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Aneuploidy Police Detect Chromosomal Imbalance Triggering Immune Crackdown!
TIGS 1384 No. of Pages 3
Spotlight
Aneuploidy Police Detect Chromosomal Imbalance Triggering Immune Crackdown! Emma V. Watson1,2 and Stephen J. Elledge1,2,* Aneuploidy is ubiquitous in cancer and plays a pivotal, early role in tumor evolution. It must therefore be avoided, and two recent papers highlight the roles of p53, senescence, and the immune system in preventing the outgrowth of aneuploid clones in tissue culture. These mechanisms are likely to synergize to maintain diploid cell populations. Aneuploidy – an abnormal number of chromosomes – underlies numerous developmental pathologies, including Down syndrome, Edward’s syndrome, Patau syndrome, and sex chromosome aneuploidies including Turner, Klinefelter, and XYY syndromes, as well as mosaic variegated aneuploidy (MVA) syndromes caused by defects in spindle assembly genes. Aneuploidy is also recognized as a driver of tumorigenesis, and patients with MVA develop childhood cancer [1]. While aneuploidy is not absolutely required for oncogenic transformation in vitro [2], aneuploidy is sufficient to induce and accelerate tumor formation, at least in the absence of p53 [3]. The mechanisms by which aneuploidy leads to tumorigenesis are currently being investigated, and contributors may include increased mutagenesis and enhanced chromosome loss rates [4] leading to increased clonal diversity and population fitness of the tumor. Additionally, chromosome arms that are gained or lost recurrently in cancer are enriched for oncogenes and tumor
suppressors, respectively [5], so the cancer genome can be sculpted by aneuploidization to optimize gene dosage. It is therefore critical for tissues to avoid the accumulation of aneuploid cells derived from spontaneous errors in mitosis or else risk a slippery slope toward oncogenic transformation. Do surveillance mechanisms exist to detect and eliminate aneuploid cells? p53, the well-known tumor suppressor and ‘guardian of the genome’ that induces cell-cycle arrest, senescence, or apoptosis in response to DNA damage has also been proposed as a ‘guardian of ploidy’ [6]. Activation of p53 and subsequent cell cycle arrest has been observed following aneuploidization in various cell lines and loss of p53 is associated with increased aneuploidy in human tumors [7]. However, it is unclear whether p53 is activated by aneuploidy itself via some unknown chromosome accounting system or whether it is activated via other conditions associated with aneuploidy (e.g., replication stress) or simply the drugs used to induce aneuploidization. Soto et al. set out to explore these questions by inducing aneuploidy in a fairly karyotypically normal, untransformed cell line (RPE1) using chemical inhibitors of centrosome function and the spindle assembly checkpoint, followed by karyotyping of cells under both p53 wild-type and p53 knockdown conditions [8]. Recently generated aneuploid cells exhibited more p53 activation than normal cells and knockdown of p53 permitted more aneuploid cells to enter mitosis. Therefore, p53 is activated by aneuploidization and induces cell-cycle arrest, but not in all cases: single-cell sequencing revealed many aneuploid cells in the cycling population, most exhibiting single whole-chromosome gains or losses. The initial population (containing many arrested cells) had more complex compound aneuploidies, with structural changes (subchromosome gains or losses). However, in p53 knockdown cells these structural
aneuploidies were found to be abundant in the cycling population. This suggests that p53 preferentially causes cells with more complex, structural aneuploidies to arrest, but is permissive of many single whole-chromosome aneuploidies. Santaguida et al. also utilized single-cell sequencing to characterize aneuploid RPE1 populations and found more complex karyotypes among the arrested cells, with an average of 20% of the genome affected [9]. Cells that were capable of cycling had only 0–5% of their genomes altered. There was also an approximately twofold increase in structural aneuploidies in the arrested population compared with cycling cells, and p53 activation only in the arrested population. To identify the causes of p53 activation, Santaguida et al. assessed whether DNA damage was increased in recently formed aneuploid cells and found that g-H2AX foci were indeed increased. Examination of DNA replication dynamics revealed that aneuploid cells