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.

The presence of extra chromosomes leads to genomic instability

“…3c ). These findings are in agreement with our previous observations that the progression through S-phase is impaired by the presence of extra chromosomes 4 .…”

“…We asked whether these expression changes might explain the phenotypes observed in aneuploid cells. To this end, we analysed protein expression data previously generated using quantitative mass spectrometry 4 . Unsupervised hierarchical clustering revealed downregulation of several replication factors in aneuploid cells ( Fig.…”

“…To generate aneuploid HCT116 and RPE1 containing an additional chromosome 3, 5, 8 or 21, microcell fusion ( Supplementary Fig. 1a ) was performed as follows 53 . In brief, murine donor cells containing additional human chromosome with a resistance gene were treated for 48 h with colchicine (final concentration 60 ng ml −1 ).…”

“…To understand the relationship between CIN and GIN, it is crucial to determine whether and how numerical aneuploidy by itself affects genome stability. Recently established model cells with defined aneuploid karyotypes have facilitated the analysis of the immediate consequences of aneuploidy per se 1 4 16 17 18 19 . Direct comparison of cognate euploid and aneuploid cells revealed that aneuploidy triggers distinct and conserved changes in gene expression 20 21 .…”

“…Direct comparison of cognate euploid and aneuploid cells revealed that aneuploidy triggers distinct and conserved changes in gene expression 20 21 . Addition of even a single extra chromosome causes profound defects in the maintenance of proteostasis as aneuploid cells are more sensitive to inhibitors of protein folding, protein translation and degradation, activate protein degradation pathways and show marked protein folding defects 3 4 5 18 22 23 . Observations in budding and fission yeasts suggested that aneuploidy impairs the fidelity of chromosome segregation, and increases mutation and recombination rates, although the underlying mechanisms remain enigmatic 17 24 25 26 .…”

“…A deviation from the normal chromosome number often markedly affects their physiology. Whole-chromosome gains or losses—called numerical aneuploidy—have particularly detrimental effects in Metazoans, as cells often suffer from impaired proliferation, increased sensitivity to proteotoxic stress and altered gene expression 1 2 3 4 . Structural aneuploidy, characterized by sub-chromosomal unbalanced rearrangements, that is, duplications or deletions of large genomic regions, also affects cellular functions.…”

Genotoxic stress in constitutive trisomies induces autophagy and the innate immune response via the cGAS-STING pathway

“…We hypothesized that this pathway might contribute to the increased TFEB activity in response to trisomy. This hypothesis is consistent with previous observations that constitutive trisomy activates the type I interferon pathway 11 , 21 , 22 . Recently, endogenous DNA damage and replication stress, which is also a hallmark of trisomic cells 3 , 13 , 19 , 20 , has been shown to cause accumulation of cytoplasmic DNA, thereby contributing to activation of the type I interferon pathway 23 , 25 , 26 , 41 .…”

“…The engineered cell lines were named according to their origin (H: HCT116; R: RPE1), the type of chromosome gain (tr: trisomic; te: tetrasomic), and the transferred chromosome (3, 5, 7, 8, 12, 18, or 21), followed by a clone number (e.g., Htr3_11 is a trisomy of chromosome 3 in HCT116, clone 11, Supplementary table 1). Proteome and transcriptome analysis revealed a general upregulation of a wide range of autophagy and lysosomal factors in these cell lines 11,21 . Detailed examination of the transcriptome and proteome data showed that specifically the targets of the transcription factor TFEB, such as LC3 (MAP1LC3B), SQSTM1, VPS18, and WIPI1, as well as cathepsins D, B and A, and other lysosome-specific proteases were upregulated in the analyzed cell lines (Fig.…”

“…Previous data revealed an increased expression of autophagosomal and lysosomal factors and increased levels of autophagosomes and lysosomes accompanied by a reliance on phagolysosome activity in somatic trisomic mammalian cells 11 , 17 . The presence of extra chromosomes leads to the production of excessive proteins that may overwhelm the capacity of lysosomal degradation, as was observed in cells acutely missegregating chromosomes 39 .…”

