Chromosome segregation errors: a double-edged sword - TI Pharma

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Abstract One of the most common hallmarks of cancer cells is aneuploidy, or an abnormal number of chromosomes. This abnormal chromosome content is a consequence of chromosome missegregation during mitosis a defect that is seen more frequently in tumor cell divisions as in normal cell divisions. In fact, a large fraction of human tumors display a chromosome instable phenotype (CIN), meaning that they very frequently missegregate chromosomes. This can cause variegated aneuploidy within the tumor tissue. It has been argued that this hallmark of cancer could be exploited in anti-cancer therapies. Here we test this hypothesis by inactivation of the mitotic checkpoint through RNAimediated depletion of an essential checkpoint component, Mps1. The mitotic checkpoint delays segregation of chromosomes during mitosis until all chromosomes are properly attached to the mitotic spindle. Its inactivation will therefore lead to increased segregation errors. Indeed, we show that this can lead to increased cell death in tumor cells. We demonstrate that increased cell death is associated with a dramatic increase in segregation errors. In addition, we show that this can inhibit tumor growth in xenografted tumors in nude mice. This suggests that inhibition of the mitotic checkpoint might represent a useful anticancer strategy. 107 Chromosome missegregations kill tumor cells 5
5 Introduction Chromosomal instability (CIN) is a common characteristic of solid tumors that is manifested as gains or losses of whole chromosomes during cell division, leading to aneuploidy 231. This propensity of tumor cells was recognized more than 100 years ago 569, leading to the hypothesis that CIN could be a driving force in tumorigenesis 232. Indeed, several lines of evidence have since suggested a causal role for CIN in tumor formation (reviewed in 570). However, the exact cause of CIN in tumors has remained largely unknown. It has become clear that a broad range of mutations in different cellular processes can result in CIN 282. Defects in mitotic checkpoint function have also been invoked as a causal event for CIN 282. The mitotic checkpoint ensures the fidelity of sister chromatid segregation over the two daughter cells by inhibiting progression to anaphase until all chromosome pairs are bi-oriented on the mitotic spindle (reviewed in 441). In various human cancers mitotic checkpoint function is compromised, and altered expression or mutations of mitotic checkpoint genes have been shown to be related to CIN and aneuploidy (reviewed in 290). Nevertheless, no evidence for checkpoint malfunctions as a direct cause of CIN in tumor cells has been found289,302,342. Moreover, the mitotic checkpoint defects that have been reported (reviewed in 282) may be well tolerated by the tumor cell population. Complete inactivation of the mitotic checkpoint, however, results in gross chromosomal missegregation and is not compatible with cell viability292-294,393. This has led to the suggestion that inhibition of the mitotic checkpoint could have therapeutic potential in cancer treatment 290. Here, we have explored the relationship between chromosome missegregations and tumor cell death by using conditional depletion of the mitotic checkpoint component Mps1. Results and Discussion Efficient Mps1 depletion severely compromises viability of tumor cells. We have previously shown that absence of Mad2, BubR1 or Mps1 causes cell death within 6 cell divisions116,172,292,299. To examine the effect of conditional inactivation of Mps1 in LS174-T colon carcinoma cells, we stably introduced a doxycycline-inducible vector encoding a small hairpin RNA (shRNA) directed against human Mps1. In addition, this line stably expresses a tetrepressor, such that the addition of tetracycline or doxycycline to the culture medium induces the expression of the Mps1 shRNA and a consequent depletion of the endogenous Mps1 protein299. A clone was selected in which expression of Mps1 was reduced by more than 90% upon addition of doxycycline to the culture medium (Fig.1A). This degree of depletion of Mps1 severely compromised the viability of the LS174-T cells, since no colonies grew out in the presence of doxycycline (Fig.1A). Thus, selective inhibition of Mps1 compromises the viability of LS174-T tumor cells. Mps1 depletion inactivates the spindle checkpoint. Mps1 is an essential component of the spindle checkpoint, and its inactivation has been shown to override the cellular response to spindle poisons, such as nocodazole and taxol 142 143 . Therefore, we next tested if RNAi-mediated depletion of Mps1 in the LS174-T cells conditionally expressing the Mps1 shRNA leads to functional inactivation of Mps1. To this end we cultured these cells in the presence or absence of doxycycline for a period of 3 days, and added 1 mM taxol to the culture medium for the last 18 hrs. of the incubation period. Taxol stabilizes microtubules, and perturbs proper spindle assembly. This will normally lead to a mitotic delay and an accumulation of cells in mitosis that is dependent on the mitotic checkpoint. Indeed, in LS174-T cells that are grown in the absence of doxycycline we find a 108
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Chromosome segregation errors: a do
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Chromosome segregation errors: a do
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Get up, stand up, stand up for your
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Chapter 1 General Introduction Anie
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cytoplasms and pinches off the memb
