UNIVERSITE PARIS SUD-11 Faculté de Médecine École Doctorale : Signalisation et Réseaux intégratifs en Biologie Discipline : Vectorologie et thérapeutiques anti-cancéreuses THÈSE DE SCIENCES pour l’obtention du grade de Docteur de l’Université Paris-Sud XI Effets des nanoparticules de siRNA-Squalène sur les oncogènes de jonction RET/PTCs dans le carcinome papillaire de la thyroïde : études moléculaires, cellulaires et investigations précliniques Présentée et soutenue par Hafiz Muhammad ALI le 22 / 04 / 2014 Président du jury: Dr. Michael Schumacher Univ. Paris Sud-11, France Rapporteur : Pr. Marie-Agnès Sari Univ. Paris Descartes-5, France Rapporteur : Dr. Bertrand Jean-Claude Univ. McGill, Canada Examinateur : Dr. Didier Desmaële Univ. Paris Sud-11, France Directeur de thèse: Dr. Liliane Massade UMR 8203 CNRS, IGR, France UNIVERSITE PARIS SOUTH-11 Faculty of Medicine Doctoral School : Signaling and integrative networks in biology Discipline : Vectorology and anti-cancer therapeutics THESIS OF SCIENCE submitted to obtain the degree of Doctor of the University of Paris-South XI Effects of siRNA-squalene nanoparticles on RET/PTCs junction oncogenes in papillary thyroid carcinoma : from molecular and cellular studies to preclinical investigations Presented by Hafiz Muhammad ALI on 22 / 04 / 2014 President of jury: Dr. Michael Schumacher Univ. Paris Sud-11, France Reporter : Pr. Marie-Agnès Sari Univ. Paris Descartes-5, France Reporter : Dr. Bertrand Jean-Claude Univ. McGill, Canada Examiner : Dr. Didier Desmaële Univ. Paris Sud-11, France Director of thesis: Dr. Liliane Massade UMR 8203 CNRS, IGR, France ACKNOWLEDGEMENTS Firstly, I would like to acknowledge and thank my PhD supervisor, Dr. Liliane Massade for accepting me in her team and providing me an opportunity to work under her guidance. I am really grateful to her as she made the thing possible for me given that she really has the ability to supervise a student. She put all the efforts to teach me the very basics of research starting from cell culture and was always beside me during the happy and hard moments of the journey to reach this destination. During the whole of my stay, her continuous support, rational ideas and dynamic supervision throughout the way, made be able to achieve this target. Her devotion to science, ability of solving problems and elegance of reasoning set an example for me to follow and also motivate me to take her guidance during my coming periods of scientific work. I am extremely grateful to all the respected members of the thesis evaluation jury. I am really thankful to Dr. Michael Schumacher (Director of Doctoral School-419, University Paris South-11) for accepting to be the President of my jury and to Dr. Marie-Agnès Sari (Professor, University Paris Descartes-7) and Dr. Bertrand Jean-Claude (McGill University Health Center, Montréal, Canada) for reviewing my thesis as reporters and helping me to ameliorate the presentation of my scientific work. I am also equally thankful to Dr. Didier Desmaële (Research Director Institut Galien, University Paris South-11, Châtenay-Malabry) for accepting to evaluate this work as an examiner. I would like to greatly acknowledge and thank to Higher Education Commission (HEC) of Pakistan for giving this opportunity to pursue my higher studies in France by granting Master-2 leading to PhD scholarship. I would also like to thank “CNRS; Centre National de la Recherche Scientifique” for their financial support during few months to complete my thesis. I would like to express my deep gratitude to Prof. Dr Patrick Couvreur and Dr. Didier Desmaële at Institut Galien, University Paris South-11, Châtenay-Malabry for their fruitful discussions about the results and various aspects of the