Shyyan M., Antoniuk S., Filonenko V. Kiyamova R. Panel of SEREX-defined antigens for breast cancer autoantibodies profile detection. Biomarkers 2016 Oct 24:1-25.
    5. Skivka LM, Fedorchuk OG, Susak YM, Susak MYa, Malanchuk OM, Rudyk MP, Nowicky YW. Physical Activity Interferes with the Immunomodulatory Effect of the Antineoplastic Drug NSC631570. Curr Pharm Biotechnol. 2015;16(1):49–59.
    6. Malanchuk OM, Panasyuk GG, Serbin NM, Gout IT, Filonenko VV. Generation and characterization of monoclonal antibodies specific to Coenzyme A. Biopolymers and Cell. 2015;31(3):187–192.
    7. Malanchuk OM. Development of monoclonal antibody against mTORC2 complex component protein Rictor. Biopolymers and Cell. 2015; 33( 5):345–350.
    8. Savinska LO, Klipa OM, Khoruzhenko AI, Shkarina KA, Garifulin OM, Filonenko VV. Generation and characterization of polyclonal antibodies specific to N-terminal extension of p85 isoform of ribosomal protein S6 kinase 1 (p85 S6K1). Biopolym. and cell. 2015;31(4):294–300.
    9. Garifulin OM, Kykot VO, Gridina NY, Kiyamova RG, Gout IT, Filonenko VV. Application of serex-analysis for identification of human colon cancer antigens. Exp Oncol. 2015 Sep;37(3):173–80.
    10. Kosach VR, Cherednyk OV, Khoruzhenko AI. Characteristic of mTOR signaling and its involvement in the regulation of cell movements through remodeling the cytoskeleton architecture. Biopolym. and cell. 2015; 31(1):5–14.
    11. Gotsulyak NYa, Kosach VR, Cherednyk OV, et al. Optimization of cell motility evaluation in scratch assay. Biopolym. Cell. 2014; 30(3):223-228.
    12. Skivka LM, Fedorchuk OG, Bezdeneznykh NO, et al. The effect of antineoplastic drug NSC631570 on immunogenicity of B16 melanoma. J Exp Integr Med. 2014;4(2):93-105.
    13. Shkarina KA, Cherednyk OV, Voloschenko II, et al. Exosomes: messengers and mediators of tumor-stromal interactions. Biopolym. Cell. 2014;30(6).
    14. Filonenko VV. PI3K/mTOR/S6K signaling pathway – new players and new functional links. Biopolym. Cell. 2013; 29(3):207–214. doi: 10.7124/bc.00081A
    15. Gudkova D, Panasyuk G, Nemazanyy I, et al. EDC4 interacts with and regulates the dephosphoCoA kinase activity of CoA synthase. FEBS Lett. 2012; 586(20):3590–5. doi: 10.1016/j.febslet.2012.08.033
    16. Savinska L, Skorokhod O, Klipa O, et al. Development of monoclonal antibodies specific to ribosomal protein S6 kinase 2. Hybridoma (Larchmt). 2012; 31(4):289–94. doi: 10.1089/hyb.2012.0032
    17. Kostianets O, Antoniuk S, Filonenko V, Kiyamova R. Immunohistochemical analysis of medullary breast carcinoma autoantigens in different histological types of breast carcinomas. Diagn Pathol. 2012 Nov 26;7:161. doi: 10.1186/1746-1596-7-161
    18. Kostianets O, Shyian M, Demidov S, et al. Serological analysis of SEREX-defined medullary breast carcinoma-associated antigens. Cancer Invest. 2012; 30(7):519–27.doi: 10.3109/07357907.2012.697231
    19. Gryshkova V, Lituiev D, Savinska L, et al Generation of monoclonal antibodies against tumor-associated antigen MX35/sodium-dependent phosphate transporter NaPi2b. Hybridoma (Larchmt). 2011;30(1):37-42. doi: 10.1089/hyb.2010.0064
    20. Goh ET, Pardo OE, Michael N, et al. Involvement of heterogeneous ribonucleoprotein F in the regulation of cell proliferation via the mammalian target of rapamycin/S6 kinase 2 pathway. J Biol Chem. 2010; 285(22):17065-76. doi: 10.1074/jbc.M109.078782
    21. Gorbenko O, Panayotou G, Zhyvoloup A, Volkova D, Gout I, Filonenko V. Identification of novel PTEN-binding partners: PTEN interaction with fatty acid binding protein FABP4. Mol Cell Biochem. 2010; 337(1-2):299-305. doi: 10.1007/s11010-009-0312-1
    22. Breus O, Panasyuk G, Gout I, Filonenko V, Nemazanyy I. CoA Synthase is phosphorylated on tyrosines in mammalian cells, interacts with and is dephosphorylated by Shp2PTP. Mol Cell Biochem. 2010; 335(1-2):195–202. doi: 10.1007/s11010-009-0255-6
    23. Gnjatic S, Cao Y, Reichelt U, Gout I, Filonenko V et.al. NY-CO-58/KIF2C is overexpressed in a variety of solid tumors and induces frequent T cell responses in patients with colorectal cancer. Int J Cancer. Int J Cancer. 2010; 127(2):381–393. doi: 10.1002/ijc.25058
    24. Kiyamova R, Kostianets O, Malyuchik S, et.al. Identification of tumor-associated antigens from medullary breast carcinoma by a modified SEREX approach. Mol Biotechnol. 2010; 46(2):105–12. doi: 10.1007/s12033-010-9285-2
    25. Breus O, Panasyuk G, Gout I, Nemazanyy I. CoA synthase is in complex with p85alphaPI3K and affects PI3K signaling pathway. Biochem Biophys Res Commun. 2009; 385(4): 581–5. doi: 10.1016/j.bbrc.2009.05.102
    26. Zhyvoloup A, Nemazanyy I, Panasyuk G, et al. Subcellular localization and regulation of coenzyme A synthase J. Biol. Chem. 2003; 278(50):50316–21. doi:10.1074/jbc.M307763200