EGFR activity as a determinant of response to EGFR-targeted therapy

Afgezien van de aanwezigheid van activerende EGFR mutaties, die in niet- kleincellige long tumoren (NKLT) gecorreleerd werden met respons aan EGF R tyrosine kinase remmers (TKR), werden er tot op heden geen voorspellen de factoren voor respons aan EGFR-TKR in andere tumoren of aan EGFR-TKR in combinatie met chemotherapie geindentificeerd. We toonden de waarde aan van de constitutieve EGFR activiteit als een pr edictieve factor voor respons aan EGFR-gerichte therapieën in colorectal e kanker (CRC) cellijnen. We toonden eveneens de predictieve waarde aan van chemotherapie-geinduceerde EGFR activatie voor de gecombineerde beha ndeling van chemotherapie met EGFR-gerichte TKR: CRC en NKLT cellijnen w aarin we een verhoging in EGFR activatie waarnamen na chemotherapie toed iening, waren gevoelig aan EGFR-gerichte TKR. Onze bevindingen suggerere n dat door het bepalen van de pEGFR expressie na chemotherapie toedienin g, CRC en NKLT patiënten die een voordeel ondervinden van de toevoeging van een EGFR-TKR aan hun chemotherapie schema, kunnen geïdentificeerd wo rden. We identificeerden verder het belang van Src-family kinases (SFK), ADAMs (a desintegrin and metalloprotease) en TGF-α als kritische mediato ren van EGFR fosforylatie na chemotherapie toediening. We suggereren dat een verandering in serum waarden van TGF-α na chemotherapie toedie ning zou kunnen gebruikt worden om een subgroep patiënten die zullen bea ntwoorden aan gecombineerde behandeling van chemotherapie met gefitinib, te identificeren. Gerichte therapieën tegen SFKs en ADAMs (ADAM-17) in combinatie met chemotherapie kunnen belangrijke therapeutische benaderin gen zijn voor de behandeling van CRC en NKLT. Tot slot toonden we de waarde aan van de combinatie van een dual EGFR/He r2 remmer met chemotherapie in CRC cellijnen en we stelden voor dat deze combinatie geïntroduceerd kan worden in plaats van of in vergelijking m et de combinatie van een EGFR specifieke remmer met chemotherapie.Chapter 1: Introduction. 1 1.1. Biology of EGFR family receptors 3 1.1.1.The Her receptors in development and normal physiology 3 1.1.2. The Her receptors and their ligands 4 1.1.2.1. EGFR 4 1.1.2.2. Her2 4 1.1.2.3. Her3 6 1.1.2.4. Her4 6 1.1.2.5. Biochemical properties of Her receptor ligands 6 1.1.2.6. Expression of Her receptors and ligands in cancer 7 1.1.3. Ligand-induced dimerization and activation 8 1.1.4. Signalling pathways activated by EGFR 10 1.1.4.1. Shc, Grb2 and Ras/MAPK pathway 11 1.1.4.2. PI3-K/Akt pathway 12 1.1.4.3. STAT pathway 13 1.1.4.4. PLC pathway 13 1.1.4.5. Nuclear EGFR as a transcriptional regulator 13 1.1.5. Internalization and recycling of the Her receptors 14 1.1.6. EGFR activation by heterologous mechanisms 15 1.1.6.1. EGFR overexpression 15 1.1.6.2. Increased expression receptor ligands 15 1.1.6.3. EGFR mutations 15 1.1.6.4. Transactivation of EGFR 15 1.2. The zinc protease superfamily and matrixins 16 1.2.1. ADAMs, multidomain proteins with multiple functions 16 1.2.2. The matrix metalloproteases 17 1.3. Non-receptor tyrosine kinase proteins 17 1.3.1. The Src-family kinases 17 1.3.2. The Abl kinase 19 1.3.3. The Jak-STAT pathway 19 1.4. The EGFR family as targets for cancer therapy 20 1.4.1. EGFR family targeted monoclonal antibodies 20 1.4.1.1. Cetuximab (C225, Erbitux) 20 1.4.1.2. Traztuzumab (Herceptin) 21 1.4.1.3. Pertuzumab (Omnitarg, 2C4) 21 1.4.2. EGFR family targeted tyrosine kinase inhibitors 22 1.4.2.1. EGFR-targeted TKIs 22 1.4.2.2. Her2-targeted TKIs 23 1.4.2.3. Pan-Her TKIs. 23 1.4.2.4. Dual Her1/Her2 TKIs 23 1.4.3. Downstream intracellular signalling targets 24 1.4.3.1. Inhibitors of the Ras/B-Raf/MAPK pathway 24 1.4.3.2. PI3K/Akt Inhibitors 24 1.5. Predictors for response to EGFR targeted therapies 24 1.5.1. EGFR and Her2 expression 24 1.5.2. Constitutive levels of pAkt and pErk1/2 24 1.5.3. EGFR gene mutations 25 1.5.4. Link between EGFR mutations, amplification and prognosis 26 1.5.5. K-Ras and B-Raf gene mutations 27 1.5.6. PTEN and PI3K gene mutations 27 1.5.7. Clinical surrogate markers 27 1.6. Aims of the study 28 Chapter 2: Materials and methods. 