Functions of the C/EBPβ isoforms in breast cancer
Sterken, Britt
DOI:
10.33612/diss.172465560
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date: 2021
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Sterken, B. (2021). Functions of the C/EBPβ isoforms in breast cancer. University of Groningen. https://doi.org/10.33612/diss.172465560
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
Appendix A: Scientific summary
Breast cancer is the most commonly diagnosed type of cancer in women and the survival outcome is strongly dependent on the stage of breast cancer at diagnosis. Therefore, it is essential to identify oncogenic factors that contribute to breast cancer development, metastasis and treatment resistance. The transcription factor C/EBPβ is known to regulate cell proliferation and differentiation in a variety of tissues. The transactivation capacity of C/EBPβ is largely determined by the ratio between the isoforms C/EBPβ-LAP and C/EBPβ-LIP. Previous studies have shown that C/EBPβ-LIP-depleted mice display improved metabolic health, a prolonged lifespan, and reduced cancer incidence. Other studies have found high expression of LIP in grade III, Estrogen receptor (ER) and Progesterone (PR) receptor negative human breast cancer, and have linked high LIP expression with increased mammary epithelial proliferation. We find that cell lines derived from triple-negative breast cancer (TNBC, lacking the Estrogen and Progesterone receptors and HER2 expression) have a very high LIP/LAP ratio. Reducing the LIP/LAP ratio by exogenous expression of LAP in TNBC cell lines reduces migration and invasion of the breast cancer cells. Moreover, the overexpression of LIP promotes cell migration of untransformed mammary epithelial cells. Transcriptomics data using TNBC cells with CEBPB knockout show a regulation of migration and extracellular matrix-related genes. Our data propose a role for the LIP/LAP ratio in the regulation of breast cancer cell migration and ECM remodelling, two key characteristics that are associated with the aggressive phenotype of TNBC cells. To test the C/EBPβ-isoform specific functions in vivo, we generate p53-deficient genetically engineered breast cancer mouse models with exogenous expression of LIP and LAP. CAG-promoter driven expression of C/EBPβ-LIP or C/EBPβ-LAP from the Col1a1-locus is epigenetically silenced, and natural variation in LIP/LAP in p53-deficient tumours does not correlate with tumour latency. Lastly, to investigate the role of the C/EBPα-isoforms in tumourigenesis, metabolism, and longevity, we create a mouse model deficient for C/EBPα-p30 by mutation of the C/EBPα-uORF. With these studies we contribute to the understanding of how the C/EBPα and C/EBPβ transcription factor isoforms affect oncogenesis and lifespan.
Appedix B: Wetenschappelijke samenvatting
Bostkanker is de meest gediagnosticeerde vorm van kanker bij vrouwen, waarbij de overlevingskans sterk afhankelijk is van het stadium waarin de kanker zich bevindt. Met name het moleculaire subtype borstkanker genaamd “triple-negatief”, waarbij de hormoonreceptoren voor oestrogeen en progesteron en de human epidermal growth factor receptor 2 afwezig zijn, is zeer agressief en kan vaak niet effectief behandeld worden met doelgerichte therapieën. Het identificeren van oncogene factoren die bijdragen aan de ontwikkeling en metastase van dit subtype borstkanker is derhalve essentieel. De transcriptiefactor C/EBPβ is een bekende regulator van mammaklierontwikkeling en beïnvloedt de celdeling in en differentiatie van verschillende weefsels. De transactivatiecapaciteit van C/EBPβ wordt grotendeels bepaald door de ratio tussen de twee isovormen C/EBPβ-LAP en C/EBPβ-LIP. Voorgaande studies hebben