From Harmony to Dissonance: An organoid-based study of chromosomal instability and aneuploidy in colorectal cancer progression

Abstract

The development of colorectal cancer (CRC) is associated with the accumulation of genetic mutations that provide cells with malignant properties. Cancer cells carry defects in genome surveillance mechanisms that lead to genetic instability, providing increased chance to generate the mutations needed for transformation. One of the main manifestations of genetic instability is aneuploidy, a karyotype that is not a multiple of the haploid set. Elevated chromosome missegregation during mitosis underlie the accumulation of aneuploidy in CRC, and this reduced mitotic fidelity gives rise to a specific from of genetic instability known as chromosomal instability (CIN). This thesis encompasses a series of studies that aim to understand the causes and consequences of CIN in CRC development. In chapter 2 we provide an in-depth discussion concerning current literature on aneuploidy and CIN in cancer biology. We discuss how aneuploidy and CIN are fundamentally different traits and how each differently influence cancer biology. Therefore, we emphasize the need to study CIN and aneuploidy individually and to that end CIN should be based on the measurement of chromosome missegregation itself. We finally provide a theoretical framework to hypothesize how aggravating CIN might be a therapeutically exploitable option in treating CIN tumors. Chapter 3 describes the first study that employs the CRISPR/Cas9 technique in combination with organoid cultures. We combine both techniques to develop an artificial CRC progression model by introducing the most commonly mutated genes in CRC that are associated with the adenoma-to-carcinoma sequence. Studying the resulting set of genetically engineered tumor progression organoids confirmed that mutation of all four genes is needed for full malignant transformation. Based on time-lapse imaging of the progression set, we furthermore find that loss of p53 induces CIN in colon organoids. We continue to use the set of progression organoids in chapter 4 to decipher the role of p53 in preventing chromosome segregation errors. We find that loss of p53 renders cells dependent on p38 activity to prevent a specific form of mitotic error that we term ‘bulky anaphase bridges’ (BABs). BABs are associated with DNA-damage and are dependent on active DNA-damage signaling in G2. Finally, we show that p53 deficient, CRC patient derived organoids depend on active p38 to prevent excessive CIN as well. This study therefore reveals previously unanticipated roles for p53 and p38 in preventing genetic instability. Furthermore, the interaction between p53 and p38 might provide therapeutic opportunities. The study described in chapter 5 is the first to assess chromosome segregation errors over the course of tumor progression. Assessment of CIN in organoids derived from benign, pre-malignant and malignant tissues from mice and men revealed that CIN is associated with malignant transformation. CIN levels do not increase from primary tumor to metastasis and do not correlate with aneuploidy scores. In chapter 6 we address the question whether each chromosome has an equal chance to missegregate. We find that a more peripheral location in the interphase nucleus correlates with increased missegregation proportions, revealing a previously unanticipated relation between interphase nuclear organization and mitotic behavior.