studies and to give various suggestions and possible solutions of scientific problems in the lab meetings during my thesis period. I am extremely thankful to Dr. Michael Schumacher (Director of Doctoral School-419, University Paris South-11) for his help and support to find the research laboratory and for Mr. Laurent Surdi (Secretary, Doctoral School-419) for her kindness, availability, assistance and reply to all queries throughout the period of my PhD. i I wish to extend my sincere thanks to all members of UMR 8203 CNRS for their support. It has been a great privilege to spend my time in this lab and all its members shall always remain dear to me. I extend my sincere thanks to Dr. Luis Mir (Director, UMR 8203 CNRS) to allow me to work in the Research Unit. I would like to thank Giorgia Urbinati from the bottom of my heart for her pieces of advice, encouragement, friendly support and the time she spent to perform animal experiments with me, to discuss my research work and to read my scientific preparations. Equally, I would like to thank Jean-remi Bertrand (for cellular microscopic analysis, useful scientific pieces of advice and various types of oriental teas), Karim Benihoud (for providing tumor pulverizer), Hubert Chapuis (for preparing and provision of nanoparticles), Nathalene Truffaux (for my experiments with IncuCyte), Franck André (for the help with flow cytometer, Elodie Grenier (to help in to perform Western blot), Marie Breton (to help find out chemicals required), Isabelle Leray (for timely provision of cellular life materials), Gaétan Cornilleau (to guide and facilitate me for working in cell culture laboratory), Estelle Daudigeos (for quantitative protein analysis), Ludivine Le Dret (to keep the cells away from mycoplasma), Wael Jdey (for cell cycle and apoptosis analysis), Mouna Raouane (for cellular internalization experiments), Andrey Maksimenko (for the part of animal experimentation), Christophe Calvet (for collaboration to publish the work in Nature Medicine), Najat Raddi (for her beautiful smiles), Aleksandra Anchim (for chocolates from Poland), Marie-Amélie De Ménorval (for discussions during the way to laboratory and to lending me her laptop for working), Charles Skarbek (for discussions in the corridor), Lea Lesueur (her kindness in sharing the daily laboratory life), Quentin Rousseau (for his everlasting good humor), Erika Clavijo (for her nice chocolates), Tobias Fox (for assisting me in laboratory experiments), Isabelle Croquison and Ysabel Descarpentries (for helping in administrative affairs), to David Castel, Marie-Anne Debily, Cathy Philippe, Carole Lecinse, Thierry Ragot, Atman Seck, Hanna Hanna, Antoine Azan and to Tarik Mohoub and Mateja Slamic (for the beautiful last period of time in their company). At this point, I would also like to thanks to Giorgia for her delicious Italian Tiramisu chocolate cakes on every occasion. I would like to really thanks to all these friends with whom I shared not only my office but a lot of emotions during these years. It was great knowing and working with such a wonderful group of people and I have very fond memories of all the times we spent together. I extend my sincere thanks to Dr. Patrick Gonin (Responsible of animal facility, IGR), Olivia Bawa (for immuno-histo chemical analysis of tumours) and the whole team of animal facility at IGR for their patience and help for the in vivo work during my PhD. ii I would like to especially thank to all my Pakistani friends and HEC scholars. A thousand thanks to Afaq Ali, Adnan, Abdul Qadir, Shoaib Ahmad, Qasim Shah for their time and friendship during their stay at IGR as well. I cannot forget the never-ending amusing general and scientific discussions with them in corridors and