Justus Liebig University Giessen MTA2/NuRD as a Regulator of NF-ƙB Signalling in Lung Cancer Progression Inaugural Dissertation submitted to the Faculty of Veterinary Medicine or Faculty of Medicine in partial fulfilment of the requirements for the PhD-Degree of the Faculties of Veterinary Medicine and Medicine of the Justus Liebig University Giessen by Nefertiti El-Nikhely of Alexandria, Egypt Giessen 2016 From Max Planck Institute for Heart and Lung Research Department of Lung Development and Remodelling Director/Chairman: Prof. Dr. med. Werner Seeger First Supervisor and Committee Member: Prof. Dr. med. Werner Seeger Co-Supervisor and Committee Member: Prof. Dr. Reinhard Dammann Date of Doctoral Defence: 01.02.2017 Dedication To my mom & To my dad To whom I owe everything I am or ever will be. Table of contents Table of contents 1 Introduction ..................................................................................................................................... 1 1.1 Lung Cancer ............................................................................................................................. 1 1.1.1 Statistics ........................................................................................................................... 1 1.1.2 Classification of lung cancer ............................................................................................ 2 1.1.3 Aetiology of lung cancer .................................................................................................. 3 1.1.4 Oncogenes KRAS and C-RAF ............................................................................................ 4 1.2 NF-ƙB signalling pathways & the role in cancer ...................................................................... 5 1.2.1 The transcription factor family NF-ƙB ............................................................................. 5 1.2.2 Canonical and non-canonical pathways .......................................................................... 8 1.2.3 Posttranslational modifications of NF-ƙB subunits ....................................................... 10 1.3 NF-ƙB cellular functions ......................................................................................................... 12 1.3.1 NF-ƙB in immune and inflammatory responses ............................................................ 12 1.3.2 NF-ƙB and cell cycle regulation and apoptosis .............................................................. 12 1.3.3 NF-ƙB in cell adhesion and angiogenesis ....................................................................... 13 1.4 IKK2 and NF-ƙB in lung carcinogenesis .................................................................................. 13 1.4.1 Expression of NF-ƙB in different cancer types .............................................................. 13 1.4.2 Targeting NF-ƙB for cancer therapy .............................................................................. 14 1.5 Epigenetic control via NuRD complex in lung cancer ............................................................ 15 1.5.1 Members of the NuRD complex .................................................................................... 15 1.5.2 Biology and function of NuRD complex in cancer ......................................................... 17 1.5.3 MTA2/NuRD in cancer ................................................................................................... 17 1.6 NF-ƙB and epigenetic control ................................................................................................ 19 2 Aims ............................................................................................................................................... 20 3 Materials and Methods ................................................................................................................. 21 3.1 Cell biological methods & cell culture ................................................................................... 21 3.1.1 Cell cultivation ............................................................................................................... 21 3.1.2 Isolation of mouse alveolar type II cells ........................................................................ 21 3.1.3 Stimulation and inhibition of NF-ƙB signalling pathway ............................................... 22 3.1.4 Transient cell transfection ............................................................................................. 22 3.1.5 Stable cell lines generation............................................................................................ 23 3.1.6 Immunocytochemistry (ICC) .......................................................................................... 24 3.1.7 Proximity ligation assay (PLA) ........................................................................................ 25 3.2 Functional assays for cells ..................................................................................................... 25 I Table of contents 3.2.1 Cell proliferation by BrdU incorporation ....................................................................... 25 3.2.2 Boyden chamber migration assay ................................................................................. 26 3.2.3 Soft agar attachment-independent colony formation assay ........................................ 26 3.3 Molecular biology & biochemical methods ........................................................................... 27 3.3.1 RNA isolation ................................................................................................................. 27 3.3.2 Reverse transcription for cDNA synthesis ..................................................................... 27 3.3.3 Quantitative real time polymerase chain reaction (qRT PCR) ....................................... 28 3.3.4 Protein isolation ............................................................................................................ 30 3.3.5 Co-Immunoprecipitation (Co-IP) ................................................................................... 30 3.3.6 Western blotting ........................................................................................................... 31 3.3.7 Chromatin Immunoprecipitation .................................................................................. 31 3.3.8 Fractionation of nuclear lysate by sucrose gradient ..................................................... 32 3.3.9 Cloning of pCDH-IKK2CA ................................................................................................. 33 3.4 Activity assays ........................................................................................................................ 34 3.4.1 NF-Gluciferase reporter assay ....................................................................................... 34 3.4.2 NF-ƙB activity assay ....................................................................................................... 34 3.4.3 HDACs activity assay ...................................................................................................... 34 3.5 Histological techniques ......................................................................................................... 35 3.5.1 Haematoxylin & Eosin staining (H&E staining) .............................................................. 35 3.5.2 Immunohistochemistry (IHC) ........................................................................................ 35 3.5.3 Immunofluorescence ..................................................................................................... 36 3.6 Animal experiments .............................................................................................................. 37 3.6.1 Transgenic animal model............................................................................................... 37 3.6.2 Breeding and Induction ................................................................................................. 37 3.6.3 Genotyping .................................................................................................................... 38 3.6.4 Subcutaneous xenograft (autochthonous) model ........................................................ 38 3.6.5 Magnetic resonance imaging of the lung ...................................................................... 39 3.6.6 Lung compliance ............................................................................................................ 39 3.7 Statistical analysis .................................................................................................................. 40 4 Results ........................................................................................................................................... 41 4.1 NF-ƙB is increased in cancer cells .......................................................................................... 41 4.1.1 NF-ƙB is classically regulated and activated via IKK2 .................................................... 44 4.1.2 Overexpression of C-Raf activates IKK2-regulated NF-ƙB signalling pathway .............. 45 4.1.3 Changing IKK2 in alveolar type II cells affects tumour growth in SpC C-Raf BxB mice .. 46 4.1.1 Analysis of the tumour microenvironment in IKK2-regulated SpC C-Raf BxB mice ...... 49 II Table of contents 4.1.2 IKK2 in the lung affects its role as a metastatic niche ..................................................