laten zien dat muizen met een verlaging van deze LIP/LAP-ratio een verbeterde metabole gezondheid, een verlengde levensduur en een verlaagde kankerincidentie hebben. De overexpressie van LIP in borstepitheelcellen in de muis leidt tot hyperplasie- en in sommige gevallen neoplasie in de mammaklieren. Borstkankerweefsel van graad III hormoonreceptor-negatieve tumoren laat een verhoogde expressie van LIP ten opzichte van LAP zien. De functies van LIP en LAP in borstkankerontwikkeling blijven evenwel tot dusver grotendeels onbekend. In dit proefschrift laten we zien dat cellijnen afgeleid van triple-negatieve borstkanker een relatief hoge LIP/LAP-ratio hebben. De exogene expressie van LAP leidt tot een verminderde migratie en invasie van borstkankercellen. Transcriptoomdata laten zien dat C/EBPβ verschillende genen reguleert die betrokken zijn bij de celmigratie en de extracellulaire matrix en waarschijnlijk bijdraagt aan een “partial EMT”. We laten zien dat LAP de transcriptie van extracellulaire matrix genen stimuleert. Onze studie suggereert dat beide LIP en LAP een rol in borstkankerwikkeling hebben en mogelijk de metastase bevorderen. Om dit te bestuderen in vivo maken we p53-deficiënte borstkanker muizenmodellen met exogene LIP- of LAP-expressie. CAG-promotor-gedreven expressie van LIP en LAP wordt door DNA-methylatie verhinderd. LIP en LAP komen allebei tot expressie in alle p53- deficiënte
mammatumoren; echter correleert de LIP/LAP-ratio en niet met de latentietijd. Tot slot maken we een muizenmodel zonder expressie van de korte isovorm van de verwante factor C/EBPα (C/EBPα-p30) om de invloed op metabolisme, veroudering en kanker te bestuderen. Met deze studies dragen we bij aan de kennis rondom de functies van de C/EBPα-en C/EBPβ-isovormen in veroudering en het ontstaan van kanker.
Appendix C: Lay summaries
English
Breast cancer is the most commonly diagnosed cancer type in women, with its survival outcome being strongly dependent on the stage the breast cancer at the time of diagnosis. Therefore, the identification of oncogenic factors contributing to breast cancer development, metastasis and treatment resistance is urgent. C/EBPβ is a factor that binds to DNA and thereby regulates the expression of genes. C/EBPβ comes in two different forms called LAP and C/EBPβ-LIP that have opposing roles. Previous studies have shown that a balance between LIP and LAP is essential for the general maintenance of organs in the body, and that elimination of LIP in the body results in increased health- and lifespan and reduced tumourigenesis. High expression of LIP has been associated with increased proliferation of cells in the breast and elevated levels of LIP have been found in an aggressive subtype of breast cancer. In this study, we show that the LIP/LAP ratio is very high in tumour cells that lack the expression of hormone receptors, which are more difficult to treat because existing targeted therapies are usually not effective. Overexpression of LAP in those tumour cells inhibits their migration and invasion, whereas overexpression of LIP in normal (non-cancerous) breast cells increases their migration. These results indicate that LIP might promote breast cancer metastasis and LAP might counteract metastasis. To test this in a body, we generate breast cancer mouse models that have high expression of LIP or LAP in the tumours. However, the overexpression of LIP and LAP in the tumours is silenced. The natural expression of LIP and LAP is consistently present in the mammary tumours, but the LIP/LAP ratio does not correlate to tumour latency. Therefore, different genetic mouse models should be generated to study if and how LIP and LAP contribute to breast cancer development and metastasis.
