Global analysis of genome, transcriptome and proteome reveals the response to aneuploidy in human cells

Abstract

Genomic, transcriptomic and proteomic profiles of human aneuploid cells reveal that mRNA levels increase with gene copy number, but protein levels are partially compensated. Aneuploid cells also exhibit common alterations in several pathways, including an activation of autophagy.

Aneuploidy: implications for protein homeostasis and disease

“…Autophagy can be monitored by assessing the number of light chain 3 (LC3)-positive foci, or by assaying the abundance of LC3-II, the autophagosome-specific lipidated form of LC3. Aneuploid human cell lines created by chromosome transfer and trisomic MEFs harbor significantly increased numbers of LC3 foci and LC3-II protein levels relative to the euploid control lines ( Stingele et al, 2012 ). p62 protein abundance is also significantly increased in aneuploid human cell lines, suggesting an increase in p62-dependent autophagy in aneuploid human cell lines ( Stingele et al, 2012 ).…”

“…Aneuploid human cell lines created by chromosome transfer and trisomic MEFs harbor significantly increased numbers of LC3 foci and LC3-II protein levels relative to the euploid control lines ( Stingele et al, 2012 ). p62 protein abundance is also significantly increased in aneuploid human cell lines, suggesting an increase in p62-dependent autophagy in aneuploid human cell lines ( Stingele et al, 2012 ). Because the effects on autophagy were detected in many aneuploid cell lines irrespective of the chromosomes that were supernumerary, higher levels of autophagosomes seems to be a cellular response to aneuploidy in mammalian cell lines, most likely deployed to manage the increased abundance of misfolded proteins.…”

“…Several previous studies have suggested that aneuploidy places strain on protein quality-control systems. As mentioned above, budding yeast, mouse and human aneuploid cells exhibit expression of cellular stress response genes ( Torres et al, 2007 ; Sheltzer et al, 2012 ; Stingele et al, 2012 ), and this response includes upregulation of protein chaperones ( Sheltzer et al, 2012 ). Human aneuploidies generated by chromosome transfer in vitro were found to have a transcriptional stress signature that shows upregulation of lysosome-mediated degradation and p62-dependent autophagy ( Stingele et al, 2012 ; Stingele et al, 2013 ).…”

“…As mentioned above, budding yeast, mouse and human aneuploid cells exhibit expression of cellular stress response genes ( Torres et al, 2007 ; Sheltzer et al, 2012 ; Stingele et al, 2012 ), and this response includes upregulation of protein chaperones ( Sheltzer et al, 2012 ). Human aneuploidies generated by chromosome transfer in vitro were found to have a transcriptional stress signature that shows upregulation of lysosome-mediated degradation and p62-dependent autophagy ( Stingele et al, 2012 ; Stingele et al, 2013 ). Furthermore, many haploid S. cerevisiae strains harboring an additional chromosome (disomic yeast strains) were found to be sensitive to chemical compounds that impair protein quality control; many disomic yeast strains are sensitive to the proteasome inhibitor MG132, the ribosome poison cycloheximide, and the Hsp90 inhibitors radicicol and geldanamycin ( Torres et al, 2007 ).…”

“…It is important to note that, although aneuploid yeast and human cells are able to elicit both a heat shock response and an unfolded protein response when prompted by heat stress or endoplasmic reticulum (ER) stress, they do not elicit these responses under normal (unchallenged) growth conditions ( Oromendia et al, 2012 ; Stingele et al, 2012 ). This indicates that there is a proteotoxicity threshold required to elicit the acute heat shock or unfolded protein response that aneuploid cells do not meet.…”

“…Stingele and colleagues looked at the relationship between gene dosage and protein levels in human aneuploid cells ( Stingele et al, 2012 ). Analysis of the transcriptome and proteome of aneuploid human cells generated by chromosome transfer showed that most genes are expressed in proportion to their copy number, and proteins are translated in strong correlation with the abundance of mRNA, resulting in a dramatic change in cellular protein composition with aneuploidy ( Stingele et al, 2012 ). However, as in aneuploid yeast, human aneuploid cells were also found to maintain a subset of proteins (enriched for complex subunits) at stoichiometric levels even if gene copy number was altered.…”