have slower replication forks, more fork stalling, and prolonged presence of PCNA foci indicating a delay in S phase. In addition, ultrafine anaphase DNA bridges were observed in aneuploid cells, which may indicate unresolved replication stress. Santaguida et al. conclude that DNA replication stress in severely aneuploid cells results in DNA damage and p53 activation. Santaguida et al. further show that arrested aneuploid cells exhibit features of senescence and activate an interesting pattern of inflammation and innate immune signals. In addition to interferon signaling, which commonly results from DNA damage-induced senescence, arrested aneuploid cells [79_TD$IF]5activated the cGAS–STING pathway, which responds to cytosolic DNA and activates IRF3 and NF-kB. Arrested aneuploid cells exhibited elevated secretion of the cytokines IL-6, IL-8, and CCL2 and increased surface expression of proteins that are recognized by natural killer (NK) cells. Consequently, arrested aneuploid RPE1 cells
Trends in Genetics, Month Year, Vol. xx, No. yy
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TIGS 1384 No. of Pages 3
were readily killed by NK cells in vitro damage, an aneuploidy patrol system is needed to prevent accelerated genomic whereas euploid RPE1 cells were not. instability and karyotypic evolution toward These studies taken together paint a pic- tumorigenesis. p53 activation, senesture in which complex aneuploidy causes cence, and NK cells may comprise such (and is then further exacerbated by) repli- a system. Simple whole-chromosome cation stress, which triggers the DNA aneuploidies with less that 5% of the damage response, p53 activation, senes- genome unbalanced do not tend to set cence, and an innate immune response off the DNA damage/p53 alarm. There(Figure 1). This enables recognition and fore, it seems that p53 does not function elimination of senescent aneuploid cells explicitly as a chromosomal accountant by NK cells. Due to the cyclic, feed-for- but rather serves to protect against the ward relationship between aneuploidy proliferation of genomically unstable and replication stress-induced DNA aneuploidies, which more readily evolve
Cells
Monosomy Disomy Trisomy Tetrasomy
Cells
Ploidy state
Chromosome
Chromosome
‘Simple’ single, whole-chromosome aneuploidies <5% genome affected
‘Complex’ paral whole-chromosome aneuploidies >5% genome affected
Mild
Replicaon stress and DNA damage
Severe
p53
DNA damage response
DNA repair Cell-cycle progression
Cell-cycle arrest
Senescence
SASP + immune signals
NK cell detecon and killing Figure 1. Severe Aneuploidy Leads to p53 Activation and Senescence. Soto et al. and Santaguida et al. show by single-cell sequencing that aneuploidization involving single whole-chromosome changes often does not trigger p53 activation or cell-cycle arrest (broken orange path) whereas aneuploidization involving multiple whole- and/or partial-chromosome changes does (dark-blue path). Newly formed cells with complex karyotypes exhibit replication stress and DNA damage, which triggers p53 activation and senescence as well as an immune response leading to natural killer (NK) cell-mediated killing. SASP, senescence-associated secretory phenotype
2
Trends in Genetics, Month Year, Vol. xx, No. yy
toward oncogenic transformation than stable diploids. A key question not addressed by these studies is whether cells are susceptible to senescence and NK surveillance only during the transition from euploidy to aneuploidy or remain sensitive once the cells have adjusted to the aneuploid state. This could be tested by conditionally inhibiting p53 during the aneuploidization phase then releasing p53 inhibition in the complexkaryotype cycling population to determine whether p53 is capable of triggering arrest well past chromosome missegregation. How do some genomically unstable tumors with complex karyotypes bypass the p53/senescence barrier and evade the immune system? One simple mechanism of bypass is p53 mutation; indeed, p53 loss is correlated with increased aneuploidy in human tumors [7]. While p53 mutation and senescence bypass may also mask some of the senescence-associated immune responses exhibited by aneuploid cells, other innate immune responses, such as the cytosolic DNA-triggered cGAS–STING pathway, may be controlled independently. Therefore, aneuploid tumors may need to evolve other ways to silence these responses and evade recognition by NK cells. It appears aneuploid tumors do this quite well, since immune cell infiltration is negatively correlated with tumor aneuploidy [10]. Perhaps aneuploidization via an intermediary tetraploid state [11] is a less dramatic route toward a tumorigenic karyotype: individual chromosome loss or gain would affect a smaller percentage of the tetraploid genome and therefore be less likely to unbalance the replication machinery, cause replication stress, and activate p53 and senescence. This would enable incremental refinement of tumor karyotypes. 1 Howard Hughes Medical Institute, Department of Genetics, Harvard University Medical School, Boston,