29 2.1. Materials 31 2.2. Cell culture 31 2.3. MTT cell Viability Assay 32 2.4. Crystal Violet assay 32 2.5. Flow cytometric analysis and cell death measurement 32 2.6. Detection of cell surface EGFR and Her2 expression 33 2.7. Western Blotting 33 2.8. Quantitative real-time PCR 34 2.9. Epidermal Growth Factor Receptor sequencing 34 2.10. siRNA transfections 35 2.11. Statistical analysis 35 Chapter 3: Sensitivity of CRC and NSCLC cells to EGFR- targeted therapy alone. 37 3.1. Introduction 39 3.2 Correlation between sensitivity to gefitinib or cetuximab and constitutive levels of pEGFR 39 3.3. Correlation between sensitivity to gefitinib and inhibition of pEGFR, pHer2, pAkt and pErk1/2 by gefitinib 44 3.4. Discussion 47 Chapter 4: Interaction between gefitinib and chemotherapy in CRC cells. 49 4.1. Introduction 51 4.2. Evaluation of the gefitinib/chemotherapy combination 51 4.2.1. Gefitinib in combination with oxaliplatin 51 4.2.2. Gefitinib in combination with 5-FU 57 4.2.3. Gefitinib in combination with SN-38 60 4.3. Effect of chemotherapy on EGFR phosphorylation 62 4.4. Discussion 66 Chapter 5: Interaction between gefitinib and chemotherapy in NSCLC cells. 69 5.1 Introduction 71 5.2. Evaluation of the gefitinib/chemotherapy interaction 71 5.2.1. Gefitinib in combination with cisplatin 71 5.2.2. Gefitinib in combination with taxol 75 5.3. Effect of chemotherapy on EGFR phosphorylation 77 5.4. Discussion 79 Chapter 6: Mechanism of increased EGFR activation following chemotherapy. 83 6.1 Introduction 85 6.2 Effect of chemotherapy on EGFR and SFK phosphorylation and expression 86 6.3. Effect of SFK inhibition and gefitinib on constitutive and activated EGFR and SFK phosphorylation following chemotherapy 86 6.4. Evaluation of the interaction between the SFK inhibitor PP2 and chemotherapy 88 6.5. Effect of metalloproteinase inhibition on EGFR and SFK phosphorylation following chemotherapy 93 6.6. Evaluation of the interaction between GM6001 and chemotherapy 94 6.7. Evaluation of the role of ADAM-17 in basal and activated EGFR and SFK phosphorylation following chemotherapy 94 6.8. Effect of the EGFR monoclonal antibody cetuximab on constitutive and chemotherapy-activated pEGFR and pSFK levels 96 6.9. Effect of TGF-alpha inhibition on constitutive and chemotherapy-activated pEGFR and pSFK levels 97 6.10. Evaluation of the role of ROS in chemotherapy-activated pEGFR and pSFK levels 99 6.11. Discussion 100 Chapter 7: Sensitivity of CRC to Her2 and dual Her1/Her2 inhibition. 105 7.1. Introduction 107 7.2. Correlation between the sensitivity of CRC cells to the DKI or Her2I and constitutive levels of pEGFR 107 7.3.Characterization of the dual EGFR/Her2 TKI (DKI, M880588) 109 7.3.1. Effect of the DKI on pEGFR/pHer2/pAkt/pErk1/2 109 7.3.2. Interaction between DKI and chemotherapy 110 7.3.2.1. Interaction between DKI and oxaliplatin 110 7.3.2.2. Interaction between DKI and 5-FU 113 7.3.2.3. Interaction between DKI and SN-38 115 7.4. Characterization of the specific Her2 TKI (Her2I, M578440) 117 7.4.1. Evaluation of the antiproliferative activity of the Her2I in combination with chemotherapy 117 7.4.2. Comparison of the effect of the Her2I, DKI or gefitinib on chemotherapy-induced apoptosis in CRC cells 119 7.4.3. Effect of Her2I on Akt activation 121 7.5. Interaction between the PI3-K inhibitor LY294002 and chemotherapy 122 7.6. Discussion 123 Chapter 8: Conclusions and future directions. 127 8.1. Summary 129 8.2. General conclusions 134 8.3. Future directions 135 Samenvatting. 138 References. 143status: publishe