“…Indeed, usually, subunits that are endogenously expressed in excess because of aneuploidy retain stoichiometric proportions within multimeric complexes ( Torres et al, 2007 ; Torres et al, 2010 ). Stingele and colleagues looked at the relationship between gene dosage and protein levels in human aneuploid cells ( Stingele et al, 2012 ). Analysis of the transcriptome and proteome of aneuploid human cells generated by chromosome transfer showed that most genes are expressed in proportion to their copy number, and proteins are translated in strong correlation with the abundance of mRNA, resulting in a dramatic change in cellular protein composition with aneuploidy ( Stingele et al, 2012 ).…”

“…These phenotypes include a cell cycle delay in G1 ( Torres et al, 2007 ; Stingele et al, 2012 ; Thorburn et al, 2013 ), metabolic alterations ( Williams et al, 2008 ; Li et al, 2010 ), genomic instability ( Sheltzer et al, 2011 ; Zhu et al, 2012 ) and proteotoxicity ( Torres et al, 2007 ; Tang et al, 2011 ; Oromendia et al, 2012 ; Stingele et al, 2012 ). Furthermore, most aneuploid cells studied to date exhibit a transcriptional signature associated with slow growth and stress ( Torres et al, 2007 ; Sheltzer et al, 2012 ; Stingele et al, 2012 ; Foijer et al, 2013 ). In Drosophila , activation of the stress kinase Jun N-terminal kinase (JNK) occurs in epithelial cells upon chromosome mis-segregation ( Dekanty et al, 2012 ).…”

A Transcriptional and Metabolic Signature of Primary Aneuploidy Is Present in Chromosomally Unstable Cancer Cells and Informs Clinical Prognosis

“…A recent report has analyzed the consequences of transferring additional chromosomes into HCT116 cells, a karyotypically stable (INH=0) colon cancer line (29). I acquired microarray data from HCT116 cells that had two extra copies of chromosome 5 introduced via microcell-mediated chromosome transfer (MMCT).…”

“…Whole-chromosome aneuploidy alters the copy numbers of hundreds or thousands of genes after a single mitotic event. Human cells lack a global dosage compensation system for autosomal aneuploidy; most copy number variation has proportional effects on the levels of mRNA's and proteins encoded on the aneuploid chromosome (29). Thus, it has been hypothesized that aneuploidy induces a fitness cost by wasting energy on the transcription, translation, and degradation of unnecessary proteins, and by interfering with the formation or function of stoichiometry-sensitive protein complexes (49).…”

“…Consumption/release rates of metabolites in the NCI60 panel were acquired from (28). Gene expression values from tetrasomic HCT116 cells were acquired from (29). Gene expression values from normal human tissues were acquires from the Gene Expression Omnibus (GSE1133).…”

Transcriptional effects of copy number alterations in a large set of human cancers

“…We have previously shown that the effects of CNAs on gene expression levels are often overshadowed by other (non-genetic) effects that are not associated with specific single CNAs 9 . For example, Stingele et al showed that aneuploidy invokes a general cellular mRNA response which is not the result of any specific CNAs but only the presence of aneuploidy 14 . In addition, tumor cells in vivo may develop transcriptional adaptive mechanisms to survive under selective pressure, which may be different from the adaptive mechanisms observed in vitro.…”

“…We obtained gene expression profiles of an experimental model of aneuploidy introduced in HCT116 cells 14 . Differences on the mRNA level and TACNA level were calculated between the parental HCT116 cells (n = 3) and an isogenic model with chromosome 5 tetrasomy (n = 3) ( Supplementary Fig.…”

Copy number variation study in Japanese quail associated with stress related traits using whole genome re-sequencing data

“…We have identified several hundred genes that were fully deleted in HS and LS lines of quail, which supports a phenomenon of perpetual gene turnover in the two quail populations and their genetic differences. Duplication of whole genes has been known to impact gene expression by altering gene dosage [50, 51]. If a duplication of a gene is adaptive, it is usually favored and retained more frequently in a population [1].…”