MA, USA [78_TD$IF]2 Division of Genetics, Brigham and Women’s Hospital, Boston, MA, USA
TIGS 1384 No. of Pages 3
*Correspondence: [email protected] (S.J. Elledge). http://dx.doi.org/10.1016/j.tig.2017.07.007 References 1. Hanks, S. et al. (2004) Constitutional aneuploidy and cancer predisposition caused by biallelic mutations in BUB1B. Nat. Genet. 36, 1159–1161 2. Zimonjic, D. et al. (2001) Derivation of human tumor cells in vitro without widespread genomic instability. Cancer Res. 61, 8838–8844
4. Passerini, V. et al. (2016) The presence of extra chromosomes leads to genomic instability. Nat. Commun. 7, 10754
8. Soto, M. et al. (2017) p53 prohibits propagation of chromosome segregation errors that produce structural aneuploidies. Cell Rep. 19, 2423–2431
5. Davoli, T. et al. (2013) Cumulative haploinsufficiency and triplosensitivity drive aneuploidy patterns and shape the cancer genome. Cell 155, 948–962
9. Santaguida, S. et al. (2017) Chromosome mis-segregation generates cell-cycle-arrested cells with complex karyotypes that are eliminated by the immune system. Dev. Cell 41, 638–651.e5
6. Aylon, Y. and Oren, M. (2011) p53: guardian of ploidy. Mol. Oncol. 5, 315–323 7. Clausen, O.P.F. et al. (1998) Association of p53 accumulation with TP53 mutations, loss of heterozygosity at 17p13, and DNA ploidy status in 273 colorectal carcinomas. Diagn. Mol. Pathol. 7, 215–223
10. Davoli, T. et al. (2017) Tumor aneuploidy correlates with markers of immune evasion and with reduced response to immunotherapy. Science 355, eaaf8399 11. Storchova, Z. and Kuffer, C. (2008) The consequences of tetraploidy and aneuploidy. J. Cell Sci. 121, 3859–3866
3. Fujiwara, T. et al. (2005) Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells. Nature 437, 1043–1047
Trends in Genetics, Month Year, Vol. xx, No. yy
3
Spotlight
Aneuploidy Police Detect Chromosomal Imbalance Triggering Immune Crackdown! Emma V. Watson1,2 and Stephen J. Elledge1,2,* Aneuploidy is ubiquitous in cancer and plays a pivotal, early role in tumor evolution. It must therefore be avoided, and two recent papers highlight the roles of p53, senescence, and the immune system in preventing the outgrowth of aneuploid clones in tissue culture. These mechanisms are likely to synergize to maintain diploid cell populations. Aneuploidy – an abnormal number of chromosomes – underlies numerous developmental pathologies, including Down syndrome, Edward’s syndrome, Patau syndrome, and sex chromosome aneuploidies including Turner, Klinefelter, and XYY syndromes, as well as mosaic variegated aneuploidy (MVA) syndromes caused by defects in spindle assembly genes. Aneuploidy is also recognized as a driver of tumorigenesis, and patients with MVA develop childhood cancer [1]. While aneuploidy is not absolutely required for oncogenic transformation in vitro [2], aneuploidy is sufficient to induce and accelerate tumor formation, at least in the absence of p53 [3]. The mechanisms by which aneuploidy leads to tumorigenesis are currently being investigated, and contributors may include increased mutagenesis and enhanced chromosome loss rates [4] leading to increased clonal diversity and population fitness of the tumor. Additionally, chromosome arms that are gained or lost recurrently in cancer are enriched for oncogenes and tumor
suppressors, respectively [5], so the cancer genome can be sculpted by aneuploidization to optimize gene dosage. It is therefore critical for tissues to avoid the accumulation of aneuploid cells derived from spontaneous errors in mitosis or else risk a slippery slope toward oncogenic transformation. Do surveillance mechanisms exist to detect and eliminate aneuploid cells? p53, the well-known tumor suppressor and ‘guardian of the genome’ that induces cell-cycle arrest, senescence, or apoptosis in response to DNA damage has also been proposed as a ‘guardian of ploidy’ [6]. Activation of p53 and subsequent cell cycle arrest has been observed following aneuploidization in various cell lines and loss of p53 