The Relationship Between MITF and Cell Cycle In Melanoma Lára Stefansson Ritgerð til meistaragráðu Háskóli Íslands Læknadeild Námsbraut í Líf- og læknavísindi Heilbrigðisvísindasvið 1 Samspil MITF og frumhringsins í sortuæxli Lára Stefansson Ritgerð til meistaragráðu í Líf- og læknavísindum Umsjónarkennarar: Eiríkur Steingrímsson Meistaranámsnefnd: Eiríkur Steingrímsson, Margrét Helga Ögmundsdóttir, Valerie Fock Læknadeild Námsbraut í Líf- og læknavísindum Heilbrigðisvísindasvið Háskóla Íslands Maí 2019 2 The Relationship Between MITF and Cell Cycle in Melanoma Lára Stefansson Thesis for the degree of Master of Science Supervisors: Eiríkur Steingrímsson Masters committee: Eiríkur Steingrímsson, Margrét Helga Ögmundsdóttir, Valerie Fock Faculty of Medicine Department of Biomedical Sciences School of Health Sciences May 2019 3 Ritgerð þessi er til meistaragráðu í Líf- og læknavísindum og er óheimilt að afrita ritgerðina á nokkurn hátt nema með leyfi rétthafa. © Lára Anna Stefansson 2019 Prentun: Háskólaprent Reykjavík, Ísland, 2019 4 Ágrip Umritunarþátturinn MITF er lykilprótein í þroskun og starfsemi litfruma. Sýnt hefur verið að MITF stjórnar tjáningu gena sem stjórna frumuhringnum svo sem p21 og CDK2. Í þessari ritgerð voru tengsl MITF og frumuhringsins í sortuæxlisfrumum rannsökuð auk þess sem áhrif MITF á tjáningu tiltekinna gena voru skoðuð. Sortuæxlisfrumur voru samstilltar, sýni tekin á reglulegum fresti og staða frumanna og tjáning MITF og annarra gena í frumuhringnum greind með frumuflæðisjá, Western blot greiningu og qPCR. Niðurstöðurnar sýndu að styrkur MITF próteinsins jókst þegar frumurnar voru flestar í G2/M fasa, en mRNA gildin voru óbreytt sem bendir til aukinnar próteinþýðingar MITF eða aukins stöðugleika þess á þessu stigi frumuhringsins. Nýlegar rannsóknir á rannsóknastofunni benda til að MITF stjórni tjáningu ýmissa áhugaverðra gena sem tengjast frumuhringnum. Tjáning nokkurra þessara gena var skoðuð í 501mel og A375P sortuæxlisfrumum eftir að MITF hafði verið yfirtjáð eða slegið niður í . Það að slá niður MITF hafði marktæk áhrif á tjáningu CABLES1 gensins auk þess sem tjáning CDK2 minnkaði. Yfirtjáning MITF hafði áhrif á tjáningu þeirra gena sem tekin voru fyrir, en reyndist hún ekki vera tölfræðilega marktæk. Áhrif þess að CDK2 og LZTS1 á tjáningu bæði MITF mRNA og próteins voru skoðuð með því að slá niður genin tvö með siRNA. Í ljós kom að þegar CDK2 var slegið niður varð marktæk lækkun á bæði MITF mRNA og próteini. Þegar LZTS1 var slegið niður minnkaði hins vegar tjáningin á MITF próteininu en ekki mRNA sameindinni. Þetta bendir til þess að CDK2 hafi áhrif á tjáningu MITF í afturkasti (feedback loop) en að LZTS1 hafi áhrif á þýðingu eða stöðugleika MITF próteinsins. 5 Abstract Microphthalmia-associated transcription factor (MITF) is the master regulator of melanocytes and is known to directly bind to the promoter regions of the genes encoding cell cycle regulators, such as p21 and CDK2. This thesis focuses on the relationship between MITF and the cell cycle in melanoma and tests the role of novel MITF target genes. A melanoma cell line, 501mel, was synchronized, samples were taken and analyzed via flow cytometry, Western blot and qPCR. The results showed that MITF protein levels increased when the cells were mainly in G2/M phase whereas mRNA levels were unaffected, suggesting a change in protein synthesis or stability during this stage. The relationship between MITF and several novel targets was determined using MITF knockdown and overexpression in 501mel or A375P cell lines. Knockdown of MITF showed a significant decrease on CABLES1 expression, with a trend of decreasing CDK2 expression. Overexpression