Characterization of the small molecule kinase inhibitor SU11248 (Sunitinib, SUTENT) in vitro and in vivo [Elektronische Ressource] : towards response prediction in cancer therapy with kinase inhibitors / Michaela Bairlein

Bairlein

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 30
• Langue: Deutsch
• Poids de l'ouvrage: 13 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Genetik





Characterization of the Small Molecule Kinase Inhibitor
SU11248 (Sunitinib/ SUTENT
in vitro and in vivo
- Towards Response Prediction in Cancer Therapy with Kinase Inhibitors




Michaela Bairlein



Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung,
Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades
eines
Doktors der Naturwissenschaften
genehmigten Dissertation.



Vorsitzender: Univ. -Prof. Dr. K. Schneitz
Prüfer der Dissertation: 1. Univ.-Prof. Dr. A. Gierl
2. Hon.-Prof. Dr. h.c. A. Ullrich
(Eberhard-Karls-Universität Tübingen)
3. Univ.-Prof. A. Schnieke, Ph.D.


Die Dissertation wurde am 07.01.2010 bei der Technischen Universität München eingereicht und durch die
Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 19.04.2010
angenommen.





























FOR MY PARENTS
1 Contents
2 Summary ................................................................................................................................................................... 5
3 Zusammenfassung .................................................................................................................................................... 6
4 Introduction .............................. 8
4.1 Cancer ............................................................................................................................................................... 8
4.1.1 The hallmarks of cancer ........................... 8
4.2 Protein kinases and cancer .............................. 11
4.3 Protein kinase inhibitors in targeted cancer therapy ........................................................................................ 18
4.3.1 Classes of small-molecule protein tyrosine kinase inhibitors ................................. 22
4.3.2 Multi-targeted small-molecule protein kinase inhibitors ........................................ 24
4.4 Sunitinib malate .............................................................................................................. 24
4.5 A closer look at targeted cancer therapy and small-molecule kinase inhibitors .............. 27
4.5.1 The advantages and drawbacks of targeted cancer therapy with kinase inhibitors ................................. 27
4.5.2 Challenges for multi-targeted kinase inhibitors ...................................................................................... 28
4.5.3 Drug resistance –single versus multi-targeted small-molecule protein kinase inhibitors ....................... 28
4.6 Drug discovery of selective kinase inhibitors.................................................................................................. 30
5 Aims of this PhD thesis .......................................... 32
6 Materials and Methods .......................................................................................................... 34
6.1 Materials .......................................................................................................................................................... 34
6.1.1 Laboratory chemicals, biochemicals and inhibitors ............... 34
6.1.2 Chemicals for SILAC and MS-analysis .................................................................................................. 34
6.1.3 Enzymes .................................................................................................................................................. 35
6.1.4 “Kits“ and other materials ..................... 35
6.1.5 Growth factors and ligands .................... 35
6.2 Media .............................................................................................................................................................. 35
6.2.1 Bacterial media ...................................... 35
6.2.2 Cell culture media .. 35
6.3 Stock solutions and commonly used buffers ................................................................... 36
6.4 Cells ................................................................................................ 36
6.4.1 Eukaryotic cell lines ............................... 36
6.5 Antibodies and recombinant proteins .............................................................................................................. 38
6.5.1 Primary antibodies ................................. 38
6.5.2 Secondary antibodies .............................................................................................. 38
6.6 Oligonucleotides ............................................................................................................................................. 38
6.6.1 siRNA oligonucleotides .......................... 38
6.7 Methods ........................................................................................................................................................... 39
6.7.1 Cellular Assays ....................................... 39
6.7.2 Affinity chromatography and mass spectrometry ................................................... 40
6.7.3 Molecular methods ................................................................. 42
7 Results ..................................................................................................... 44
7.1 Profiling of SU11248 activity in cancer cells .................................................................................................. 44
7.1.1 Effect of SU11248 on cancer cell proliferation ...................... 44
7.1.2 Effect of SU11248 on cancer cell apoptosis and cell cycle distribution ................................................. 52
7.1.3 Effect of SU11248 on cancer cell migration and invasion ..................................... 56
7.1.4 Morphological changes of cancer cells after SU11248 treatment .......................... 60
7.2 Target-selectivity profiling of SU11248 in cancer cell lines and metastatic renal cell carcinoma (mRCC)
tumors ................................................................................................................................................................. 62
7.2.1 Workflow of drug target profiling by affinity chromatography and mass spectrometry ........................ 63
7.2.2 Target identification and functional classification of SU11248 protein interaction partners ................. 65
7.3 Binding-affinity-analysis of SU11248 towards qualitatively identified cellular targets ................................. 72
7.4 Quantification of target-dissociation constants directly from cells ................................. 82
7.4.1 Workflow of target affinity measurement based on quantitative mass spectrometry combined with
affinity purification experiments ............................................................................................................ 82
7.5 In vitro binding studies and cellular kinase assays .......................... 87
7.6 Quantification of relative target amount binding to SU11248 in sensitive and insensitive cell lines .................
......................................................................................................................................................................... 89
7.7 Gene expression analysis of SU11248 sensitive and less responsive cancer cell lines of different cancer types ..
......... 93
7.8 Phosphoproteomic analysis of cancer cell lines treated with SU11248 .......................................................... 97
7.9 Functional characterization of SU11248 targets by knock-down experiments ............. 108
7.9.1 High affinity SU11248 targets – their relevance for functional processes in cancer cell lines ...................
.............................................................................................................................................................. 108
7.9.2 Functional correlation of target expression and SU11248 efficacy in cancer cell lines ....................... 113
7.10 Functional characterization of the receptor tyrosine kinase ROS1 ................................ 116
7.10.1 ROS1 plays a key role in cancer cell proliferation and survival ........................................................... 116
7.10.2 ROS1 expression seems to correlate with chemoresistance to Taxol treatment of cancer cell lines from
different tumor types ............................................................................................ 120
7.10.3 Identification of ROS1 interaction partners with mass spectrometry ................................................... 122
7.10.4 Screening of compound libraries against the receptor tyrosine kinase ROS1- identification and
characterization of a small-molecule kinase inhibitor of ROS1 kinase activity in vitro ....................... 126
8 Discussion ..................................