No potential conflicts of interest were disclosed.
2.7 Acknowledgement
We thank J.J. Molenaar for providing the IMR-32 cell line carrying an inducible CDK2 shRNA and K. Astrahantseff for manuscript editing.
Supplementary information see appendix of the thesis.
47 Manuscript I
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3 Manuscript II
The following manuscript was accepted by Cancer Letters, 08.11.2012.
MYCN-mediated overexpression of mitotic spindle regulatory genes and loss of p53-p21 function jointly support the survival of tetraploid neuroblastoma cells Sina Gogolin1, Richa Batra2*, Nathalie Harder2*, Volker Ehemann3*, Tobias Paffhausen1*, Nicolle Diessl2#, Vitaliya Sagulenko1, Axel Benner4, Stephan Gade4, Ingo Nolte5, Karl Rohr2, Rainer König2, Frank Westermann1
1Division of Tumor Genetics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
2Department of Bioinformatics and Functional Genomics, University of Heidelberg, BIOQUANT, IPMB and German Cancer Research Center (DKFZ), Im Neuenheimer Feld 267, 69120 Heidelberg, Germany
3Department of Pathology, University of Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany
4Division of Biostatistics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580/581, 69120 Heidelberg, Germany
5Division of Molecular Genetics, Cancer Genome Research, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
6Small Animal Clinic, University of Veterinary Medicine Hannover, Bünteweg 9, 30559 Hannover, Germany
* contributed equally
# current address: Department of Genomics and Proteomics Core Facility, High Throughput Sequencing, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany
Corresponding author Dr. Frank Westermann
German Cancer Research Center (DKFZ) Im Neuenheimer Feld 280
69120 Heidelberg, Germany E-mail: f.westermann@dkfz.de Phone: +49 (0) 6221 423275 FAX: +49 (0) 6221 423277
Running title: metaphase-anaphase checkpoint overactivation and loss of p53-p21 support tetraploidization in neuroblastoma cells
Contributions to the study:
- GO term/cluster enrichment analysis, pharmacological treatment of neuroblastoma cell lines and generating ploidy data overview for subsequent statistical analysis
- flow cytometric analyses of neuroblastoma cell lines together with PD Dr.
Volker Ehemann
- siRNA screnning: improvement of the training set, manual validation of classification results and interpretation of cellular phenotype analyses
- interpretation of results
- generating the theoretical model and drafting of the manuscript together with PD Dr. Frank Westermann
53 Manuskript II
3.1 Abstract
High-risk neuroblastomas often harbor structural chromosomal alterations, including amplified MYCN, and usually have a near-di/tetraploid DNA index, but the mechanisms creating tetraploidy remain unclear. Gene-expression analyses revealed that certain MYCN/MYC and p53/pRB-E2F target genes, especially regulating mitotic processes, are strongly expressed in near-di/tetraploid neuroblastomas. Using a functional RNAi screening approach and live-cell imaging, we identified a group of genes, including MAD2L1, which after knockdown induced mitotic-linked cell death in MYCN-amplified and TP53-mutated neuroblastoma cells. We found that MYCN/MYC-mediated overactivation of the metaphase-anaphase checkpoint synergizes with loss of p53-p21 function to prevent arrest or apoptosis of tetraploid neuroblastoma cells.
3.2 Introduction
Aneuploidy and chromosomal instability are hallmarks of most if not all cancers and play an essential role in tumor formation and progression [1]. Neuroblastoma, the most common solid extracranial tumor in early childhood, is characterized by contrasting clinical courses, ranging from low-risk to high-risk disease. To adjust therapy and improve prognosis, markers have been identified, such as loss of chromosome arm 1p/11q or MYCN amplification, that are associated with an aggressive disease and poor overall survival [2,3,4,5]. Several studies have further shown an association of tumor ploidy and outcome in neuroblastoma [6,7,8,9]. Thus, near-diploid and near-tetraploid neuroblastomas are associated with poor outcome, whereas near-triploid/near-pentaploid tumors are associated with low-risk disease and may even undergo spontaneous maturation or regression [10,11]. Near-triploid/near-pentaploid neuroblastomas lack structural chromosomal alterations, whereas near-diploid and near-tetraploid neuroblastomas are frequently associated with structural chromosomal alterations, suggesting that numerical, whole-chromosome aneuploidy and ploidy changes associated with chromosomal instability may arise from at least two different mechanisms.
To date, several models have been proposed about how aneuploidy might arise in cancer. One extensively investigated mechanism in recent years is the development of aneuploidy through a tetraploid genetically unstable intermediate [12,13]. An association between unscheduled tetraploidy, cell transformation and tumor formation has been shown at least in mice [14]. The appearance of tetraploid cells is characteristic especially for the pre-malignant condition Barrett’s esophagus [15] and early stages of cervical carcinogenesis [16] but can also be detected in several other cancers independent of the tumor stage [17]. Tetraploidy can arise via cell fusion, cytokinesis failure, endoreduplication or mitotic slippage. Prolonged activation of the anaphase checkpoint resulting from overexpressed metaphase-anaphase checkpoint genes, such as Mad2 [18], has been shown to provoke mitotic slippage associated with incomplete cytokinesis resulting in tetraploidization [19]. The main function of the metaphase-anaphase checkpoint is to inhibit the anaphase-promoting complex or cyclosome (APC/C) until the spindles have properly attached
55 Manuskript II
to all kinetochores, thereby preventing chromosome mis-segregation. Only one unattached kinetochore is sufficient to stop anaphase by Mad2 activation, which subsequently forms complexes with Cdc20, BubR1 and Bub3. These complexes prevent APC/C-mediated ubiquitylation of securin and, consequentially, separase-mediated cohesin degradation [20]. Deregulation of mitotic checkpoint genes, including MAD2L1, either by overexpression, reduced expression or mutation has been reported for many cancers, including neuroblastoma [1,21,22].
Recently, it has been suggested that overactivation of the metaphase-anaphase checkpoint might trigger aneuploidy induction in cells with a defective G1-S arrest as a consequence of non-functional pRB and p53-p21 [23]. Both the p53-p21 and pRB axes are deregulated especially in relapse neuroblastomas and tumor-derived cell lines through genetic aberrations that include TP53 mutation and/or amplification of MDM2, CDK4 or CCND1 [24,25,26]. Furthermore, MYCN impairs the p53-p21 and pRB pathway through transcriptional inhibition of p21 and upregulation of MDM2 and CDK4, which are direct p53 and pRB inhibitors, respectively [27,28,29,30]. An association between tetraploidy and loss of p53 function has been described for Barrett’s esophagus [31] and, more recently, for medulloblastoma [32]. Whether impaired p53-p21/pRB-mediated checkpoints might further contribute to tetraploidy in neuroblastoma cells and whether deregulation of the metaphase-anaphase checkpoint contributes to the development of aneuploidy in neuroblastomas has not been investigated to date.
To address both points, we analyzed the expression of MYCN/MYC, p53 and pRB-E2F target genes, which are primarily involved in cell cycle regulation or neuronal differentiation, in association with tumor DNA index and structural chromosomal aberrations in a large cohort of primary neuroblastomas. We then used a functional siRNA screening approach combined with a live-cell imaging microscopy-based readout in two neuroblastoma cell lines with either low MYCN expression and functional p53 or amplified MYCN and mutated TP53 to determine the consequences of metaphase-anaphase checkpoint gene repression.
3.3 Material and methods