The role of Nanos3 in tumor progression Evi De Keuckelaere Thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Science: Biochemistry and Biotechnology 2017-2018 Promoters: Prof. Dr. em. Frans Van Roy and Prof. Dr. Geert Berx The research described in this doctoral thesis was performed at the Department of Biomedical Molecular Biology, Faculty of Sciences, Ghent University, and at the Inflammation Research Center (IRC), Flanders Institute for Biotechnology (VIB). Evi De Keuckelaere was supported by a personal fellowship of the Research Foundation Flanders (FWO-Vlaanderen). No part of this thesis may be reproduced or used in any way without prior written permission of the author. © Evi De Keuckelaere 2018 The role of Nanos3 in tumor progression By Evi De Keuckelaere Molecular and Cellular Oncology Lab Department of Biomedical Molecular Biology, Ghent University Cancer Research Institute Ghent (CRIG) Inflammation Research Center, Flanders Institute for Biotechnology (VIB) Technologiepark 927 B-9052 Gent-Zwijnaarde, Belgium Academic year: 2017-2018 Promoters: Prof. Dr. em. Frans Van Roy1, 2 Prof. Dr. Geert Berx1, 3 Examination committee: Chair: Prof. Dr. Peter Brouckaert1 Secretary: Prof. Dr. Wim Declercq1, 2 Examination board Prof. Dr. Kris Vleminckx1, 3, 4 Prof. Dr. Philippe Birembaut5, 6 Prof. Dr. Pieter Mestdagh7 Prof. Dr. Nadine Van Roy7 1 Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium 2 VIB Inflammation Research Center, Ghent University, Ghent, Belgium 3 Cancer Research Institute Ghent, Ghent University, Belgium 4 Center for Medical Genetics, Ghent University, Ghent, Belgium 5 INSERM UMR-S 903, SFR CAP-Santé, University of Reims Champagne-Ardenne, France 6 Laboratory of Histology, CHU Reims, France 7 Department of Pediatrics and Medical Genetics, Ghent University, Ghent, Belgium SUMMARY SUMMARY Nanos genes encode RNA-binding proteins (RBPs) that form a post-transcriptional repressor complex together with a Pumilio protein. This complex regulates transcript stability and translation upon binding Nanos regulatory/response elements (NREs), Pumilio-binding elements (PBEs), or both in the 3’UTR of mRNA targets. Nanos proteins belong to a highly conserved protein family found in both vertebrates and invertebrates. Since a general overview of the extent of this conservation was missing, we created a phylogenetic tree based on the C-terminal zinc finger domain, which is the only conserved domain among all nanos proteins. The normal physiologic expression of nanos proteins is mainly restricted to the gonads and the brain. As such, they have generally been studied with regard to their role in germ cell development. However, in humans, Nanos1 and Nanos3 are both upregulated in non-small cell lung cancers (NSCLCs). In NSCLC cell lines, overexpression and silencing of Nanos3 are linked to epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET), respectively. This occurs partly by negatively influencing E-cadherin transcription and stimulating vimentin expression. In this project, we investigated the expression of Nanos3 in several human cancer cell lines and we made use of NSCLC cell lines with and without Nanos3 overexpression to identify new mRNA targets. To examine the contribution of Nanos3 to tumor progression in vivo we generated a conditional Nanos3-expressing mouse, which was crossed with mice from established lung cancer mouse models and a prostate cancer mouse model. Prostate cancer was chosen since preliminary data in our group demonstrated a correlation between Nanos3 expression levels and tumor aggressiveness in human prostate cancer tissues. Both lung cancer and prostate cancer are frequently diagnosed cancers that are responsible for around 23% of the cancer deaths worldwide. Any research that focuses on better understanding the tumorigenesis of these cancers can be relevant in the search for better treatments for these cancers. In both LSL-KRasG12D;p53fl/fl;CCSP-rtTA+/-;TetO-Cre+/- and LSL-KRasG12D;CCSP-rtTA+/-;TetO-Cre+/- NSCLC models, after doxycycline induction, mice with ectopic Nanos3 expression (Nanos3LSL/- I SUMMARY ;LSL-KRasG12D;p53fl/fl;CCSP-rtTA+/-;TetO-Cre+/- and Nanos3LSL/-;LSL-KRasG12D;CCSP-rtTA+/-;TetO- Cre+/-) died earlier than the control mice (LSL-KRasG12D;p53fl/fl;CCSP-rtTA+/-;TetO-Cre+/- and LSL-KRasG12D;CCSP-rtTA+/-;TetO-Cre+/-). Mice from the LSL-KRasG12D;p53fl/fl;CCSP-rtTA+/-;TetO- Cre+/- NSCLC mouse model were used to further investigate this. The only notable difference between the lungs of both genotypes seemed to be the enhanced bronchiolar dysplasia observed in the Nanos3-expressing mice compared to the control mice. Several primary cell lines were established from the lung of a control and a Nanos3 NSCLC mouse. These were used