FACULTY OF SCIENCE Candidate biomarkers of resistance to retinoids in cell lines derived from neuroblastoma Ph.D. Dissertation VIERA DOBROTKOVÁ Supervisor: RNDr. Petr Chlapek, DiS., Ph.D. Department of Experimental Biology Brno 2019 Bibliographic Entry Author: Mgr. Viera Dobrotková Faculty of Science, Masaryk University Department of Experimental Biology Title of Thesis: Candidate biomarkers of resistance to retinoids in cell lines derived from neuroblastoma Degree Programme: Biology Field of Study: Molecular and Cellular Biology Supervisor: RNDr. Petr Chlapek, DiS., Ph.D. Academic Year: 2018/2019 Number of Pages: 115+0 Keywords: Neuroblastoma; Retinoids; Candidate biomarkers; Resistance; Sensitivity; Differentiation therapy Bibliografický záznam Autorka: Mgr. Viera Dobrotková Přírodovědecká fakulta, Masarykova univerzita Ústav experimentální biologie Název práce: Kandidátní biomarkery rezistence k retinoidům u buněčných linií derivovaných z neuroblastomu Studijní program: Biologie Studijní obor: Molekulární a buněčná biologie Vedoucí práce: RNDr. Petr Chlapek, DiS., Ph.D. Akademický rok: 2018/2019 Počet stran: 115+0 Klíčová slova: Neuroblastom; Retinoidy; Kandidátní biomarkery; Rezistence; Senzitivita; Diferenciační terapie Abstract Retinoids are key compounds in differentiation therapy, mainly in acute promyelocytic leukemia and high-risk neuroblastoma. Despite their proven effectiveness in preventing tumor relapse and improving patient survival, resistance to this treatment represents the most limiting factor in achieving an adequate clinical response. The aim of this study was to analyze the expression of five candidate biomarkers of responsiveness to retinoids that were recently discussed as promising in neuroblastoma. Analysis was performed in neuroblastoma cell lines, and the results were compared with data obtained from immunohistochemistry analysis of corresponding formalin-fixed paraffin-embedded samples. HOXC9 negativity was found to correlate with poor response to retinoids, and HOXC9 and PBX1 upregulation after retinoid treatment could predict sensitivity to retinoids in investigated cell lines. At the protein level, PBX1 downregulation was observed in retinoid-sensitive cell lines, and subsequent immunohistochemical analysis revealed that higher expression of PBX1 was significantly associated with a poorer response to chemotherapy and worse clinical outcome, supporting the usefulness of this biomarker in clinical practice. Abstrakt Retinoidy jsou klíčovou složkou diferenciační terapie, zejména u akutní promyelocytární leukémie a u vysoce rizikového neuroblastomu. Navzdory prokázané účinnosti při prevenci relapsu nádorů a zlepšování přežití pacientů představuje rezistence na tuto léčbu faktor, který nejvíce limituje dosahování adekvátní klinické odpovědi. Cílem této práce bylo analyzovat expresi pěti kandidátních biomarkerů senzitivity nebo rezistence k retinoidům, které byly u neuroblastomu v posledních letech popsány. Analýza byla prováděna na neuroblastomových buněčných liniích, výsledky byly porovnány s daty získanými z imunohistochemické analýzy odpovídajících formalínem fixovaných parafínových vzorků. Bylo zjištěno, že HOXC9 negativita koreluje s rezistencí k retinoidům a zvýšení exprese HOXC9 a PBX1 po podání retinoidů byl spojen se senzitivitou k těmto látkám. Na úrovni proteinů byla u senzitivních buněčných linií jako odpověď na působení retinoidů pozorována snížená exprese PBX1. Podle výsledků imunohistochemické analýzy byla vyšší exprese PBX1 spojena s horší odpovědí pacientů na chemoterapii a závažnějším průběhem onemocnění, což podporuje potenciální význam tohoto biomarkeru také v klinické praxi. Acknowledgements I would like to express my sincere gratitude to my supervisor RNDr. Petr Chlapek, DiS., Ph.D. for his support, patience, and motivation during my studies. My sincere thanks also go to prof. Renata Veselská, Ph.D., M.Sc. for her professional suggestions and kind encouragements, especially during the most difficult season of my study. I would also like to thank to my amazing colleagues from the Laboratory of Tumor Biology, who taught me to “keep calm and smile”. I would also like to express my gratitude to MUDr. Marta Ježová, Ph.D. for immunohistochemical analysis of tumor samples and to prof. MUDr. Jaroslav Štěrba, Ph.D. and MUDr. Pavel Mazánek for providing us with the clinical data and tumor samples. Last but not least, I would like to warmly thank to my husband, parents, sisters and all beloved ones for their great support, patience and love throughout my studies. Thank you! This research was supported by the project No. 15-34621A from the Ministry of Healthcare of the Czech Republic, by the project Translation Medicine No. LQ1605 from the National Program of Sustainability II (MEYS CR) and by the