Systems approaches identify the consequences of monosomy in somatic human cells

“…Previous work on trisomic human cells uncovered that the protein abundance of approximately 25% of proteins encoded on the trisomes was adjusted to closely match the disomic abundance, and this was most strikingly prominent for proteins subunits of macromolecular complexes 25 , 26 . Comparison of the abundance of subunits of macromolecular complexes, as defined in the CORUM database, with the non-CORUM proteins revealed only a subtle shift towards diploid levels, suggesting that this mechanism is not important in monosomic cells (Fig.…”

“…For further analysis comparing genomic, transcriptomic, and proteomic datasets, the DNA and mRNA datasets were matched to the corresponding protein entries and merged into a single table (Supplementary Data 1 ). To compare monosomic and trisomic cell lines, proteome data of trisomic RPE cell lines 25 was merged to the dataset. Chromosome/scaffold name, gene start (bp), gene stop (bp) and Ensembl gene stable ID (ENSG) were annotated through BioMart.…”

Suppressing Aneuploidy-Associated Phenotypes Improves the Fitness of Trisomy 21 Cells

“…To investigate whether the acquisition of an extra copy of a given chromosome affects the nucleus in human cells, we analyzed the nuclear morphology of a series of human trisomic cell lines constructed by micronuclei-mediated chromosome transfer into RPE-1 cells (Stingele et al, 2012). As observed in primary fibroblasts, we found that up to 40% of the RPE-1 cells harboring extra copies of chromosome 7 (RPE-1 7/3), chromosome 8 (RPE-1 8/3), or chromosome 21 (RPE-1 21/3) also display abnormal nuclei compared with their control counterparts (Figures S6E and S6F).…”

Aneuploidy tolerance caused by BRG1 loss allows chromosome gains and recovery of fitness

“…Proteotoxic stress associated with imbalances in copy number in aneuploid cells means that proteasome activity and regulation are important for tolerating aneuploidy 3 , 7 , 23 – 25 . The core 20 S proteasome can associate with the 19 S regulatory caps to form the 26 S proteasome, which facilitates the degradation of ubiquitin-targeted proteins.…”

Altering microtubule dynamics is synergistically toxic with spindle assembly checkpoint inhibition

“…CIN and the resulting aneuploidy lead to a deregulated transcriptome and proteome (Tang et al, 2011;Stingele et al, 2012;Foijer et al, 2013Foijer et al, , 2014 and can provoke cell cycle delay, senescence, or apoptosis (Giam & Rancati, 2015;Andriani et al, 2016;Santaguida et al, 2017;Chunduri & Storchová, 2019). Furthermore, ongoing CIN can lead to further DNA damage (Zhang et al, 2015;MacKenzie et al, 2017).…”

“…We, therefore, reasoned that targeting RNA or protein processing, transcriptional regulation, apoptosis, or DNA repair might be particularly toxic to aneuploid cells and cells exhibiting a CIN phenotype. As CIN and aneuploidy are different concepts (Schukken & Foijer, 2018) and have different consequences for cells (Stingele et al, 2012;Andriani et al, 2016;Schukken & Foijer, 2018) aneuploidy and CIN might impose different therapeutic vulnerabilities. To test this, we performed two small-scale drug screens, one to identify compounds that selectively prevent the propagation of aneuploid cells and another to identify small molecules that selectively targets CIN cells.…”

“…"n" referrers to the number of mitotic events per condition. " # " refers to that the same data are also used in Fig 2E. Aneuploid cells are sensitive to compounds that hyperactivate the cellular metabolism When we screened for compounds that selectively prevent expansion of aneuploid cells, we found that ZLN005, a transcriptional stimulator of PGC-1α, was significantly more toxic to doubletrisomic RPE1 Ts12 Ts5 cells (Stingele et al, 2012) than control cells. PGC-1α is a master regulator of mitochondrial biogenesis and energy metabolism and its activation is, thus, expected to increase mitochondrial respiration.…”

“…In this study, we explored whether cells exhibiting CIN or stable aneuploidy displayed selective vulnerabilities to particular drugs. As CIN and aneuploidy trigger a number of responses in cells, including, but not limited to proteotoxic stress (Oromendia et al, 2012;Stingele et al, 2012), a deregulated cellular metabolism (Williams et al, 2008;Tang et al, 2011), a DNA damage response (Zhang et al, 2015;MacKenzie et al, 2017), senescence (Andriani et al, 2016), and apoptosis (Ohashi et al, 2015), we selected a small library of Food and Drug Administrationapproved drugs or compounds in clinical trials targeting aneuploidy/ CIN-related responses.…”

