Gene expression analysis identifies the Activin/Inhibin signaling pathway as a target of CSN5/JAB1 in colorectal epithelial cancer cells Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation Vorgelegt von Diplom Biologe Thomas Wilhelm Hennes aus Erkelenz Berichter: Universitätsprofessor Dr. rer. nat. Jürgen Bernhagen Universitätsprofessor Dr. rer. nat. Joost T. van Dongen Tag der mündlichen Prüfung: 28.09.2017 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar. Acknowledgements This thesis was conducted and written at the Institute of Biochemistry and Molecular Cell Biology. I want to thank Univ.-Prof. Dr. Jürgen Bernhagen for giving me the opportunity to work on this interesting and challenging topic. His guidance, scientific advices and trust during the whole progress of this thesis was vital for the successful completion. I thank Univ.-Prof. Dr. Jost van Dongen for reviewing my thesis and accepting the Co- Referat. His ideas and comments were always very helpful and contributed to a successful completion of my thesis. I want to thank Dr. rer. nat. Anke Schütz for the supervision of my work and reviewing of my thesis. Furthermore, I want to thank the whole CSN5 group. Birgitt Lennartz for all the technical support and know-how she shared with me. Sandra Jumpertz for sharing and solving the problems of our topic, the useful discussions and the helping hands whenever I needed them. I appreciate the time working in this group. Also I would like to thank Dr. rer. nat. Bernd Denecke and the IZKF chip facility for performing the GeneChip microarray and helping with some first analysis of the data. I want to thank Dr. rer. nat. Ivan Gesteira Costa Filho from the Division of Computational Biology for the help with the further bioinformatical analysis of the microarray data sets. I want to thank all my colleagues at the Bernhagen lab for their support during the last years. Especially I would like to thank Josefine Soppert, Sandra Krämer, Setareh Alampour Rajabi, Sebastian Borosch and all the other temporary members of our little office community. When things did not work out as planned, they helped with technical support, good advices and helpful discussions or when everything failed even with their humor. They and all the other members of the lab ensured that I had a great time, which I will enjoy looking back. Furthermore, I want to thank my parents, brothers and sister. Without them I would not be able to go my way. Their support during the whole time was fantastic and made this thesis possible. At the end I want to thank especially Sarah Lenz for encouraging and motivating me even in hard times. Her constant support was invaluable for the successful completion of this thesis. I II Table of contents Acknowledgements ................................................................................................... I Table of contents ..................................................................................................... III Abbreviations ........................................................................................................... VI 1) Introduction ....................................................................................................... 1 1.1) The human digestive system ......................................................................... 1 1.2) The colorectal carcinoma .............................................................................. 4 1.3) The constitutive photomorphogenesis 9 signalosome (CSN) ........................ 6 1.3.1) CSN5/ JAB1 ............................................................................................ 9 1.3.2) CSN(5) as regulator of protein degradation .......................................... 10 1.3.3) The involvement of the CSN in posttranslational modifications............. 12 1.3.4) CSN in cellular homeostasis and cancer .............................................. 12 1.4) Activins/Inhibins ........................................................................................... 15 1.4.1) Activin/Inhibin expression and secretion ............................................... 15 1.4.2) Activin/Inhibin signaling pathways ......................................................... 17 1.4.3) Activin/Inhibin in cellular homeostasis and cancer ................................ 20 1.5) Aim of the study ........................................................................................... 22 2) Material and methods ...................................................................................... 23 2.1) Material ........................................................................................................ 23 2.1.1) Chemicals and reagents ....................................................................... 23 2.1.2) Buffer and solutions .............................................................................. 26 2.1.3) Cell lines ............................................................................................... 27 2.1.4) Equipment and consumables ................................................................ 27 2.1.4.1) Equipment ...................................................................................... 27 2.1.4.2) Consumables ................................................................................. 29 2.2) Methods ....................................................................................................... 30 III 2.2.1) Cell culture ............................................................................................ 30 2.2.1.1) Cultivation of mammalian cells ....................................................... 30 2.2.1.2) Cell counting ................................................................................... 30 2.2.1.3) Freezing and thawing of mammalian cells ...................................... 31 2.2.1.4) Knockdown of protein expression in mammalian cells ................... 31 2.2.1.5) Overexpression of proteins in mammalian cells ............................. 32 2.2.2) Molecular biological methods ................................................................ 32 2.2.2.1) Ribonucleic acid (RNA)-Isolation .................................................... 32 2.2.2.2) Complementary DNA (cDNA)-synthesis ......................................... 33 2.2.2.3) Quantitative real-time polymerase chain reaction (RT-qPCR) ........ 34 2.2.2.4) RNA-Isolation and expression analysis using microarray approach 35 2.2.2.5) Protein-Isolation out of whole cell lysates and sample preparation for gelelectrophoresis .......................................................................... 36 2.2.2.6) Protein-Isolation out of supernatants using TCA-precipitation and sample preparation ......................................................................... 37 2.2.2.7) SDS-Polyacrylamid (SDS-PAA) gel electrophoresis and Western blot ....................................................................................................... 38 2.2.3) Cell biological assays ............................................................................ 40 2.2.3.1) Proliferation assay .......................................................................... 40 2.2.3.2) Stimulation of CaCo2 cells with supernatants and/or recombinant protein ............................................................................................ 41 2.2.4) Bioinformatical GeneChip Analysis ....................................................... 42 3) Results ............................................................................................................. 44 3.1) siRNA-knockdown of CSN5 changes the gene expression pattern in SW480 cells ............................................................................................................. 44 3.2) CSN5 influences the expression of activin/inhibin subunits ......................... 53 3.3) Knockdown of CSN5 do not alter the protein level of inhibin A subunits ... 55 3.4) Overexpression of CSN5 does not affect the expression of activin/inhibin subunits ....................................................................................................... 56 IV 3.5) The knockdown of CSN1 or CSN2 does not alter the expression of inhibin A .................................................................................................................... 57 3.6) Knockdown of different CSN subunits influences the protein stability of the CSN ............................................................................................................. 60 3.7) Cullin1 seems to compensate the effect of CSN5 on inhibin A but not inhibin α mRNA expression ......................................................................... 61 3.8) The influence of CSN5 on activin/inhibin subunits seems to be mediated by transcriptional regulation ............................................................................. 63 3.9) Inhibition of proteasomal degradation reduces inhibin A protein level ....... 65 3.10) Increased expression of inhibin A seems to increase the secretion of activins/inhibins ........................................................................................ 66 3.11) The influence of CSN5 on CRC cell proliferation seems to be at least partly mediated via activin/inhibin signaling ......................................................