Tetraplodization, however, was not only observed in neuroblastomas harboring mutated TP53 but also in primary tumors with functional p53 but deregulated MYCN.
We further investigated this apparent interplay between deregulated MYCN and the metaphase-anaphase checkpoint in cells with functional p53-p21 in the WAC2-neuroblastoma cell model, which stably expresses a MYCN transgene driven by a CMV promoter in a wild-type TP53 genetic background. WAC2 cells are characterized by a near-diploid DNA index and express high levels of MYCN protein [33,34], which results in metaphase-anaphase checkpoint overactivation. The parental SH-EP cell line, which is characterized by single-copy MYCN, barely detectable MYCN levels and a lower metaphase-anaphase checkpoint activation status, was used as a control. To test the effect of metaphase-anaphase checkpoint deregulation in a functional p53-p21 background, we stably transfected the WAC2 and SH-EP cell lines with a doxycycline-inducible shRNA targeting the metaphase-anaphase checkpoint regulator, MAD2L1. The SH-EP-MYCN neuroblastoma cell model was not suitable for these experiments because these cells already express a tetra-/doxycycline inducible system. MAD2L1 was effectively repressed upon
shMAD2L1 induction in both SH-EP and WAC2 cells, as demonstrated by western blotting (Fig. 4A). The cell proportions in different phases of the cell cycle were similar for SH-EP-shMAD2L1 and WAC2-shMAD2L1 cells with or without MAD2L1 knockdown and to control cells expressing scrambled shRNA. To mimic a weakened p53-p21 checkpoint, we treated both cell lines with vincristine, which disrupts microtubule formation and reduces nuclear accumulation of p53, consequently preventing the transcriptional activation of p21 [62]. Vincristine treatment resulted in G2/M arrest of 4N cells in both WAC2-shMAD2L1 and SH-EP-shMAD2L1 cells expressing MAD2L1 (Fig. 4B and Suppl. Fig. 5). Combined inhibition of the p53-p21 axis (vincristine treatment) and the metaphase-anaphase checkpoint (MAD2L1 silencing) resulted in approximately 40% of the WAC2-shMAD2L1 cells being 8N, indicative of cycling tetraploid cells (Fig. 4B and Suppl. Fig. 5A). Only about 5% of SH-EP-shMAD2L1 cells were 8N following combined vincristine treatment and MAD2L1 silencing (Suppl. Fig. 5B). These results strengthen our hypothesis that deregulated MYCN is associated with tetraploidization when p53-p21 signaling is weakened and the metaphase-anaphase checkpoint unbalanced.
To further verify induction of tetraploidization in WAC2-shMAD2L1 cells, centromeric regions of chromosome 6 and 8 were labeled with fluorescent dyes (Cy3.5 and FITC, respectively) and 2-color FISH was performed. In addition to cells with two signals for each centromer, indicative of normal diploid cells, interphase nuclei with four or eight centromeric signals, indicative of cycling tetraploid cells, were also observed after MAD2L1 repression and vincristine treatment in WAC2-shMAD2L1 cells (Fig. 4C).
For complete confirmation, 4-color FISH for chromosome 3, 6, 8 and 18 centromeres was also performed, and at least 250 interphase nuclei were manually counted from WAC2-shMAD2L1 cell cultures treated with vincristine and expressing MAD2L1 or not. The numerical index of centromeric signals was used as a direct indicator for nuclear DNA content. The fraction of 8N cells reached 8.7% 18h after shMAD2L1 induction and vincristine treatment. After 36h, the 8N fraction had increased from 8.7% to 30.1% (Suppl. Table 5). Interphase nuclei of 8N cells appeared strongly enlarged with an asymmetrical shape in contrast to small, round diploid cells (Fig. 4C and 4D). We also detected 3.5% 16N cells 36h after induction and treatment.
75 Manuskript II
Vincristine treatment alone resulted in 5.1% 8N cells after 36h of treatment (Suppl.
Table 5). Immunofluorescence imaging using a CREST antibody that unspecifically binds to centromeric regions further supported the observation of polyploidization after MAD2L1 knockdown and vincristine treatment (Fig. 4D).
80
77 Manuskript II
Figure 4 MAD2L1 silencing after vincristine treatment induces tetraploidization in near-diploid neuroblastoma cells. (A) Western blot showing MAD2L1 and MYCN expression in whole-cell lysates from WAC2 and SH-EP cells stably transfected with shRNA targeting MAD2L1. (B) Flow cytometric analysis of cell cycle and ploidy in WAC2-shMAD2L1 36h cell cultures after treatment. Curves are paired with bar-graph quantifications (below) for each treatment group. (C) 2-color FISH of WAC2-shMAD2L1 after 36h of vincristine treatment and MAD2L1 shRNA induction using centromeric probes for chromosome 6 and 8 (pink and green, respectively) and counterstained with DAPI (blue). Representative images from 250 interphases are shown. (D) Merged immunofluorescence images of WAC2-shMAD2L1 stained for centromers with CREST antibodies (green) and DNA (blue) to visualize altered nuclear size after combined MAD2L1 silencing and vincristine treatment.
