University of Groningen
The consequences of aneuploidy and chromosome instability
Schukken, Klaske Marijke
DOI:
10.33612/diss.135392967
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Publication date: 2020
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Schukken, K. M. (2020). The consequences of aneuploidy and chromosome instability: Survival, cell death and cancer. University of Groningen. https://doi.org/10.33612/diss.135392967
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1
1
Chapter 1
General introduction
and chapter outlines
1
General Introduction and Chapter Outlines
8 CHAPTER 1 General Introduction Chromosomal Instability (CIN) is the process that causes cells to change their chromosome number or structure over time. The process of CIN can lead to aneuploidy, which is defined as the presence of extra or missing chromosomes in a cell. While related, CIN and aneuploidy are different concepts1, as CIN is an ongoing process and aneuploidy is a state. To better understand the differences between CIN and aneuploidy, imagine a car factory. This factory uses an instruction manual to create cars. However, if there is a faulty copy machine which produces copies of manuals with extra or missing pages, the resulting car will also be affected; there may be cars with extra wheels, or missing screws, with extra engines or missing doors. Some of these cars might still be able to deal with their alterations and drive on the road, but many others will not. In this scenario, the bad copy machine which creates altered copies of the manual is CIN, while the cars with extra or missing components are the aneuploid cells. CIN and aneuploidy are generally detrimental to cell growth1–3, and almost always cause lethality in embryonic development4–6. Despite this, 3 out of 4
cancers are aneuploid7,8, CIN is considered a hallmark of cancer9,10, and
patients with aneuploid cancers have a lower survival rate1,11–13.This
seeming paradox can be better understood by carefully examining the circumstances surrounding cell death or cell survival as a result of the CIN phenotype. The fate of CIN and aneuploid cells are strongly influenced by cell type14,15, grade and type of CIN14,16, the resulting aneuploid
karyotype7,17, mutational background14,18–21, the immune system22,23 and
whether cells reside in vivo vs in vitro 14,15,24,25. Part of the reason that CIN cancers are so difficult to target, is because CIN is an ongoing source of change that allows for selection and evolution9,26. A single CIN tumor can be made up of several different aneuploid clones27, which facilitates resistance to cancer therapies26,28, and tumor recursion29. Furthermore, CIN has been linked to chronic inflammation and metastasis22,30. One potential method to target CIN cancers is by increasing the rate of chromosome mis‐segregation to a level beyond what can be tolerated by the cell population16,31,32. However, the level of CIN necessary to kill cancer cells in vivo is not known, as there are no models to properly measure CIN in living tissues. Furthermore, CIN tolerance is highly dependent on the tissue type15,18, further complicating the efforts to target CIN in vivo. The aim of this thesis is to further explore the consequences of CIN and aneuploidy, so as to better understand their role in cancer progression and aid in the creation of cancer therapies targeting CIN and aneuploidy. We do so by reviewing the differences between the two, showing that CIN and aneuploid cells are vulnerable to different drugs, reporting on the engineering of two novel mouse models to observe the occurrence and fate of either CIN or aneuploid cells in vivo, and further exploring the tissue specific consequences of CIN. Thesis Outline
In Chapter 2 the consequences of CIN and aneuploidy are reviewed1. While
similar, CIN and aneuploidy are different concepts with different consequences for the cell. This chapter further discusses the seeming paradox of how aneuploidy and CIN are detrimental to cell growth and function1–3, yet present in the majority of cancers7,8. Here we look at the
effects of aneuploidy and the effects of CIN separately, and review how CIN and aneuploidy are assessed in the field. Aneuploidy can be measured in dead cells, making it easier to assess in cell culture and tissues. CIN, on the other hand, can only be observed in actively proliferating cells, making it much more difficult to examine in vivo with the mouse models currently available. In Chapter 3 we set up a screen in an effort to identify drugs which selectively target CIN or aneuploid cells. A cohort of drug‐like molecules which were either already being used in the clinic or are being evaluated in clinical trials were applied to both control and stable aneuploid drugs. Cells were grown and growth curves were analyzed relative to their cell line controls to find drugs which selectively inhibited aneuploid cells. Then a similar screen was set up with CIN cells; CIN was induced in otherwise stable cells by knocking down Mad2, an essential part of the Spindle Assembly Checkpoint (SAC). We found one drug that selectively inhibited aneuploid cells, and two drugs that selectively inhibited CIN cells, and followed up on one of the CIN‐targeting drugs, Bosutinib. This drug was found to be a Src inhibitor33,34 which deregulated the spindle network and
GENERAL INTRODUCTION AND CHAPTER OUTLINES 9
1
General Introduction Chromosomal Instability (CIN) is the process that causes cells to change their chromosome number or structure over time. The process of CIN can lead to aneuploidy, which is defined as the presence of extra or missing chromosomes in a cell. While related, CIN and aneuploidy are different concepts1, as CIN is an ongoing process and aneuploidy is a state. To better understand the differences between CIN and aneuploidy, imagine a car factory. This factory uses an instruction manual to create cars. However, if there is a faulty copy machine which produces copies of manuals with extra or missing pages, the resulting car will also be affected; there may be cars with extra wheels, or missing screws, with extra engines or missing doors. Some of these cars might still be able to deal with their alterations and drive on the road, but many others will not. In this scenario, the bad copy machine which creates altered copies of the manual is CIN, while the cars with extra or missing components are the aneuploid cells. CIN and aneuploidy are generally detrimental to cell growth1–3, and almost always cause lethality in embryonic development4–6. Despite this, 3 out of 4cancers are aneuploid7,8, CIN is considered a hallmark of cancer9,10, and