“…TFEB-dependent transcription and autophagy is activated in constitutive trisomies. Previously, we established isogenic series of human cell lines derived from HCT116 or RPE1 that carry an extra copy of one or more chromosomes 11,38 . The engineered cell lines were named according to their origin (H: HCT116; R: RPE1), the type of chromosome gain (tr: trisomic; te: tetrasomic), and the transferred chromosome (3, 5, 7, 8, 12, 18, or 21), followed by a clone number (e.g., Htr3_11 is a trisomy of chromosome 3 in HCT116, clone 11, Supplementary table 1).…”

“…CCL-247) with pBOS–H2B–GFP (BD Pharmingen) according to the manufacturer’s protocols 63 . Trisomic and tetrasomic cell lines were generated by microcell-mediated chromosome transfer as previously described 11 . The cell line Hte5_1 was kindly provided by Minoru Koi, Baylor University Medical Centre, Dallas, TX, USA.…”

“…Transcriptome and proteome analysis. Genome-wide proteome and transcriptome expression profiling of HCT116-and RPE1-derived aneuploid cell lines was previously performed 11,21 . As previously described, bioinformatics analysis of the proteomic and transcriptomic data was performed using Perseus (1.6.2.3) as part of the MaxQuant Software Package.…”

“…Cells with extra chromosomes also upregulate autophagy and lysosomal genes and show increased levels of LC3-positive autophagosomes 11 , 21 . Moreover, murine aneuploid cells were selectively sensitive to treatment with chloroquine, a drug that impairs lysosomal function and thus blocks autophagic degradation 17 .…”

“…Building on our previous knowledge of the consequences of chromosome gain in mammalian cells and recent findings that the activation of both autophagy and the innate immune response might be tightly linked, we sought to determine the cellular mechanisms that contribute to the constitutive upregulation of autophagy and lysosomal pathways in trisomic cells. To this end, we used a series of trisomic cell lines derived from RPE1 (a diploid, immortalized retinal pigmented epithelial cell line) and HCT116 (a diploid colorectal cancer cell line) generated by microcell-mediated chromosome transfer 11 . Strikingly, we show that activation of autophagy and the lysosomal pathway in constitutively trisomic cells is independent of mTORC1 status.…”

“…Recently established model mammalian somatic cell lines with defined chromosome gains have allowed in vitro studies of the effects of aneuploidy on cellular function [9][10][11] . These studies revealed that the gain of a single chromosome triggers a uniform and conserved deregulation of specific pathways that primarily affect cell proliferation and genome and proteome homeostasis 7,12,13 .…”

Aneuploidy-driven genome instability triggers resistance to chemotherapy

“…We then removed the drug and monitored their proliferation over time either in the absence ( Figure 1A-C) or in the presence of a chemotherapeutic agent ( Figure 1D-G). In agreement with previous reports Williams et al 2008;Sheltzer et al 2017;Stingele et al 2012;Santaguida et al 2017;Tang et al 2011), induction of CIN led to decreased proliferation ( Figure 1B, C). To test the effects of CIN on cell proliferation in the presence of a chemotherapeutic agent, cancer cell lines were exposed to a battery of chemotherapeutic drugs, after reversine removal ( Figure 1D, E).…”

Extensive protein dosage compensation in aneuploid human cancers

“…To visualize these expression patterns, we generated density plots of the expression differences of subsets of genes that were encoded on aneuploid chromosomes (Figure 3B). Consistent with previous results 24,26,41,42 , we observed that ribosomal genes, RNA processing genes, spliceosome components, and other genes that encode protein complex subunits were enriched among genes that were buffered upon chromosome gain. Interestingly, we also found that these proteins tended to be buffered upon chromosome loss, and decreased in expression less than other proteins encoded on lost chromosomes.…”

“…Consistent with prior publications 24,41 , we observed that ribosomal and rRNA processing terms were strongly negatively correlated with cellular aneuploidy scores at both the RNA and protein level (Figure 4B, S4B, Supplementary File 3). Indeed, the two most significantly negatively correlated proteins are the ribosome-associated proteins RPL22L1 and RNF138 (Figure 4C).…”

“…Consistent with previous results 41,42,62,63 , we found that protein complex subunits exhibited significant buffering when they were encoded upon a gained chromosome arm. This trend is exemplified by ribosomal complex proteins 24,26 , which showed highly-significant dosage compensation. Interestingly, we discovered that protein complex subunits were also significantly buffered upon chromosome arm loss.…”