“…HT29 cells were growth in McCoy media supplemented with 10% FBS and Pen/Strep as above. Aneuploid cells RPE1 with trisomy 12 and trisomy 5 (Ts12 Ts5) and RPE1 hTert cells were kindly provided by the Storchova laboratory (Stingele et al, 2012). Most drugs used in this study were synthesized by Syncom B.V., except for AZD8055 (Sigma-Aldrich), EPZ015666 (Sigma-Aldrich) and SKI-1 (Abcam).…”

“…For this purpose, we selected a collection of drug-like molecules from a list of drugs already being used in the clinic or in advanced-stage clinical trials. Compounds were further selected for their potential role in targeting CIN or aneuploid cells, such as targeting cell survival (Dekanty et al, 2012;Foijer et al, 2013), proliferation (Williams et al, 2008;Ben-david et al, 2014;Gogendeau et al, 2015;Sheltzer et al, 2017), protein processing (Oromendia et al, 2012;Stingele et al, 2012), DNA repair (Bakhoum et al, 2014(Bakhoum et al, , 2018, transcriptional deregulation (Upender et al, 2004;Stingele et al, 2012), and cellular metabolism (Williams et al, 2008;Tang et al, 2011) as these processes are typically deregulated in aneuploid cells. Indeed, our screen for aneuploidy-targeting compounds revealed a compound targeting cellular metabolism, validating earlier findings from the Amon laboratory (Tang et al, 2011).…”

Altering microtubule dynamics is synergistically toxic with inhibition of the spindle checkpoint

“…Fig. 1A , (Stingele et al , 2012)), to the same drug-treatment regime and compared proliferation between diploid and aneuploid RPE1 cells (Sup. Data 1) using an Incucyte high content imager.…”

“…We therefore reasoned that targeting RNA or protein processing, transcriptional regulation, apoptosis, or DNA repair might be particularly toxic to aneuploid cells and cells exhibiting a CIN phenotype. As CIN and aneuploidy are different concepts (Schukken & Foijer, 2017) and have different consequences for cells (Schukken & Foijer, 2017; Stingele et al , 2012; Andriani et al , 2016), aneuploidy and CIN might impose different therapeutic vulnerabilities. To test this, we performed two small-scale drug screens, one to identify compounds that selectively kill aneuploid cells and another to identify small molecules that selectively kill CIN cells.…”

“…CIN and the resulting aneuploidy lead to a deregulated transcriptome and proteome (Stingele et al , 2012; Foijer et al , 2013, 2014; Tang et al , 2011b), and can provoke senescence (Andriani et al , 2016; Santaguida et al , 2017) or apoptosis (Giam & Rancati, 2015). Furthermore, ongoing CIN can lead to further DNA damage (Zhang et al , 2015; MacKenzie et al , 2017).…”

“…When we screened for compounds that selectively kill aneuploid cells, we found that ZLN005, a transcriptional stimulator of PGC-1α, was significantly more toxic to double-trisomic RPE1 Ts12 Ts5 cells (Stingele et al , 2012) than control cells. PGC-1α is a master regulator of mitochondrial biogenesis and energy metabolism and its activation is thus expected to increase the cellular metabolism.…”

“…In this study, we explored whether cells exhibiting CIN or stable aneuploidy displayed selective vulnerabilities to particular drugs. As CIN and aneuploidy trigger a number of responses in cells, including, but not limited to proteotoxic stress (Oromendia et al , 2012; Stingele et al , 2012), a deregulated cellular metabolism (Tang et al , 2011b; Williams et al , 2008), a DNA damage response (Zhang et al , 2015; MacKenzie et al , 2017), senescence (Andriani et al , 2016) and apoptosis (Ohashi et al , 2015), we selected a small library of FDA-approved drugs or compounds in clinical trials targeting aneuploidy/CIN-related responses.…”

