THESIS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (Ph.D.) Molecular interactions of ErbB receptor tyrosine kinases and integrin β1: implications for tumor therapy by Miklós Petrás Supervisor: Prof. János Szöllősi, D.Sc. Álmos Klekner, Ph.D. UNIVERSITY OF DEBRECEN DOCTORAL SCHOOL OF MOLECULAR MEDICINE DEBRECEN, 2013 Molecular interactions of ErbB receptor tyrosine kinases and integrin β1: implications for tumor therapy Miklós Petrás Ph.D. Thesis UNIVERSITY OF DEBRECEN MEDICAL AND HEALTH SCIENCE CENTER DEPARTMENT OF BIOPHYSICS AND CELL BIOLOGY DEPARTMENT OF NEUROSURGERY DEBRECEN, 2013. 2 Ph. D. thesis data Title: Molecular interactions of ErbB receptor tyrosine kinases and integrin β1: implications for tumor therapy Author: Miklós Petrás, M.D. Supervisors: Prof. János Szöllősi, D.Sc. Álmos Klekner, M.D., Ph.D. Doctoral School: "Molecular Medicine" at the University of Debrecen Head of Doctoral School: Prof. László Csernoch, D.Sc. Doctoral examination committee: Chairman: Prof. László Csernoch, D.Sc. Members: Prof. Ferenc Erdődi, D.Sc. Beáta Bugyi, Ph.D. Place and time of doctoral examination: Debrecen, 3 May 2013 Doctoral thesis committee: Chairman: Prof. László Csernoch, D.Sc. Reviewers: Prof. István Jóna, D.Sc. József Tóvári, Ph.D. Members: Prof. Ferenc Erdődi, D.Sc. Beáta Bugyi, Ph.D. Place and time of thesis defence: Debrecen, 3 May 2013 Key words: ErbB (EGFR), integrin, tyrosine kinase, FRET, trastuzumab, PI-3K/Akt, breast cancer, astrocytoma (glioblastoma) 3 "Because strait is the gate, and narrow is the way, which leadeth unto life, and few there be that find it." Matthew 7:14 Bible - King James Version, 1611 Oxford King James Bible (Authorized Version), 1769 4 CONTENTS ABBREVIATIONS ..................................................................................................................................... 7 1. INTRODUCTION................................................................................................................................... 9 2. MOLECULAR PATHOLOGY OF TUMORIGENESIS............................................................................. 11 2.1. The ErbB family of receptor tyrosine kinases.............................................................................11 2.2. The integrin family of cell adhesion molecules ..........................................................................13 2.3. Molecular markers and signal transduction pathways.................................................................15 2.4. Associations of ErbBs and interactions with integrin 1 ............................................................18 2.4.1. Homo- and heterodimerization of ErbB molecules ......................................................................... 18 2.4.2. Molecular interactions between ErbB and integrin 1 molecules .................................................. 19 2.5. Analysis of multi-faceted molecular interactions of membrane proteins....................................19 3. ERBB MOLECULES IN TUMOR DEVELOPMENT – THE MEDICAL ASPECT ..................................... 21 3.1. Alterations of ErbB2 in breast cancer .........................................................................................21 3.1.1. Epidemiology and diagnosis of breast cancers - a general outline................................................. 21 3.1.2. Treatment options for breast cancer ............................................................................................... 22 3.1.3. Therapy resistance to trastuzumab in the management of breast cancers ...................................... 23 3.2. Alterations of ErbB1 in astrocytoma...........................................................................................25 3.2.1. Epidemiology and diagnosis of astrocytomas - a general outline................................................... 25 3.2.2. Treatment options for astrocytoma.................................................................................................. 27 3.2.3. Therapy resistance to irradiation in the management of astrocytomas........................................... 28 4. OBJECTIVES...................................................................................................................................... 30 5. MATERIALS AND METHODS............................................................................................................. 31 5.1. Antibodies ...................................................................................................................................31 5.2. Labeling of cells with antibodies.................................................................................................32 5.3. Cell cultures.................................................................................................................................32 5.4. Fresh frozen sections...................................................................................................................33 5.5. Cellular Model Systems ..............................................................................................................33 5.5.1. Chromosome 7 transferred U251 NCI subclones............................................................................ 33 5.5.2. Stable erbB1 gene transfected U251 NCI subclones....................................................................... 34 5.6. Determination of radiosensitivity................................................................................................35 5.7. Flow cytometric determination of cell surface receptor expressions ..........................................36 5.8. Assessing the molecular interactions of ErbB and integrin molecules .......................................36 5.8.1. Fluorescence resonance energy transfer (FRET)............................................................................ 36 5.8.2. Flow cytometric fluorescence resonance energy transfer measurements (FCET) .......................... 37 5.8.3. Confocal microscopy....................................................................................................................... 38 5.8.4. Calculation of molecular colocalization ......................................................................................... 39 5.8.5. Acceptor photobleaching method (abFRET)................................................................................... 39 5.8.6. Donor photobleaching method (dbFRET)....................................................................................... 42 5.9. Stimulation of cells with EGF, heregulin and trastuzumab.........................................................43 5.10. Western blot analysis ................................................................................................................44 5.11. Statistical analysis .....................................................................................................................44 5 6. RESULTS............................................................................................................................................ 45 6.1. Interactions of ErbBs and integrin β1 on breast and gastric cancer cell lines.............................45 6.1.1. Lower ErbB2 expression is accompanied by higher integrin β1 levels........................................... 45 6.1.2. Colocalization of ErbB2, integrin β1 and lipid rafts upon treatments ............................................ 46 6.1.3. Crosslinking of integrin β1 or lipid rafts disrupts their colocalizations.......................................... 48 6.1.4. ErbB2 - integrin β1 interaction is independent of integrin β1 expression level .............................. 49 6.1.5. ErbB2-integrin β1 interaction do not alter trastuzumab-mediated phosphorylation ...................... 51 6.2. Associations of ErbBs are dynamically modulated by integrin β1 molecules -..........................52 application of the newly established two-sided FRET method..........................................................52 6.2.1. Excess integrin β1 inversely correlates with ErbB2 level on trastuzumab resistant cells............... 52 6.2.2. ErbB2 interactions are shifted towards heteroassociations with integrin β1.................................. 53 6.2.3. Model system for two-sided FRET (tsFRET)................................................................................... 54 6.2.4. Application of tsFRET: ErbB2 homoassociations are disintegrated by integrin β1 ....................... 57 6.2.5. Cell function related distribution of membrane receptors............................................................... 59 6.3. Molecular interactions of ErbB1 and integrin β1 molecules reliably predict clinical outcome and correlate with radioresistance of astrocytic tumors ..............................................................61 6.3.1. Higher ErbB1 and integrin β1 levels correlate with pronounced radioresistance.......................... 61 6.3.2. Overexpression of ErbB1 alone is also accompanied by higher integrin β1 levels......................... 62 6.3.3. Integrin β1 contributes to survival, whereas ErbB1 promotes colony forming ability.................... 62 6.3.4. Increased integrin β1 is critical for radioresistance avertible by inhibition of PI-3K .................... 63 6.3.5. ErbB1 interactions are shifted towards heteroassociations with integrin β1.................................. 64 FCET measurements: shift of balance towards ErbB1-integrin β1 heteroassociations............................ 64 tsFRET