in an allograft experiment and showed that Nanos3 expression did not seem to influence the ectopic tumor growth. Both mice, injected with either control or Nanos3-expressing lung tumor-derived (LuTD) cell lines, demonstrated lung metastases. Although lymph node metastases were observed in both mice injected with control or Nanos3-expressing LuTD cell lines, these metastases were bigger and differentiated in mice injected with the latter. Prostate-specific ectopic Nanos3 expression did not appear to effect the survival of Hi-Myc transgenic mice, as comparable prostate cancer progression was observed in Hi-Myc control mice and Nanos3-expressing mice. To further look into the mechanisms and possible pathways involved in the function of Nanos3 we used several approaches in search of interaction partners of Nanos3, including MAPPIT, co-immunoprecipitation and proximity-dependent biotin identification (BioID). This revealed DDX1 and several Argonaute proteins as new interaction partners of Nanos3. DDX1 seems to be involved in Nanos3-mediated post-transcriptional regulation. The interaction of Nanos3 with members of the Argonaute family further establishes the link between Nanos proteins and the miRNA regulatory network. Additionally, other interesting complexes, involved in mRNA regulation, were identified in the Nanos3 proxeome. In summary, the Nanos genes constitute a highly conserved gene family with a role in germ cell development. Recently, evidence for ectopic expression of Nanos proteins in human cancer is growing. We have clearly shown that expression of Nanos3 influences the survival of NSCLC mice and uncovered a link between Nanos3 expression and lymph node metastatic propensity. Further research will be necessary to further elucidate the molecular functions II SUMMARY of Nanos proteins and the involved molecular interaction partners, and to verify whether Nanos proteins are potent promoters of tumor formation and progression. III SAMENVATTING SAMENVATTING Nanos genen coderen voor RNA-bindende eiwitten die een posttranscriptioneel repressorcomplex vormen met een Pumilio eiwit. Dit complex reguleert de stabiliteit van de mRNA sequentie en de translatie na het binden van Nanos-regulatorische elementen, Pumilio-bindende elementen, of beide in de 3’UTR van de mRNA doelwitten. Nanos eiwitten behoren tot een sterk geconserveerde eiwitfamilie die zowel in gewervelde als in ongewervelde dieren voorkomt. Gezien een algemeen overzicht van de omvang van deze conservatie nog niet gerapporteerd was, hebben we een fylogenetische boom gemaakt op basis van het C-terminale zinkvinger domein, wat tevens het enige geconserveerde domein is van alle nanos eiwitten. De normale fysiologische synthese van nanos eiwitten is voornamelijk beperkt tot de geslachtsklieren en de hersenen. Zodoende worden ze voornamelijk bestudeerd met betrekking tot hun rol in de ontwikkeling van kiemcellen. In mensen worden Nanos1 en Nanos3 echter beide opgereguleerd in niet-kleincellige longcarcinomen (NSCLCs). In cellijnen van dit soort kanker zijn overexpressie en neerregulatie van Nanos3 respectievelijk gelinkt aan epitheliale-mesenchymale transitie (EMT) en mesenchymale-epitheliale transitie (MET). Dit gebeurt gedeeltelijk door de negatieve regulatie van E-cadherine transcriptie en het stimuleren van vimentine expressie. In dit project gingen we de expressie van Nanos3 na in verschillende humane kankercellijnen en maakten we gebruik van NSCLC cellijnen met en zonder overexpressie van Nanos3 met als doel nieuwe mRNA doelwitten te identificeren. Om de bijdrage van Nanos3 na te gaan in “in vivo tumorprogressie” hebben we een conditionele Nanos3-expresserende muis gecreëerd. Deze muis werd vervolgens gekruist met muizen van gevestigde muismodellen voor longkanker en een muismodel voor prostaatkanker. Dit laatste werd gekozen aangezien voorafgaande data in onze groep een correlatie aantoonde tussen Nanos3 expressie en de kwaadaardigheid van de tumor in humane prostaatkankerweefsels. Longkanker en prostaatkanker zijn vaak gediagnosticeerde kankers en zijn wereldwijd verantwoordelijk voor ongeveer 23% van alle sterfgevallen die te wijten zijn aan kanker. Elk onderzoek dat zich richt op een beter begrip van het ontstaan en de progressie van tumoren IV SAMENVATTING van deze kankers kan relevant zijn bij het zoeken naar betere behandelingen voor deze kankers. In beide onderzochte NSCLC modellen stierven muizen met ectopische expressie van Nanos3 (Nanos3LSL/-;LSL-KRasG12D;p53fl/fl;CCSP-rtTA+/-;TetO-Cre+/- en Nanos3LSL/-;LSL-KRasG12D;CCSP- rtTA+/-;TetO-Cre+/-), na doxycycline inductie, eerder dan de controle muizen (LSL- KRasG12D;p53fl/fl;CCSP-rtTA+/-;TetO-Cre+/- en LSL-KRasG12D;CCSP-rtTA+/-;TetO-Cre+/-). Muizen van het