during tea at Afaq’s residence. I cannot forget to mention my friends outside IGR for their support and time we spent together during my stay in France. I would like to especially thank to Zahid Mustafa, Junaid Ali Khan, Zahid Safi, Ch Iftikhar Ahmed, Rana Iftikhar Ahmad, Habib Magsi, Zeeshan Ali and Moazam. My stay in France would have been much less exciting and pleasurable if it was without the company of these great friends. At this point, again I express thanks to HEC who put all us together by granting financial assistance to complete PhD studies in France. Besides, I would also like to thank all other friends in Pakistan who always encouraged and supported me with useful discussions during and before my PhD. I want to thank my M.Sc. (Hons.) supervisor, Anas Sarwar (Prof. Anatomy, University of Agriculture, Faissalabad) who guided me to start my research and teaching career under his supervision. Equally, I thanks to my colleagues at Faculty of Veterinary Science, University of Agriculture Faisalabad and College of Veterinary Sciences, Islamia University Bahawalpur, where I served as lecturer before coming to France. Special thanks to Jahanzeb Ansari (Govt. Poultry farm, Bahawalpur), Rana Nisar Khan (Prof. Parasitology) and Najib-ur-Rahman (Prof. Theriogenology), Rao Zahid Abbass, Sohail Sajid and Kashif Saleemi at University of Agriculture, Faissalabad and Masood Akhtar (Prof. and Dean, Faculty of Veterinary Sciences, Bahauddin Zakariya University, Multan) who always welcomed, encouraged and appreciated me during my stays in Pakistan. I am highly grateful to my family who always supported me during my stay in France but words cannot express my feeling of appreciation and gratitude to my mother. I am highly grateful to my mother, my sisters and brother for their prayers and motivation in every aspect of my life. Whatever I have achieved till now and shall be achieving is just because of them. Finally, thanks to France who received me to learn science and to visit its nice people, pleasant culture and beautiful cities and monuments. I will miss you as well, as all the people who were with me during this important period of my life. I’m almost crying!!!! Hafiz Muhammad ALI iii ABBREVIATIONS ANTS 1-aminonaphthalene-3, 6, 8-trisulfonic acid ARA70 Androgen receptor-associated protein 70 AS-ONs Antisense oligonucleotides ATA American Thyroid Association ATC Anaplastic (undifferentiated) thyroid carcinoma BID Twice daily C Control CBR Clinical beneficial response CCDC6 Coiled-coil domain-containing gene 6 CEACAM1 Carcino-embryonic antigen-related cell adhesion molecule-1 Chol Cholesterol CLSM Confocal laser scan microscopic CPTC Conventional PTC CR Partial response Ct Cycle threshold CTCA Cancer Treatment Centers of America DBVs Designer biomimetic vectors DMEM Dulbecco’s modified eagle medium DSBs Double-strand breaks DTC Differentiated TC ECM Extracellular matrix EGFR Epidermal growth factor receptor eIF2α Eukaryotic initiation factor 2α ERP Estrogen receptor related protein FAM 6-CarboxyFluorescein-Aminohexyl Amidite FCS Fetal calf serum FDA Food and Drug Administration of USA FISH Fuorescence in situ hybridization FNAC Fine-needle aspiration cytology FoxP3 Forkhead box P3 FRET-SE Fluorescence Resonance Energy Transfer-Sensitized Emission FVPTC Follicular variant PTC GALA Glutamic-Alanine-Leucine-Glutamic acids GDNF Glial cell line derived neurotrophic factor GFR Growth factor receptor GnRH Gonadotrophin-releasing hormone GPI Glycosyl-phosphatidyl-inositol GR Glucocorticoid-R iv HBME-1 Human mesothelial cell marker 1 I131 Radioactive iodine 131 IgG Immunoglobulin G IκB-α Nuclear factor-κB LDL Low-density lipoprotein LIMD2 LIM kinase domain containing 2 LM-PCR Ligation-mediated PCR LTB