Nederlands
Bostkanker is de meest gediagnosticeerde vorm van kanker bij vrouwen, waarbij de overlevingskans sterk afhankelijk is van het stadium waarin de kanker zich bevindt. Om deze reden is het identificeren van oncogene factoren die bijdragen aan de ontwikkeling en het uitzaaien van borstkanker essentieel. C/EBPβ is een factor die bindt aan DNA en de expressie van genen reguleert. C/EBPβ komt voor in twee verschillende vormen: C/EBPβ-LAP en C/EBPβ-LIP, die functionele tegenhangers van elkaar zijn. Voorgaande studies hebben laten zien dat een balans tussen LIP en LAP belangrijk is voor het normaal functioneren van het lichaam en dat genetische eliminatie van LIP leidt tot een verbeterde gezondheid, een verlengde levensduur en minder kanker bij muizen. Daarnaast is een verhoogde LIP expressie gevonden in een agressief subtype borstkanker. In dit proefschrift laten we zien dat triple negatieve borstkankercellen een hoge LIP/LAP-ratio hebben. Dit is met name interessant omdat de meeste doelgerichte therapieën niet effectief zijn voor de behandeling van dit subtype borstkanker. We laten zien dat het inbrengen van extra LAP in de borstkankercellen zorgt voor een verminderde invasie/verspreiding van deze cellen, en het inbrengen van LIP in normale borstcellen de migratie/verspreiding van de cellen versnelt. Deze experimenten suggereren dat LIP uitzaaiingen bevordert, en dat LAP deze vermindert. Om dit te onderzoeken maken wij muizenmodellen met borstkanker, waarin selectief LIP of LAP in de kankercellen wordt verhoogd om op deze manier de functies van LIP en LAP in borstkanker en uitzaaiingen te bestuderen. De extra LIP of LAP expressie in tumoren wordt echter onderdrukt in deze modellen. LIP en LAP zijn wel beide consistent aanwezig in de mammatumoren, maar deze LIP/LAP-ratio in de tumoren correleert niet met de tumorlatentie bij muizen. Vervolgexperimenten met genetische borstkankermodellen en veranderde LIP/LAP-ratios zijn nodig om de functies van LIP en LAP in borstkanker en uitzaaiingen te bestuderen.
Appendix D: Acknowledgements
This study has been carried out at the European Research Institute for the Biology of Ageing (ERIBA, UMCG, Rijksuniversiteit Groningen) and additionally funded by the Dutch Cancer Society/ Koningin Wilhelmina Fonds voor de Nederlandse Kankerbestrijding (KWF), De Stichting De Cock-Hadders and the Graduate School for Medical Sciences (GSMS). Therefore, I would like to thank all involved funding bodies to allow this research project to develop.
I would like to express my gratitude towards my supervisor Cor Calkhoven. Cor has given me the opportunity to do a bachelor project in his lab, after which he hired me as doctoral candidate in 2016. The relaxed atmosphere is characteristic of the Calkhoven lab which is driven by the calm and solution-oriented character of Cor and the selection of smart, funny and relaxed people he hires. Cor, thanks for the fantastic time in your lab and all help over the past 4.5 years!
A special thanks to members of my PhD committee Marco Demaria and Folkert Kuipers for the yearly feedback on my projects. Marco, our meetings and your critical questions have helped me greatly to develop my project. Folkert, thank you for the project related discussions, tips for structuring the project and occasional brainstorm sessions.
In addition, I would like to thank further members of the reading committee Helen Morisson and Floris Foijer. Floris, thank you for always showing curiosity in the project and suggestions.
I would like to thank Bart van der Sluis and Jan van Deursen for help with the generation of the C/EBPα mouse.
A huge thanks to my fellow lab members Christine, Gertrud, Hidde and Josi. Christine – your critical eye has greatly helped me with fine-tuning presentations and writing the thesis. Gertrud, your experimental support has greatly helped my project especially during the last year. From RNA-seq library prep to invasion assays, you arranged it! Hidde, thank you for all the discussions we had over the past 3 years. You were always there to help, give feedback, and think along and
that has been very valuable to me and I think also everyone else in the lab. Josi, thank you for your scientific feedback during my final year, and also the many science-unrelated discussions in the office have brought me many very welcomed breaks in between the lab work! Finally, I would like to thank Amr for all the fun and conversations we had in the office, and the help at the beginning of my PhD. This brings me to members of the ground floor, Anita, Mandy, Wytse, Leen, Renee, Lale, Ellen, Esther, Ale, Simone, Boshi, Jas, Nynke, Jamil, Michaela, Eleni, Willem, Sebastian, Thijmen and Martha. I would like to thank all members for creating the great environment at the ERIBA ground floor. Our coffee corner breaks have entertained me so much over the past 4.5 years and have facilitated many pubquiz nights and occasional karaoke nights. Apart from these social aspects, I would like to highlight the ERIBA ground floor spirit of fitness which has contributed to my PhD because - healthy mind, healthy body they say! I would like to express a special thanks to Ale, for keeping me fit at 7 am at the Trainmore or the swimming pool, and also for introducing me to analysis of RNA-seq data in R. Also a special thanks to members of the Ripped Six Pack app group for the group effort for ripped abs. A very special thanks to Anita, Wytse, Mandy, Renee, Leen, Josi and Nynke for our great running trips. From Groningen to Stockholm and 8ks to marathons, the running spirit and support were always there to compete in some fantastic races! And a special thanks to you Jas, for keeping an eye out for a new job for me in Scotland!