is associated with increased aneuploidy in human tumors [7]. However, it is unclear whether p53 is activated by aneuploidy itself via some unknown chromosome accounting system or whether it is activated via other conditions associated with aneuploidy (e.g., replication stress) or simply the drugs used to induce aneuploidization. Soto et al. set out to explore these questions by inducing aneuploidy in a fairly karyotypically normal, untransformed cell line (RPE1) using chemical inhibitors of centrosome function and the spindle assembly checkpoint, followed by karyotyping of cells under both p53 wild-type and p53 knockdown conditions [8]. Recently generated aneuploid cells exhibited more p53 activation than normal cells and knockdown of p53 permitted more aneuploid cells to enter mitosis. Therefore, p53 is activated by aneuploidization and induces cell-cycle arrest, but not in all cases: single-cell sequencing revealed many aneuploid cells in the cycling population, most exhibiting single whole-chromosome gains or losses. The initial population (containing many arrested cells) had more complex compound aneuploidies, with structural changes (subchromosome gains or losses). However, in p53 knockdown cells these structural
aneuploidies were found to be abundant in the cycling population. This suggests that p53 preferentially causes cells with more complex, structural aneuploidies to arrest, but is permissive of many single whole-chromosome aneuploidies. Santaguida et al. also utilized single-cell sequencing to characterize aneuploid RPE1 populations and found more complex karyotypes among the arrested cells, with an average of 20% of the genome affected [9]. Cells that were capable of cycling had only 0–5% of their genomes altered. There was also an approximately twofold increase in structural aneuploidies in the arrested population compared with cycling cells, and p53 activation only in the arrested population. To identify the causes of p53 activation, Santaguida et al. assessed whether DNA damage was increased in recently formed aneuploid cells and found that g-H2AX foci were indeed increased. Examination of DNA replication dynamics revealed that aneuploid cells have slower replication forks, more fork stalling, and prolonged presence of PCNA foci indicating a delay in S phase. In addition, ultrafine anaphase DNA bridges were observed in aneuploid cells, which may indicate unresolved replication stress. Santaguida et al. conclude that DNA replication stress in severely aneuploid cells results in DNA damage and p53 activation. Santaguida et al. further show that arrested aneuploid cells exhibit features of senescence and activate an interesting pattern of inflammation and innate immune signals. In addition to interferon signaling, which commonly results from DNA damage-induced senescence, arrested aneuploid cells [79_TD$IF]5activated the cGAS–STING pathway, which responds to cytosolic DNA and activates IRF3 and NF-kB. Arrested aneuploid cells exhibited elevated secretion of the cytokines IL-6, IL-8, and CCL2 and increased surface expression of proteins that are recognized by natural killer (NK) cells. Consequently, arrested aneuploid RPE1 cells
Trends in Genetics, Month Year, Vol. xx, No. yy
1
TIGS 1384 No. of Pages 3
were readily killed by NK cells in vitro damage, an aneuploidy patrol system is needed to prevent accelerated genomic whereas euploid RPE1 cells were not. instability and karyotypic evolution toward These studies taken together paint a pic- tumorigenesis. p53 activation, senesture in which complex aneuploidy causes cence, and NK cells may comprise such (and is then further exacerbated by) repli- a system. Simple whole-chromosome cation stress, which triggers the DNA aneuploidies with less that 5% of the damage response, p53 activation, senes- genome unbalanced do not tend to set cence, and an innate immune response off the DNA damage/p53 alarm. There(Figure 1). This enables recognition and fore, it seems that p53 does not function elimination of senescent aneuploid cells explicitly as a chromosomal accountant by NK cells. Due to the cyclic, feed-for- but rather serves to protect against the ward relationship between aneuploidy proliferation of genomically unstable and replication stress-induced DNA aneuploidies, which more readily evolve
Cells
Monosomy Disomy Trisomy Tetrasomy
Cells
Ploidy state
Chromosome
Chromosome
‘Simple’ single, whole-chromosome aneuploidies <5% genome affected
‘Complex’ paral whole-chromosome aneuploidies >5% genome affected
Mild