patients with aneuploid cancers have a lower survival rate1,11–13.This
seeming paradox can be better understood by carefully examining the circumstances surrounding cell death or cell survival as a result of the CIN phenotype. The fate of CIN and aneuploid cells are strongly influenced by cell type14,15, grade and type of CIN14,16, the resulting aneuploid
karyotype7,17, mutational background14,18–21, the immune system22,23 and
whether cells reside in vivo vs in vitro 14,15,24,25. Part of the reason that CIN cancers are so difficult to target, is because CIN is an ongoing source of change that allows for selection and evolution9,26. A single CIN tumor can be made up of several different aneuploid clones27, which facilitates resistance to cancer therapies26,28, and tumor recursion29. Furthermore, CIN has been linked to chronic inflammation and metastasis22,30. One potential method to target CIN cancers is by increasing the rate of chromosome mis‐segregation to a level beyond what can be tolerated by the cell population16,31,32. However, the level of CIN necessary to kill cancer cells in vivo is not known, as there are no models to properly measure CIN in living tissues. Furthermore, CIN tolerance is highly dependent on the tissue type15,18, further complicating the efforts to target CIN in vivo. The aim of this thesis is to further explore the consequences of CIN and aneuploidy, so as to better understand their role in cancer progression and aid in the creation of cancer therapies targeting CIN and aneuploidy. We do so by reviewing the differences between the two, showing that CIN and aneuploid cells are vulnerable to different drugs, reporting on the engineering of two novel mouse models to observe the occurrence and fate of either CIN or aneuploid cells in vivo, and further exploring the tissue specific consequences of CIN. Thesis Outline
In Chapter 2 the consequences of CIN and aneuploidy are reviewed1. While
similar, CIN and aneuploidy are different concepts with different consequences for the cell. This chapter further discusses the seeming paradox of how aneuploidy and CIN are detrimental to cell growth and function1–3, yet present in the majority of cancers7,8. Here we look at the
effects of aneuploidy and the effects of CIN separately, and review how CIN and aneuploidy are assessed in the field. Aneuploidy can be measured in dead cells, making it easier to assess in cell culture and tissues. CIN, on the other hand, can only be observed in actively proliferating cells, making it much more difficult to examine in vivo with the mouse models currently available. In Chapter 3 we set up a screen in an effort to identify drugs which selectively target CIN or aneuploid cells. A cohort of drug‐like molecules which were either already being used in the clinic or are being evaluated in clinical trials were applied to both control and stable aneuploid drugs. Cells were grown and growth curves were analyzed relative to their cell line controls to find drugs which selectively inhibited aneuploid cells. Then a similar screen was set up with CIN cells; CIN was induced in otherwise stable cells by knocking down Mad2, an essential part of the Spindle Assembly Checkpoint (SAC). We found one drug that selectively inhibited aneuploid cells, and two drugs that selectively inhibited CIN cells, and followed up on one of the CIN‐targeting drugs, Bosutinib. This drug was found to be a Src inhibitor33,34 which deregulated the spindle network and
10 CHAPTER 1 significantly increased CIN in SAC deficient cells. We found that this synergistic toxicity is the result of increased CIN beyond a tolerable level and may be a viable method to target CIN cancer cells in the future. Chapter 4 covers the creation and initial validation experiments of a novel mouse model called the “CIN tracker”. This mouse model allows for inducible, tissue specific expression of three fluorophores to label key mitotic processes: H2B‐GFP to label the chromatin, CenpB‐mCherry to label kinetochores and mTurquoise2‐αTubulin to label the mitotic spindle, respectively. Indeed, we succeeded in imaging a live mouse after inducing fluorescence and observed H2B and tubulin expression within the epidermis. This new mouse model will allow us to observe chromosome segregation and mis‐segregation in vivo, and image cells over time to monitor their fate after mis‐segregation in vivo. The development of another mouse model, the “AneuTracker mouse”, is described in Chapter 5. While the CIN tracker allows us to view chromosome mis‐segregation during mitosis in vivo, AneuTracker mice allow us to image the copy number state of one targeted chromosome in interphase and immediately assess the copy number state of this AneuTracker chromosome. To engineer this mouse model, we investigated two techniques used to fluorescently label chromosome loci: Tetracycline Repetitive Element (TRE) repeats combined with fluorescent tetR35, or endogenous genomic repeats targeted by fluorescent dCas936,37. After comparing the two techniques, we found that the TRE/tetR approach was most feasible and therefore next engineered the AneuTracker mouse taking this approach. We found that Mouse Embryonic Fibroblasts (MEFs) made from these mice show clear foci within the nucleus when transduced with fluorescent binding proteins. In the future, this aneuploid model can be used to observe aneuploidy in living tissues, and track the fate of aneuploid cells over time. We further investigate the consequences of CIN in vivo in Chapter 6. We knockout the SAC to induce CIN in mammary tissue and find that this has no effect on overall mouse survival. This is in accordance with previous studies showing that some tissues can tolerate ongoing CIN long term15,38. Intriguingly, we find that the consequences of CIN in a tumor prone background are highly dependent upon the type of CIN induction, as different mutations can either accelerate or have no significant effect on tumorigenesis. Next, we look at the consequences of inducing high grade CIN by knocking out the SAC systemically in adult mouse. We find that systemic inactivation of the SAC is acutely lethal to adult mice. While several tissues were unaffected by four days of SAC alleviation, the intestinal villi showed severe degradation leading to significant and acute weight loss in the mice, presumably because this is one of the tissues with the highest turnover of cells. We therefore conclude that the response to CIN can vary dramatically per tissue type. Finally, Chapter 7 summarizes and discusses the major findings of this work and addresses the future research directions and potential applications of our findings.
GENERAL INTRODUCTION AND CHAPTER OUTLINES 11