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which sister-chromatids are not pro
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Non-MCC members, but important mito
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completely separated. Following cle
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Two striking features of cancer cel
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predisposition at a very young age,
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that aberrant spindle formation inc
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(Fig.7C). As discussed in section 4
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Thesis outline Chromosome segregati
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286,395,407,408 Increase (thymus) I
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2 Abstract Various types of chromos
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2 34 A C D E % of cells Control 100
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2 assess whether cytokinesis induce
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2 A Asynchronous Monastrol release
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2 for 14 hours (unless indicated ot
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2 Immuno Blotting Cells were lysed
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2 Supplemental figures 44 A B C % o
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2 A MCF7 B C 53BP1 foci/cell 46 % o
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2 A siLuc siATM C 48 p-S1981 ATM me
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2 50 A B C % of EdU positive cells
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Chapter 3 Studying Chromosomal inst
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Introduction Aneuploidy is a common
Page 58 and 59: activity by ~80% 170-172 . With the
Page 60 and 61: A WT/CiMKi T649A Embryo’s: Figure
Page 62 and 63: control treated MEFs (unpaired t te
Page 64 and 65: Materials and Methods Cloning targe
Page 66 and 67: - Loop out BamHI site with site-dir
Page 68 and 69: Primers: pGH_pA_FW#2: TCTATGGCTTCTG
Page 70 and 71: was prepared using standard procedu
Page 72: 71 Cre-inducible Mps1 knock-in mous
Page 75 and 76: 4 Abstract Most of the current drug
Page 77 and 78: 4 Two other interesting mitotic tar
Page 79 and 80: 4 inhibition of the anti-apoptotic
Page 81 and 82: 4 cluster their centrosomes. This
Page 83 and 84: 4 of checkpoint function can be ach
Page 85 and 86: 4 4.5 Disadvantages of exploiting m
Page 88 and 89: Chapter 5 Elevating the frequency o
Page 90 and 91: Introduction Error-free chromosome
Page 92 and 93: Partial depletion of Mps1 or BubR1
Page 94 and 95: death is not induced in the short t
Page 96 and 97: had survived growth for 7 days in t
Page 98 and 99: The following antibodies have been
Page 100 and 101: Supplemental data Supplemental figu
Page 102 and 103: A BubR1 α-tubulin Hela-TetRBubR1 -
Page 104 and 105: - doxycycline + doxycycline - doxyc
Page 106 and 107: A B C percentage of cells % PI posi
Page 110 and 111: clear accumulation of cells in mito
Page 112 and 113: the mice did not receive any doxycy
Page 114 and 115: 113 Chromosome missegregations kill
Page 116 and 117: Chapter 6 Aneuploid tumor cells are
Page 118 and 119: Introduction Equal segregation of t
Page 120 and 121: S1A, data not shown). Based on thes
Page 122 and 123: A B U2OS + H2B-YFP percentage of ce
Page 124 and 125: tumor cells, leaving diploid cells
Page 126 and 127: A B p-S10-Histone H3 DAPI C µmol/l
Page 128 and 129: Supplemental Figure-1. Protein bioc
Page 130 and 131: Bioavailability of compound-5 after
Page 132: A B U2OS (7.5nM) Hela (10nM) RPE-1
Page 135 and 136: 7 Abstract Taxanes, such as docetax
Page 137 and 138: 7 of mitotic aberrations. These dat
Page 139 and 140: 7 A Low CFP lifetime High CFP lifet
Page 141 and 142: 7 docetaxel treatment in vitro resu
Page 143 and 144: 7 chromosome unstable, which means
Page 145 and 146: 7 Cells (~50.000/well) were plated
Page 147 and 148: 7 Supplemental data Supplemental fi
Page 149 and 150: 7 148
Page 151 and 152: 8 Thesis summary Unequal separation
Page 153 and 154: 8 exerted through contraction of th
Page 155 and 156: 8 1.4 NoCut: preventing the inducti
Page 157 and 158: 8 tumorformation using intravital i
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Addendum References Nederlandse sam
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41. McEwen, B.F., et al., CENP-E is
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2010. 6(5): p. 359-68. 124. Liu, S.
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210. Stucki, M., et al., MDC1 direc
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tumors in mice. Cancer Res, 2007. 6
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adiotherapy in breast cancer. Breas
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470. Marcus, A.I., et al., Mitotic
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559. Kienitz, A., et al., Partial d
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Nederlandse Samenvatting Celdeling,
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verdeling van tumorcellen compleet
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Publications A. Janssen, R.H.Medema
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En dan zijn er nu nog een aantal li
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promotie-stukjes! Je bent een voorb
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edankt voor al jullie hulp bij de i
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Chromosome segregation errors: a double-edged sword - TI Pharma

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