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Micronuclei-based model system reveals functional consequences of chromothripsis in human cells
(2019)
Cancer cells often harbor chromosomes in abnormal numbers and with aberrant structure. The consequences of these chromosomal aberrations are difficult to study in cancer, and therefore several model systems have been developed in recent years. We show that human cells with extra chromosome engineered via microcell-mediated chromosome transfer often gain massive chromosomal rearrangements. The rearrangements arose by chromosome shattering and rejoining as well as by replication-dependent mechanisms. We show that the isolated micronuclei lack functional lamin B1 and become prone to envelope rupture, which leads to DNA damage and aberrant replication. The presence of functional lamin B1 partly correlates with micronuclei size, suggesting that the proper assembly of nuclear envelope might be sensitive to membrane curvature. The chromosomal rearrangements in trisomic cells provide growth advantage compared to cells without rearrangements. Our model system enables to study mechanisms of massive chromosomal rearrangements of any chromosome and their consequences in human cells.
Chromosomal aberrations are manifold changes in the configuration of the DNA. Each cell in a tumor
may accumulate different karyotype changes, making it challenging to determine the causes and
consequences of this instability. Therefore, model systems have been developed in the past to
generate and study specific genome alterations. In this thesis, I present the results of my studies on
three types of chromosomal aberrations, all of which may contribute to tumor development or
progression.
Chromothripsis is a phenomenon that describes a one-off massive chromosomal disruption and
reassembly, perhaps arising via DNA damage micronuclei (MN). MN are small DNA-packed nuclear
envelopes. I tested potential causes of DNA damage in MN and found that the rupture of the MN
envelope and the entry of cytosolic fractions increase DNA damage in MN. Furthermore, I addressed
the question of what physiological consequences cell lines with an additional rearranged chromosome
have compared to those with an intact extra chromosome. Strikingly, the cells with more
rearrangements showed a functional advantage resulting in an improved fitness potential.
However, the engineering of polysomic cell lines with fully intact additional chromosomes increases
various cellular stress responses and reduces the proliferation capacity. To investigate how cancer cells
overcome the detrimental consequences of aneuploidy, I explored physiological adaptations of model
cells with a defined additional chromosome that underwent in vivo and in vitro evolution. Interestingly,
unfavorable phenotypes of aneuploid cells, such as the replication stress, were mitigated upon
evolution. Furthermore, I examined the replication on single molecule resolution, showing alteration
after evolution that might underlie the replication stress bypass or tolerance.
In contrast to these unbalanced forms of genomic aberrations, whole genome doubling (WGD) leads
to a full doubled chromosome set, which was shown to evolve into aneuploid karyotypes by
chromosomal instability (CIN), frequently by losing chromosomes. Cells that underwent WGD
accumulate DNA damage in the S phase. I performed a single molecule analysis on the DNA during the
first cell cycle after WGD to elucidate how the DNA damage arises and found that the number of active
origins is not sufficient to replicate the doubled amount of DNA in the first S phase after WGD faithfully.
This starts a genome-destabilizing cascade that eventually promotes tumorigenesis, metastasis, and
poor patient outcome.
Taken together, these studies provide insights into the causes and consequences of three types of
genomic aberrations: chromothripsis, polysomy, and WGD. However different these phenomena may
be, they share one common feature – they contribute to tumor development and progression.
Therefore, elucidating the aberrant cell functions caused by genomic aberrations contributes to a
better understanding of a cancer cell's nature and will perhaps help to find new cancer therapy targets.
Loss of USP28 and SPINT2 expression promotes cancer cell survival after whole genome doubling
(2021)
Background
Whole genome doubling is a frequent event during cancer evolution and shapes the cancer genome due to the occurrence of chromosomal instability. Yet, erroneously arising human tetraploid cells usually do not proliferate due to p53 activation that leads to CDKN1A expression, cell cycle arrest, senescence and/or apoptosis.
Methods
To uncover the barriers that block the proliferation of tetraploids, we performed a RNAi mediated genome-wide screen in a human colorectal cancer cell line (HCT116).
Results
We identified 140 genes whose depletion improved the survival of tetraploid cells and characterized in depth two of them: SPINT2 and USP28. We found that SPINT2 is a general regulator of CDKN1A transcription via histone acetylation. Using mass spectrometry and immunoprecipitation, we found that USP28 interacts with NuMA1 and affects centrosome clustering. Tetraploid cells accumulate DNA damage and loss of USP28 reduces checkpoint activation, thus facilitating their proliferation.
Conclusions
Our results indicate three aspects that contribute to the survival of tetraploid cells: (i) increased mitogenic signaling and reduced expression of cell cycle inhibitors, (ii) the ability to establish functional bipolar spindles and (iii) reduced DNA damage signaling.