project “Podpora výzkumné činnosti studentů molekulární biologie a genetiky” No. 5 (MUNI/A/0877/2016), 6 (MUNI/A/0824/2017), and 7 (MUNI/A/0958/2018). Declaration Hereby I declare that I worked on this Ph.D. dissertation on my own under the supervision of RNDr. Petr Chlapek, DiS., Ph.D., and I used only primary and secondary literature, which is properly cited and listed in the References. Brno 16.7.2019 …………………. Viera Dobrotková © Viera Dobrotková, Masaryk University, 2019 Publications related to thesis and author´s contribution disclosure Ph.D. thesis is based on 4 manuscripts on which the author has a share (Viera Dobrotková, VD; maiden name Sláviková). Manuscript 1 Dobrotkova, V., Chlapek, P., Jezova, M., Adamkova, K., Mazanek, P., Sterba, J., Veselska, R., 2019. Prediction of neuroblastoma cell response to treatment with natural or synthetic retinoids using selected protein biomarkers. PLOS ONE 14, e0218269. VD realized most of the experiments, prepared the original draft and finalized the text after coauthorsʼ comments. Manuscript 2 Dobrotkova, V., Chlapek, P., Mazanek, P., Sterba, J., Veselska, R., 2018. Traffic lights for retinoids in oncology: molecular markers of retinoid resistance and sensitivity and their use in the management of cancer differentiation therapy. BMC Cancer 18, 1059. VD conceived and composed the review and finalized the text after coauthorsʼ comments. Manuscript 3 Chlapek, P., Slavikova, V., Mazanek, P., Sterba, J., Veselska, R., 2018. Why Differentiation Therapy Sometimes Fails: Molecular Mechanisms of Resistance to Retinoids. Int J Mol Sci 19. VD citically edited and commented the draft version of this manuscript. Manuscript 4 (In review) Veselska, R., Jezova, M., Kyr, M., Mazanek, P., Chlapek, P., Dobrotkova, V., Sterba, J., 2019. Comparative analysis of putative prognostic and predictive markers in neuroblastomas: High expression of PBX1 is associated with a poor response to induction therapy. Manuscript in review. VD participated in data analyses and manuscript preparation. Contents List of abbreviations .......................................................................................................... 10 1 Introduction .................................................................................................................... 13 2 Background ..................................................................................................................... 14 2.1 Neuroblastoma ........................................................................................................... 14 2.1.1 Diagnosis ............................................................................................................ 17 2.1.2 Etiology and genetic predispositions .................................................................. 17 2.1.3 Staging and risk stratification ............................................................................. 20 2.1.4 Treatment ............................................................................................................ 24 2.2 Differentiation therapy .............................................................................................. 27 2.2.1 Retinoids ............................................................................................................. 28 2.2.2 Mechanisms of resistance to retinoids ................................................................ 33 2.2.3 Downstream protein markers of retinoid resistance ........................................... 37 3 Objectives ........................................................................................................................ 41 4 Materials and methods ................................................................................................... 42 4.1 Tumor samples and derivation of cell lines ............................................................... 42 4.2 Chemicals .................................................................................................................. 45 4.3 MTT assay ................................................................................................................. 45 4.4 RT-PCR ..................................................................................................................... 46 4.5 Immunoblotting ......................................................................................................... 48 4.6 Retinoid treatment ..................................................................................................... 49 5 Results .............................................................................................................................. 51 5.1 Effect of retinoids on cell proliferation ....................................................................