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The P53/P73 - P21cip1 Tumor Suppressor Axis Guards Against Chromosomal Instability by Restraining CDK1 in Human Cancer Cells

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The P53/P73 - P21cip1 Tumor Suppressor Axis Guards Against Chromosomal Instability by Restraining CDK1 in Human Cancer Cells Oncogene (2021) 40:436–451 https://doi.org/10.1038/s41388-020-01524-4 ARTICLE The p53/p73 - p21CIP1 tumor suppressor axis guards against chromosomal instability by restraining CDK1 in human cancer cells 1 1 2 1 2 Ann-Kathrin Schmidt ● Karoline Pudelko ● Jan-Eric Boekenkamp ● Katharina Berger ● Maik Kschischo ● Holger Bastians 1 Received: 2 July 2020 / Revised: 2 October 2020 / Accepted: 13 October 2020 / Published online: 9 November 2020 © The Author(s) 2020. This article is published with open access Abstract Whole chromosome instability (W-CIN) is a hallmark of human cancer and contributes to the evolvement of aneuploidy. W-CIN can be induced by abnormally increased microtubule plus end assembly rates during mitosis leading to the generation of lagging chromosomes during anaphase as a major form of mitotic errors in human cancer cells. Here, we show that loss of the tumor suppressor genes TP53 and TP73 can trigger increased mitotic microtubule assembly rates, lagging chromosomes, and W-CIN. CDKN1A, encoding for the CDK inhibitor p21CIP1, represents a critical target gene of p53/p73. Loss of p21CIP1 unleashes CDK1 activity which causes W-CIN in otherwise chromosomally stable cancer cells. fi Vice versa 1234567890();,: 1234567890();,: Consequently, induction of CDK1 is suf cient to induce abnormal microtubule assembly rates and W-CIN. , partial inhibition of CDK1 activity in chromosomally unstable cancer cells corrects abnormal microtubule behavior and suppresses W-CIN. Thus, our study shows that the p53/p73 - p21CIP1 tumor suppressor axis, whose loss is associated with W-CIN in human cancer, safeguards against chromosome missegregation and aneuploidy by preventing abnormally increased CDK1 activity. Introduction perpetual missegregation of whole chromosomes or large parts thereof during mitosis [4]. An important type of Chromosomal instability (CIN) is a major form of genome mitotic errors contributing to whole chromosome mis- instability representing a hallmark of cancer [1, 2]. While segregation in human cancer cells are so-called lagging structural alterations are caused by structural chromosome chromosomes that are caused by unresolved merotelic instability (S-CIN), changes in the chromosome numbers, microtubule-kinetochore attachments [5]. Various referred to as aneuploidy, are a result of whole chromo- abnormalities in chromosomally unstable cancer cells can some instability (W-CIN). A majority of cancer cells contribute to the generation of lagging chromosomes shows aneuploidy that is strongly associated with tumor- including supernumerary centrosomes [6, 7] or hyper- igenesis, tumor progression, therapy resistance, and poor stable microtubule-kinetochore attachments [8, 9]. More prognosis [3]. W-CIN is definedbyanelevatedrateof recently, we have demonstrated that abnormally increased microtubule plus end assembly rates within mitotic spin- dles result in the induction of W-CIN in cancer cells Supplementary information The online version of this article (https:// [10–16]. In fact, increased microtubule assembly rates doi.org/10.1038/s41388-020-01524-4) contains supplementary cause merotelic microtubule-kinetochore attachments, material, which is available to authorized users. lagging chromosomes, and chromosome missegregation * Holger Bastians [10](Fig.1a). [email protected] Despite the strong link between increased mitotic micro- tubule assembly rates and W-CIN, the cancer-associated 1 Georg-August University Göttingen, Göttingen Center for genetic alterations leading to abnormal microtubule dynamics Molecular Biosciences (GZMB) and University Medical Center Göttingen (UMG), Institute of Molecular Oncology, Section for are not well understood. So far, only a few tumor suppressors Cellular Oncology, D-37077 Göttingen, Germany and oncogenes including BRCA1, CHK2, AURKA,and CEP72 2 University of Applied Sciences Koblenz, Department of were shown to be involved in triggering W-CIN by Mathematics and Technology, D-53424 Remagen, Germany increasing mitotic microtubule assembly rates [10–15]. The p53/p73 - p21CIP1 tumor suppressor axis guards against chromosomal. 