I would like to express my gratitude to the Jonkers lab at the NKI. In particular Jos, for a great collaboration and hospitality in your lab. In addition I would like to thank Peter, for great discussions and great feedback on the project. I would like to thank Anne Paulien, Eline en Timo for all mouse-related work, teaching me how to work with mice and the nice conversations we had in the mouse house! I would also like to thank Koen, Lisette and Millie in particular, for their elaborate help and feedback with my project. Next, I would like to thank the group for their warm welcome. Outside of the lab I have greatly enjoyed the borrels, parties and even some running events! Unfortunately the mouse models did not work as hoped but I’ve had such a great time in the lab!
Naast het onderzoek was er natuurlijk ook tijd nodig voor ontspanning en daarom wil ik graag mijn familie bedanken, Oma, Arjan, Papa, Mama, Max, Bo Fay en Wolf. De ultieme ontspanning blijft een biertje/aperolletje op de veranda of een hardlooprondje door het bos! Max, Bo, Fay en Wolf – dank voor de vele potjes Cards Against Humanity, filmavonden en boswandelingen met de kerel. Daarnaast wil ik speciaal mijn ouders bedanken – jullie mentale stimulatie vanaf jonge leeftijd heeft mij gedreven om dit punt te bereiken. Jullie werkhouding en enthousiasme voor het verkrijgen, uitwisselen en overdragen van kennis heeft mij geïnspireerd om hetzelfde te willen doen (desalniettemin in een iets ander vakgebied ;)).
Last I would like to thank Tobias. Apart from the great scientific help with my PhD project you have always been by my side to support me. I know you in the lab as a driven, knowledgeable scientist who asks exactly the right questions and always helps everyone around him and you keep inspiring me to do the same. Outside of the lab, life is so much more fun with you and I look forward to the years ahead!
Britt Amber Sterken
Date of birth: 19-01-1992, Groningen, The Netherlands Cancer Research UK Beatson Institute, United Kingdom
• Postdoctoral scientist in the lab of Professor Sara Zanivan • Breast cancer, cancer-associated fibroblasts, epigenetics University of Groningen, The Netherlands
• PhD student in the lab of Professor Cor Calkhoven • Gene regulation, breast cancer, cell migration University of Edinburgh, United Kingdom
• MSc by Research Biomedical Sciences (Life Sciences)
• Pre-implantation embryology and reprogramming, reproductive biology
University of Groningen, The Netherlands • BSc Pharmacy
• BSc Honours College
Manuscripts
Sterken, B.A., Ackermann, T., Zuidhof, H.R., Kortman J.G.A., Broekhuis, M., Müller, C., … Calkhoven, C.F. C/EBPβ isoform-specific regulation of migration and invasion in triple-negative breast cancer cells. (In revision at npj Breast Cancer)
Ackermann, T., Hartleben, G., Müller, C., Mastrobuoni, G., Groth, M., Sterken, B. A., … Calkhoven, C. F. (2019). C/EBPβ-LIP induces cancer-type metabolic reprogramming by regulating the let-7/LIN28B circuit in mice. Communications Biology, 2(1), 208. https://doi.org/10.1038/s42003-019-0461-z