Lymphotoxin beta MAGE Melanoma-associated antigen MAPK Mitogen-activated protein kinase MEN Multiple endocrine neoplasis MKIs Multi-kinase inhibitors MMPs Matrix metalloproteinases MRI Magnetic resonance imaging MRT Magnetic resonance tomography MTC Medullary thyroid carcinoma MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide NBCS New born calf serum NcoA4 Nuclear receptor coactivator-4 NGF Nervous (central and peripheral) growth factor NHEJ Non-homologous end joining NPs Nanoparticles NTRK-1 Neurotrophic receptor-tyrosine kinase-1 ONs Oligonucleotide OSE Ovarian surface epithelial Pax-8 Paired box-containing gene-8 PBS Phosphate Buffered Saline PCCL3 Parental well differentiated rat thyroid cell line cells pCND Prophylactic central compartment neck dissection PD Progressive disease PDGFR Platelet derived growth factor receptor pDNA Plasmid DNA PET Positron emission tomography scans PFS Progression free survival PI3K-mTOR Phosphatidyl-inositol-3 Kinase-mammalian target of rapamycin PKR Protein kinase R POPC 1-pahnitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPG 1-palmnitoyl-2-oleoyl-phosphatidylglycerol PPAR-γ Peroxisome proliferator-activated receptor-γ PR Partial response v PTC Papillary thyroid carcinoma PTMC Papillary thyroid micro-carcinoma PTPRC Protein tyrosine phosphatase receptor type Concentration PTPRJ Protein Tyrosine Phosphates Receptor type-J QD Once daily R8-MEND Octa-arginine peptide-multifunctional envelope-type nano device RAI Radioactive iodine RES Reticulo-endothelial system RET REarranged during Transfection RFG Ret fused gene RISC RNA induced silencing complex RNAi RNA interference RPL13A Ribosomal Protein 13A RT-qPCR Real Time quantitative Reverse Transcription Polymerase Chain Reaction SD Stable disease SDS Sodium dodecyl sulphate shRNAs Short hairpin RNAs siRNA small interfering RNA SQ Squalene T3 Tri-iodothyronine T4 Tetra-iodothyronine TCPTC Tall-cell PTC TEM Transmission electron microscopy Tf-FITC L Transferrins-fluorescein isothiocyanate liposomes TG Thyroglobulin TH Thyroid hormone TKR Tyrosine kinase receptor TLR3 Endosomal toll-like receptor-3 TPO Thyroid peroxidase TRH Thyrotropin releasing hormone TSH Thyroid stimulating hormone TSH-R TSH receptor TTF-1 Thyroid transcription factor-1 u-PA Urokinase-type plasminogen activator VEGFR Vascular endothelial growth factor receptor WB Western blot vi TABLE OF CONTENTS Aim of the thesis……………………………………………….…………………………..01 1.1. Thyroid gland…………………………………………………………………..……..03 1.1.1. Anatomical location and structure of the thyroid gland……………….……03 1.1.2. Histological structure of the thyroid gland…………………………….……03 1.1.3. Function of the thyroid gland………………………………………….……04 1.2. Thyroid cancer………………………………………………………………………..06 1.2.1. Oncogene and oncogenesis…………………………………………………06 1.2.2. Incidence and distribution of thyroid cancer………………………………..06 1.2.3. Forms and types of thyroid cancer………………………………………….07 1.2.4. Diagnosis of thyroid cancer…………………………………………………08 1.2.5. Treatment of thyroid cancer………………………………………………...10 1.2.5.1. Classical treatment of thyroid cancer………………………..10 1.2.5.2. Demerits and disadvantages of classical treatment………….11 1.2.5.3. Inhibition of tyrosine kinase receptor………………………..12 1.2.5.4. Immuno-therapy in thyroid cancer…………………………..15 1.3. Papillary thyroid carcinoma……………………………………………….…………16 1.3.1. Incidence of papillary thyroid carcinoma…………………………………...16 1.3.2. Prognosis of papillary thyroid carcinoma…………………………………...17 1.3.3. Risk factors and causative agents of PTC…………………………………...17 1.3.3.1. Role of ionizing or therapeutic radiation exposure…………..17 1.3.3.2. Age effect in PTC development……………………………...19 1.3.3.3. Sex effect in PTC development………………………………20 1.3.3.4. Miscellaneous factors involved in PTC progression…………21 1.3.4. Genetic rearrangements occurring in PTC…………………………………...21 1.3.5. Role of gene positioning in PTC progression………………………………..23 1.4. RET proto-oncogene……………………………………………………………….