437 low-dose a Taxol cancer-relevant triggers? increased microtubule lagging chromosomes whole chromosome plus end growth during anaphase missegregation chromosomal instability (W-CIN) HCT116 + DNp73α b clone 1 clone 2 clone 3 - - - Dox + + + p73α (endogenous) DNp73α TA DBD OD SAM TID p73α p73β (endogenous) p53 DBD OD SAM TID DNp73α β-actin c 25 **** **** **** **** **** **** 20 15 (μm/min) 16.6 16.8 20.0 16.9 16.3 20.2 16.5 16.3 19.7 16.3 10 m/min m/min m/min m/min m/min m/min m/min m/min m/min m/min n=30 n=30 n=30 n=30 n=30 n=30 n=30 n=30 n=30 n=30 + -++-++-++Dox microtubule polymerisation rate - --+--+--+0.2 nM Taxol HCT116 clone 1 clone 2 clone 3 HCT116 + DNp73 d DNA CENP-C α-tubulin merge 10 HCT116 + DNp73α cells (clone 1) 8 ** ** * ** ** * 6 HCT116 + DNp73α 4 (clone 1) + Dox 2 proportion of anaphase 0 with lagging chromosomes (%) + - ++ -++ -++ Dox HCT116 + DNp73α - --+ --+ --+ 0.2 nM Taxol (clone 1) HCT116 clone 1 clone 2 clone 3 + Dox + 0.2 nM Taxol HCT116 + DNp73 e 30 generations ± Dox ± 0.2 nMTaxol evolvement of chromosome number variability = W-CIN single cells single cell clones metaphase spread f HCT116 + DNp73α (clone 1) g 40 ber - m 100 Dox Dox + 0.2 nM Taxol 30 80 e nu m lls (%) e c 60 20 omoso 40 r 10 20 proportion of of proportion deviating from (%) modal deviating 0 0 cells with ch 2 6 3 3 5 7 0 -++Dox 45 46 48 4 4 4 44 47 40 42 47 4 41 43 44 45 4 48 48 41 42 4 44 4 46 40 41 --+0.2 nM Taxol number of chromosomes per metaphase spread HCT116 + DNp73α (clone 1) Interestingly, loss of the tumor suppressor gene TP53 is abnormal microtubule dynamics nor chromosome mis- strongly associated with the presence of W-CIN [17]. segregation and W-CIN [10, 18]. p53 belongs to a family of However, loss of p53 alone is neither sufficient to trigger transcription factors that also includes p73 [19]. While TP53 438 A.-K. Schmidt et al. Fig. 1 Expression of oncogenic DNp73 induces whole chromosome Material and methods instability by increasing mitotic microtubule plus end assembly rates. a Model depicting the causal relationship between increased Cell culture microtubule plus end growth rates in mitosis and the induction of chromosome missegregation constituting W-CIN. b Left panel: Model for the structure of p73 and DNp73: transactivation domain (TA), HCT116, HT29, RKO, SW480, and SW620 cells were DNA-binding domain (DBD), oligomerization domain (OD), SAM purchased from ATCC (USA), DLD-1 cells were from domain (SAM), transactivation inhibitory domain (TID). Right panel: Sigma-Aldrich (Germany). DLD-1-CDKN1A−/− cells were Doxycycline-inducible expression of DNp73 in chromosomally stable TP53−/− HCT116 cells. Three independent stable cell lines were treated with obtained from Horizon Discovery (UK). HCT116- − − doxycycline for 24 h and expression of DNp73 was detected by [27] and HCT116-CDKN1A / cells [28] were kindly western blotting. A representative western blot is shown. Note that the provided by Bert Vogelstein (Johns Hopkins University, α β two bands for endogenous p73 represent the and isoforms of the Baltimore, USA). RKO cells expressing CDKN1A in a protein. c Mitotic microtubule plus end assembly rates upon expres- sion of DNp73. HCT116-DNp73 cell lines were treated with or ponasterone A-inducible manner (RKO-p21-Pon) were without doxycycline and additionally with low-dose Taxol. As a kindly provided by Matthias Schmidt [29]. Details on cul- control HCT116 parental cells were treated with doxycycline. turing conditions are given in the Supplemental Methods. Microtubule growth rates were determined by tracking EB3-GFP comets in mitotic cells by live-cell microscopy. Scatter dot plots show average microtubule polymerization rates (20 microtubules/cell, n = Measurement of microtubule plus end assembly 30 mitotic cells from 3 independent experiments, mean ± SD, t-test). rates d Proportion of cells exhibiting lagging chromosomes in anaphase DNp73 after expression of and after restoration of proper microtubule To determine microtubule plus end assembly rates, live-cell growth rates. HCT116 parental cells were incubated with doxycycline as a control. Left: Representative examples of anaphase cells with or microscopy was performed on cells expressing GFP-tagged without lagging chromosomes (white arrows), Scale bar, 10 µm. Right: end-binding protein 3 (EB3) [10, 30] as described [11]. The graph shows the proportion of anaphase cells exhibiting lagging n = chromosomes for three independent stable cell lines ( 300 anaphase Detection of lagging chromosomes cells from three independent experiments, mean ± SD, t-test). e Scheme depicting the generation of single-cell clones in order to determine chromosome number variability as a measure of W-CIN. To quantify anaphase cells exhibiting lagging chromosomes, f Proportion of cells harboring the indicated chromosome numbers per cells were fixed, stained, and imaged as described [15]. cell (n = 50 metaphase spreads). g Proportion of cells with chromo- some numbers deviating from the modal (45 chromosomes in HCT116 cells). The calculation is based on analyses of chromosome number W-CIN analysis variability shown in f. Single-cell clones were generated to determine the evolved chromosome number variability as a measure of W-CIN. is often mutated or deleted in cancer cells, TP73 is infre- Chromosome numbers were determined from metaphase quently mutated, and thus, it is still under debate whether spreads as described
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