Replicaon stress and DNA damage
Severe
p53
DNA damage response
DNA repair Cell-cycle progression
Cell-cycle arrest
Senescence
SASP + immune signals
NK cell detecon and killing Figure 1. Severe Aneuploidy Leads to p53 Activation and Senescence. Soto et al. and Santaguida et al. show by single-cell sequencing that aneuploidization involving single whole-chromosome changes often does not trigger p53 activation or cell-cycle arrest (broken orange path) whereas aneuploidization involving multiple whole- and/or partial-chromosome changes does (dark-blue path). Newly formed cells with complex karyotypes exhibit replication stress and DNA damage, which triggers p53 activation and senescence as well as an immune response leading to natural killer (NK) cell-mediated killing. SASP, senescence-associated secretory phenotype
2
Trends in Genetics, Month Year, Vol. xx, No. yy
toward oncogenic transformation than stable diploids. A key question not addressed by these studies is whether cells are susceptible to senescence and NK surveillance only during the transition from euploidy to aneuploidy or remain sensitive once the cells have adjusted to the aneuploid state. This could be tested by conditionally inhibiting p53 during the aneuploidization phase then releasing p53 inhibition in the complexkaryotype cycling population to determine whether p53 is capable of triggering arrest well past chromosome missegregation. How do some genomically unstable tumors with complex karyotypes bypass the p53/senescence barrier and evade the immune system? One simple mechanism of bypass is p53 mutation; indeed, p53 loss is correlated with increased aneuploidy in human tumors [7]. While p53 mutation and senescence bypass may also mask some of the senescence-associated immune responses exhibited by aneuploid cells, other innate immune responses, such as the cytosolic DNA-triggered cGAS–STING pathway, may be controlled independently. Therefore, aneuploid tumors may need to evolve other ways to silence these responses and evade recognition by NK cells. It appears aneuploid tumors do this quite well, since immune cell infiltration is negatively correlated with tumor aneuploidy [10]. Perhaps aneuploidization via an intermediary tetraploid state [11] is a less dramatic route toward a tumorigenic karyotype: individual chromosome loss or gain would affect a smaller percentage of the tetraploid genome and therefore be less likely to unbalance the replication machinery, cause replication stress, and activate p53 and senescence. This would enable incremental refinement of tumor karyotypes. 1 Howard Hughes Medical Institute, Department of Genetics, Harvard University Medical School, Boston,
MA, USA [78_TD$IF]2 Division of Genetics, Brigham and Women’s Hospital, Boston, MA, USA
TIGS 1384 No. of Pages 3
*Correspondence: [email protected] (S.J. Elledge). http://dx.doi.org/10.1016/j.tig.2017.07.007 References 1. Hanks, S. et al. (2004) Constitutional aneuploidy and cancer predisposition caused by biallelic mutations in BUB1B. Nat. Genet. 36, 1159–1161 2. Zimonjic, D. et al. (2001) Derivation of human tumor cells in vitro without widespread genomic instability. Cancer Res. 61, 8838–8844
4. Passerini, V. et al. (2016) The presence of extra chromosomes leads to genomic instability. Nat. Commun. 7, 10754
8. Soto, M. et al. (2017) p53 prohibits propagation of chromosome segregation errors that produce structural aneuploidies. Cell Rep. 19, 2423–2431
5. Davoli, T. et al. (2013) Cumulative haploinsufficiency and triplosensitivity drive aneuploidy patterns and shape the cancer genome. Cell 155, 948–962
9. Santaguida, S. et al. (2017) Chromosome mis-segregation generates cell-cycle-arrested cells with complex karyotypes that are eliminated by the immune system. Dev. Cell 41, 638–651.e5
6. Aylon, Y. and Oren, M. (2011) p53: guardian of ploidy. Mol. Oncol. 5, 315–323 7. Clausen, O.P.F. et al. (1998) Association of p53 accumulation with TP53 mutations, loss of heterozygosity at 17p13, and DNA ploidy status in 273 colorectal carcinomas. Diagn. Mol. Pathol. 7, 215–223
10. Davoli, T. et al. (2017) Tumor aneuploidy correlates with markers of immune evasion and with reduced response to immunotherapy. Science 355, eaaf8399 11. Storchova, Z. and Kuffer, C. (2008) The consequences of tetraploidy and aneuploidy. J. Cell Sci. 121, 3859–3866
3. Fujiwara, T. et al. (2005) Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells. Nature 437, 1043–1047
Trends in Genetics, Month Year, Vol. xx, No. yy
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