…..24 1.4.1. Structure of RET……………………………………………………………..24 1.4.2. Function of RET……………………………………………………………...25 1.4.3. RET recombination and activation…………………………………………...26 1.4.3.1. Radiation exposure and RET recombination…………………26 1.4.3.2. Mechanism of RET recombination…………………………...27 1.4.3.3. Activation of chimeric protein………………………………..28 1.4.3.4. Effect of RET recombination on tumour progression………...29 1.5. RET mutations, translocations and oncofusions in PTC…………………………....30 1.5.1. CCDC6 / H4 / D10S170 (Coiled-coil domain-containing gene 6)…………..34 1.5.2. NcoA4 / ELE1 / RFG (Nuclear receptor co-activator 4)……………………..35 1.5.2.1. Structural properties of NcoA4……………………………………….36 1.5.2.2. Functional properties of NcoA4……………………………………...37 1.5.2.3. Role of NcoA4 as an androgen receptor co-regulator………………..38 1.5.2.4. Potential involvement of NcoA4 in PTC progression………………..39 1.6. RNA interference…………………………………………………………….…………39 1.6.1. Antisense Oligonucleotides…………………………………………………..40 vii 1.6.2. Small interfering RNAs………………………………………………………40 1.6.3. Application and uses of RNAi technology…………………………………...41 1.6.4. Merits of siRNA……………………………………………………………...43 1.6.5. Drawbacks of siRNA………………………………………………………....43 1.6.5.1. Transitory gene silencing effects……………………………………..43 1.6.5.2. Non-specific and off-target interference……………………………...44 1.6.5.3. Activation of immune response………………………………………44 1.6.5.4. Miscellaneous flaws of siRNA therapy………………………………45 1.7. Hurdles of RNAi based cancer therapies……………………………………………..45 1.8. Strategies to improve RNAi design and therapeutic effects………………………....46 1.9. Strategies to deliver the RNAi to the target tissue…………………………………...47 1.9.1. Use of viral vectors for siRNA delivery……………………………………...47 1.9.2. Use of non-viral siRNA carrier systems……………………………………...48 1.9.3. Outcomes of cationic systems for siRNA delivery…………………………...48 1.9.4. Use of neutral lipids for siRNA delivery……………………………………..49 1.9.4.1. Squalene: a neutral lipid………………………………………………50 1.9.4.2. Applications and uses of squalène……………………………………50 1.9.4.3. Squalenoylation……………………………………………………….51 1.10. Article–1: Significance and applications of nanoparticles in siRNA delivery for.….. cancer therapy………………………………………………………………………...53 1.11. Use of a fusogenic peptide Gala-Chol……………………………………………...77 1.11.1. Properties and function of Gala……………………………………................77 1.11.2. Why Gala as a choice………………………………………………………...78 1.11.3. Role of Gala location in the nanoparticles…………………………………...79 1.11.4. Role of cholesterol in Gala function…………………………………………80 1.12. Article–2: Effects of silencing RET/PTC1 oncogene in papillary thyroid carcinoma by siRNA-squalene nanoparticles with and without fusogenic companion GALA-Cholesterol…………………………………………….………81 1.13. Article–3: Effects of siRNA on RET/PTC3 junction oncogene in papillary thyroid carcinoma: from molecular and cellular studies to preclinical investigations……………………………………………………………. ………..111 1.14. Discussion……………………………………………………………………..……146 1.15. Conclusions………………………………………………………………….……...153 1.16. Perspectives………………………………………………………………….……..154 1.17. Synthèse du travail ………………………………………………………………..155 1.17.1. Introduction…………………………………………………………………155 1.17.2. Principaux résultats obtenus au cours de la thèse…………………………..158 1.17.3. Discussion……………………………………………………………….…161 1.17.4. Conclusions…………………………………………………………………164 1.17.5. Perspectives…………………………………………………………………164 1.17.6. Retombées cliniques………………………………………………………...165 1.18. References………………………………………………………………………….166 viii