“…HT29 cells were growth in McCoy media supplemented with 10% FBS and Pen/Strep as above. Aneuploid cells RPE1 with trisomy 12 and trisomy 5 (Ts12 Ts5) and RPE1 hTert cells were kindly provided by the Storchova lab (Stingele et al , 2012). Most drugs used in this study were synthesized by Syncom B.V. (Groningen, NL), except for AZD8055 (Sigma), EPZ015666 (Sigma), and SKI-1 (Abcam).…”

“…For this purpose, we selected a collection of drug-like molecules from a list of drugs already being used in the clinic, or in advanced stage clinical trials. Compounds were further selected for their potential role in targeting CIN or aneuploid cells, such as targeting cell survival (Dekanty et al , 2012; Foijer et al , 2013), proliferation (Gogendeau et al , 2015; Ben-david et al , 2014; Sheltzer et al , 2017; Williams et al , 2008), protein processing (Oromendia et al , 2012; Stingele et al , 2012), DNA repair (Bakhoum et al , 2014, 2018), transcriptional deregulation (Upender et al , 2004; Stingele et al , 2012), and cellular metabolism (Tang et al , 2011b; Williams et al , 2008) as these processes are typically deregulated in aneuploid cells. Indeed, our screen for aneuploidy-targeting compounds revealed a compound targeting cellular metabolism, validating earlier findings from the Amon lab (Tang et al , 2011a).…”

Single-chromosome Gains Commonly Function as Tumor Suppressors

“…As aneuploidy is a nearly universal occurrence in cancer, we hypothesized that the acquisition of aneuploidy in previously transformed cells, rather than in primary cells, could have distinct, protumorigenic consequences. To test this, we utilized a series of chromosomally stable human colorectal cancer cells (HCT116) into which extra chromosomes had been introduced via microcell-mediated chromosome transfer (Donnelly et al, 2014; Stingele et al, 2012). …”

“…Aneuploid chromosomes are transcribed and translated proportional to their copy number (Dephoure et al, 2014; Stingele et al, 2012; Torres et al, 2007, 2010), which can lead to stoichiometric imbalances in endogenous proteins and protein complexes (Oromendia et al, 2012; Sheltzer and Amon, 2011). To compensate, cells rely on a set of protein quality control mechanisms, including the HSF1/HSP90 folding pathway (Donnelly et al, 2014; Oromendia et al, 2012), autophagy, and proteasomal degradation (Dephoure et al, 2014; McShane et al, 2016; Santaguida et al, 2015; Stingele et al, 2012; Torres et al, 2010). The energetic cost of expressing, folding, and turning over excess proteins, as well as the downstream consequences of unmitigated protein imbalances, impose a significant fitness cost on the cell.…”

“…All animal studies and procedures were approved by the MIT Institutional Animal Care and Use Committee. Aneuploid cell lines derived from HCT116 cells were previously described in Stingele et al (2012). Further experimental details are provided in the Supplemental Information.…”

“…In order to further our understanding of the effects of aneuploidy on cell and organismal physiology, systems have been developed to generate cells with a range of aneuploid karyotypes (Pavelka et al, 2010; Stingele et al, 2012; Torres et al, 2007; Williams et al, 2008). These cells have been constructed without CIN-promoting mutations, thereby allowing the study of aneuploidy in the absence of other genetic perturbations.…”

“…These cells have been constructed without CIN-promoting mutations, thereby allowing the study of aneuploidy in the absence of other genetic perturbations. This research has demonstrated the existence of a set of phenotypes that are shared among many different aneuploid cells and are largely independent of the specific chromosomal alteration: aneuploid cells display reduced fitness (Stingele et al, 2012; Torres et al, 2007; Williams et al, 2008), are deficient at maintaining proteostasis (Donnelly et al, 2014; Oromendia et al, 2012; Tang et al, 2011), and exhibit a specific set of gene expression changes that include the downregulation of cell-cycle transcripts and the upregulation of a stress-response program (Dürrbaum et al, 2014; Sheltzer, 2013; Sheltzer et al, 2012). A crucial question, however, is in what way(s) the cellular changes induced by aneuploidy affect (and possibly drive) tumorigenesis.…”