of MITF did not significantly change expression of any of the cell cycle regulating genes checked, although there was a general increase of each gene checked. To determine if the cell cycle regulators CDK2 and LZTS1 affected MITF expression, these genes were knocked down with siRNA before collecting samples for Western blot and qPCR. Knockdown of CDK2 caused a significant decrease in MITF mRNA and protein levels, whereas knockdown of LZTS1 only caused a significant decrease in MITF protein levels. This suggests that CDK2 might regulate MITF transcription in a feedback loop and LZTS1 plays a role either in translation or stability of MITF. 6 Acknowledgements First and foremost, I would like to thank Eiríkur Steingrímsson for giving me the opportunity to work in his lab. It has been a great experience and I have learned a lot in my time. I could not have asked for a better supervisor. I would also like to thank Margrét Helga Ögmundsdóttir for always being positive and encouraging. Through the ups and downs, she is always supportive and it really helped when things were looking less than positive. I would like to thank the past and present members of the Steingrímsson lab, as they have all helped me in navigating my project. Thanks to Valerie Fock, Remina Dilixiati, Berglind Einarsdóttir, Sara Sigurbjörnsdóttir, Josué Ballesteros, Hilmar Gunlaugsson, Romain Lasseur, Melanie Allram, Anna Köck and Philipp Cerny. It is quite a list of people, but every single one of them helped me along the way and I would have been lost without them. I would also like to thank Marta Sól Alexdóttir and Kristrún Ýr Holm, who have supported me greatly throughout my project. The last people I would like to acknowledge are the ones that are completely responsible for my scientific career. Not many of my peers can say that their parents understand what they do; I am truly priviledged to have parents who not only understand, but can give constructive criticism. Thanks to my mom and dad for being my first lab supervisors and supporting me throughout my project. I hope you two like the thesis. 7 Table of Contents Ágrip. .5 Abstract. 6 Acknowledgements. .7 Table of Contents. 8 Figure Legend. .10 Table Legend. 11 List of Abbreviations. 12 1 Introduction. 13 1.1 Melanocytes. .13 1.2 Melanoma. .14 1.3 MITF. 15 1.4 Cell Cycle. .18 1.5 MITF and Cell Cycle Regulation. .21 2 Aims. .23 3 Materials and Methods. 24 3.1 Cell Culture. .24 3.1.1 General Maintenance. .24 3.1.2 DOX Induction of PiggyBac Cell Lines for qPCR. .24 3.1.3 siRNA Knockdown in 501mel. .24 3.2 Cell Cycle Synchronization. .25 3.2.1 Double Thymidine Block. .25 3.2.2 Double Thymidine Block with DOX Induction. .26 3.3 Flow Cytometry. 26 3.3.1 Lysing Cells using Vindelöv Solution. .26 3.3.2 Flow Cytometry Analysis. 26 3.4 Western Blotting. 28 3.4.1 Sample Collecting. .28 3.4.2 Gel Electrophoresis. 28 3.4.3 Transfer. .28 3.4.4 Blocking. 29 3.4.5 Staining. 29 3.4.6 Developing and Quantification. 29 3.5 RNA Expression Analysis using Quantitative Real-Time PCR. .29 8 3.5.1 RNA Isolation. 29 3.5.2 cDNA Synthesis. .30 3.5.3 qPCR. .30 4 Results. .32 4.1 Cell Cycle Synchronization and MITF. .32 4.1.1 Elevated Levels of MITF Correlated with G2/M phase. .32 4.1.2 Knockdown of MITF in 501mel has No Effect on Cell Cycle Progression. 35 4.1.3 Overexpression of GFP-MITF in 501mel has Little Effect on Cell Cycle Progression. 37 4.2 MITF Affects Expression of Cell Cycle Regulating Genes. 38 4.3 Knockdown of CDK2 or LZTS1 Changes Levels of MITF. .40 5 Discussion. 43 5.1 Higher MITF Levels in G2/M. .43 5.2 Potentially Novel Targets of MITF. 44 5.3 CDK2 and LZTS1 Expression Affects MITF Levels . ..