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The Role of Med12 in Tumorigenesis

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The Role of Med12 in Tumorigenesis Helsinki Graduate Program in Biotechnology and Molecular Biology (GPBM)/ Integrative Life Sciences (ILS) Doctoral Program THE ROLE OF MED12 IN TUMORIGENESIS Kati Kämpjärvi Department of Medical and Clinical Genetics, Medicum & Genome-Scale Biology Research Program, Research Programs Unit Faculty of Medicine University of Helsinki Helsinki, Finland Academic dissertation To be publicly discussed, with the permission of the Faculty of Medicine, University of Helsinki, in Haartman Institute, Lecture Hall 1, Haartmaninkatu 3, Helsinki, on the 18th of November 2016, at 12 noon. Helsinki 2016 Supervised by Docent Pia Vahteristo, Ph.D. Department of Medical and Clinical Genetics, Medicum Genome-Scale Biology Research Program, Research Programs Unit Faculty of Medicine University of Helsinki Helsinki, Finland Academy Professor Lauri A. Aaltonen, M.D., Ph.D. Department of Medical and Clinical Genetics, Medicum Genome-Scale Biology Research Program, Research Programs Unit Faculty of Medicine University of Helsinki Helsinki, Finland Reviewed by Docent Miina Ollikainen, Ph.D. Institute for Molecular Medicine Finland, FIMM Department of Public Health, Clinicum Faculty of Medicine University of Helsinki Helsinki, Finland Docent Katri Pylkäs, Ph.D. Laboratory of Cancer Genetics and Tumor Biology Biocenter Oulu Faculty of Medicine University of Oulu Oulu, Finland Official opponent Professor Anne Kallioniemi, M.D., Ph.D. BioMediTech University of Tampere Tampere, Finland ISBN 978-951-51-2643-6 (paperback) ISBN 978-951-51-2644-3 (PDF) htpp://ethesis.helsinki.fi Unigrafia Helsinki 2016 TABLE OF CONTENTS ORIGINAL PUBLICATIONS .......................................................................... 6 ABBREVIATIONS ............................................................................................ 7 ABSTRACT ........................................................................................................ 8 REVIEW OF THE LITERATURE ................................................................ 10 1. Tumorigenesis ....................................................................................................................... 10 2. Genetics of cancer ................................................................................................................. 11 2.1 Genomic alterations ........................................................................................................ 12 2.2 Epigenetic and other changes in tumorigenesis ............................................................... 13 2.3 Cancer genes ................................................................................................................... 14 2.3.1 Oncogenes ................................................................................................................ 14 2.3.2 Tumor suppressor genes .......................................................................................... 15 2.3.3 Stability genes .......................................................................................................... 16 2.4 Inherited susceptibility for cancer ................................................................................... 16 2.5 Genome-wide methods in cancer genetics ...................................................................... 17 3. Uterine leiomyomas ............................................................................................................... 18 3.1 Development of uterine leiomyomas .............................................................................. 19 3.2 Chromosomal alterations in uterine leiomyomas ............................................................ 20 3.3 Hereditary leiomyomatosis and renal cell cancer syndrome ........................................... 21 3.4 MED12 mutations in uterine leiomyomas ....................................................................... 22 4. MED12 .............................................................................................................................. 23 4.1 MED12 as a subunit of the Mediator complex ................................................................ 25 4.2 MED12 in signaling pathways ........................................................................................ 27 4.3 MED12 mutations in the germ line ................................................................................. 28 4.4 Somatic MED12 mutations in other tumor types? .......................................................... 29 AIMS OF THE STUDY ................................................................................... 31 MATERIALS AND METHODS .................................................................... 32 1. Study subjects and samples .................................................................................................. 32 1.1 Tumor samples (I-V) ....................................................................................................... 32 1.2 Normal tissue samples (I-III) .......................................................................................... 35 1.3 Cell lines (II, V) .............................................................................................................. 35 2. Methods .................................................................................................................................. 36 2.1 Histopathological evaluation (I, III) ................................................................................ 36 2.2 DNA and RNA extraction (I-V) ...................................................................................... 36 2.3 Sanger sequencing (I-V) ................................................................................................. 37 3 2.3.1 Mutation screening and validation (I-V) ................................................................. 37 2.3.2 Loss of heterozygosity analysis (III) ........................................................................ 37 2.4 Gene expression analysis (II, III). ................................................................................... 38 2.5 Immunoprecipitation and kinase activity assay (II) ........................................................ 38 2.6 Western blotting (II, V) .................................................................................................. 38 2.7 Tissue microarray construction and immunohistochemistry (III) ................................... 39 2.8 Creating MED12-expressing Flp-In 293 T-Rex cell lines (V) ........................................ 39 2.9 Immunofluorescence (V) ................................................................................................ 39 2.10 Affinity purification and BioID -mass spectrometry (V) ................................................ 41 2.11 In silico prediction tools and online databases (I, IV, V) ................................................ 41 2.12 Statistical analyses (II-V)................................................................................................ 42 3. Ethical issues ......................................................................................................................... 43 RESULTS ......................................................................................................... 44 1. MED12 exon 2 mutations in uterine leiomyosarcoma and colorectal cancer (I) ............. 44 1.1 MED12 exon 2 mutations occur recurrently in uterine leiomyosarcoma ........................ 44 1.2 Rare MED12 exon 2 mutations in colorectal cancer ....................................................... 44 2. Mutations in exon 1 of MED12 (II) ..................................................................................... 46 2.1 MED12 exon 1 mutations in conventional uterine leiomyomas ..................................... 46 2.2 MED12 exon 1 mutations lead to similar gene expression profile as exon 2 mutations . 46 2.3 MED12 exon 1 mutations disrupt the Mediator kinase module integrity ....................... 47 3. The role of MED12 in HLRCC patients’ uterine leiomyomas (III) .................................. 48 3.1 MED12 mutations and FH deficiency are mutually exclusive in uterine leiomyomas ... 48 3.2 MED12 mutation-positive uterine leiomyomas from HLRCC patients display similar gene expression profile as sporadic MED12 mutation-positive tumors .......................... 51 3.3 HLRCC patient with multiple MED12 mutation-positive uterine leiomyomas .............. 51 4. MED12 mutations in chronic lymphocytic leukemia (IV) ................................................. 51 4.1 Somatic MED12 mutations are recurrent in CLL ........................................................... 52 4.2 Positive MED12 mutation status is associated with poor prognosis markers in CLL ..... 52 5. Somatic MED12 nonsense mutation in T-cell acute lymphoblastic leukemia (V) ........... 52 5.1 MED12 exon 1 nonsense mutation escapes nonsense mediated mRNA decay and encodes N-terminally truncated protein .......................................................................... 53 5.2 MED12 E33X mutation abolishes the interactions between MED12 and other Mediator components ..................................................................................................................... 55 5.3 MED12 E33X mutant derivative is missing the nuclear localization signal and remains in the cytoplasm ............................................................................................................... 55 DISCUSSION ..................................................................................................
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