3 .5 Discussion
In this study, we show that overactivation of the metaphase-anaphase checkpoint acts as a pro-survival mechanism in the development of tetraploid neuroblastoma cells lacking functional p53-p21 signaling. Elevated expression of mitotic spindle regulatory genes has been shown to be associated with MYCN amplification and 1p loss in neuroblastomas [52,63,64]. This is consistent with our gene expression analyses in primary neuroblastoma tumors, which further showed that overexpression of MYCN/MYC and p53/pRB-E2F target genes, especially those involved in regulating mitotic processes, such as sister chromatid segregation, microtubule organization or metaphase-anaphase checkpoint regulation, is associated with near-di/tetraploidy and poor outcome in neuroblastoma patients. One gene we identified in this regard was MAD2L1, which is a direct MYC and E2F-1 target [22,65]. This suggests that MYCN/MYC-mediated overactivation of the metaphase-anaphase checkpoint might be causally involved in the development of near-di/tetraploidy by initially provoking sustained mitotic arrest, as shown for Mad2 overexpression [66]. Cells might escape this sustained arrest by mitotic slippage – an adaptation that consequently results in the failure of cytokinesis and tetraploidy [1].
Our observation that neuroblastoma cells lacking p53-p21 function, either through TP53 mutation or mediated by p21CIP1 knockdown, consist of diploid and tetraploid cell fractions indicates that functional p53-p21-mediated checkpoints are required to arrest these cells [53,67] and to subsequently initiate cell death or senescence programs [57]. Evidence that p53 and p21 inactivation is mainly involved in the origin of tetraploidy exists in the mouse model p53-R172P equivalent to R175P in human, which harbor an Arg-to-Pro TP53 mutation. The p53 in cells from these mice is incapable of inducing apoptosis, but still activated p21-mediated G1-S arrest [68].
Accordingly, these mice developed tumors with a diploid DNA index. Crossing the TP53-mutant mice into a p21-/- background resulted in formation of aneuploid tumors.
Some evidence exists for a potential association between tetraploidy and loss of p53-p21 functionality in neuroblastoma. Additionally to SK-N-BE(2)-C cells, other neuroblastoma cell lines harboring mutant TP53 are characterized by a near-tetraploid DNA index, including LA-N-1, NMB and NB-6 [69,70,71,72]. Whole
79 Manuskript II
genome sequencing of primary neuroblastomas revealed that at least some tetraploid tumors harbored TP53 mutations (unpublished data). This genetic alteration, although frequent in many other cancers, occurs mainly in relapse neuroblastomas and is associated with therapy resistance [55], suggesting a direct connection of p53 functionality to neuroblastoma biology.
A direct connection between the p53-p21 axis, mitotic spindle regulatory genes and tetraplodization was presented by Schvartzman, et al. using the same TP53-mutant and p21-deficient mice. He showed that p21 is a direct negative Mad2 regulator and that normalization of Mad2 expression reduced the aneuploid cell fraction in these murine tumors [73]. In cells lacking p53 and/or p21 function, overactivation of metaphase-anaphase checkpoint members might, therefore, facilitate the development and survival of tetraploid cells (Figure 5). We observed that tetraploid cell fractions increased after several passages or drug-induced DNA damage in both the TP53-mutated neuroblastoma cell line and in neuroblastoma cells silenced for p21CIP1 expression. Our siRNA screening approach also demonstrated that knockdown of genes involved in metaphase-anaphase checkpoint regulation, p53-p21 function as a consequence of TP53 mutation. These results from various in vivo and in vitro studies show that functional p53-p21 signaling is crucial to control including MAD2L1, induced mitotic-linked cell death only in neuroblastoma cells lacking the expression of metaphase-anaphase checkpoint genes and to inhibit the survival of tetraploid cells.
In summary, the results we present here reveal novel insights into how genetic aberrations of the p53-p21 axis contribute to tetraploidy in neuroblastoma cells.
These data enhance our understanding of how MYCN/MYC mediates aggressive behavior in neuroblastomas. Since overactivation of the metaphase-anaphase checkpoint supports the survival of tetraploid cells lacking p53-p21 function, targeted inhibition of certain metaphase-anaphase checkpoint members, such as MAD2L1, may provide a therapeutic option for neuroblastomas harboring genomic alterations reducing p53-p21 function.
Figure 5 Schematic model describing the role of MYCN and aneuploid checkpoints in the development of tetraploid neuroblastomas.
3.6 Acknowledgements
We thank Geoffrey M. Wahl for providing the H2B-GFP expression vector, Frank Berthold, Barbara Hero and the German Neuroblastoma Study Group for providing clinical data, and Kathy Astrahantseff for manuscript editing.