“…Previously, Stingele et al 24 created aneuploid cell lines by introducing one or more extra chromosomes into chromosomally-stable colon cancer cell lines and non-transformed epithelial cell lines. They then determined the effects of these chromosome gains on gene expression relative to their near-euploid parental cell lines.…”

“…Stable aneuploidy cell line data were retrieved from Stingele et al 2012 24 . Protein and RNA difference upon aneuploidy was calculated by taking the log2 fold change between aneuploidy clones and their near-euploid cell line controls.…”

“…If these patterns of scaling and buffering represent a fundamental consequence of aneuploidy, then we would expect to observe similar expression patterns in other aneuploid cell lines. To investigate this possibility, we examined published transcriptome and proteome data from stably-aneuploid human cell lines 24 , Down syndrome fibroblasts 57,58 , and aneuploid yeast 39 .…”

“…However, we have significantly less insight into how aneuploidy shapes the proteome of human cancers. Studies performed on single cancer cell lines harboring a few chromosome gains have suggested that human cells similarly overexpress most proteins on gained chromosomes 24,[42][43][44] , with ribosomes and some protein complex subunits exhibiting dosage compensation 24,41,42,45 . Additionally, while chromosome loss events outnumber chromosome gain events in most tumors 46,47 , the cellular effects of chromosome losses in human cancers have not been comprehensively explored 48 .…”

“…Despite these findings, aneuploidy itself has also been observed to induce substantial tumor suppressive stresses 10,22,23 . Aneuploid cells have increased metabolic requirements [24][25][26][27][28][29] , display high levels of senescence 23,[30][31][32][33] , exhibit significant genomic instability 34,35 , and are sensitive to compounds that interfere with protein folding and turnover 28,[36][37][38] . Aneuploidy-associated stresses may be caused by the deregulation of gene expression, which leads to the imbalanced production of key cellular proteins.…”

Extensive protein dosage compensation in aneuploid human cancers

“…Consistent with previous results ( Stingele et al 2012 ; Dephoure et al 2014 ; McShane et al 2016 ; Brennan et al 2019 ), we observed that ribosomal genes, RNA processing genes, spliceosome components, and other genes that encode protein complex subunits were enriched among genes that were buffered upon chromosome gain. We also found that these proteins tended to be buffered upon chromosome loss and decreased in expression less than other proteins encoded on lost chromosomes.…”

“…Consistent with prior publications ( Stingele et al 2012 ; Brennan et al 2019 ), we observed that ribosomal and rRNA processing terms were strongly negatively correlated with cellular aneuploidy scores at both the RNA and protein level ( Fig. 4 B; Supplemental Fig.…”

“…As aneuploidy has been widely reported to significantly alter the expression of genes encoded on altered chromosomes ( Pollack et al 2002 ; Tsafrir et al 2006 ; Grade et al 2007 ; Torres et al 2007 ; Gu et al 2008 ; Fontanillo et al 2012 ; Stingele et al 2012 ; Davoli et al 2013 ), we were surprised by our discovery that chromosome copy number changes affected average protein expression by only ∼10%. In light of this finding, we conducted several control analyses to investigate alternate explanations for these results.…”

“…Consistent with previous results ( Geiger et al 2010 ; McShane et al 2016 ; Gonçalves et al 2017 ; Brennan et al 2019 ; Taggart et al 2020 ; Senger and Schaefer 2021 ), we found that protein complex subunits showed significant buffering when they were encoded upon a gained chromosome arm. This trend is exemplified by ribosomal complex proteins ( Stingele et al 2012 ; Dephoure et al 2014 ), which showed highly significant dosage compensation. A recent study showed that protein complex subunits located on nonaneuploid chromosomes tend to co-regulate with their co-complex partners on aneuploid chromosomes in human tumor samples ( Senger et al 2022 ).…”

“…If these patterns of scaling and buffering represent a fundamental consequence of aneuploidy, then we would expect to observe similar expression patterns in other aneuploid cell lines. To investigate this possibility, we examined published transcriptome and proteome data from stably aneuploid human cell lines ( Stingele et al 2012 ), Down syndrome fibroblasts ( Letourneau et al 2014 ; Liu et al 2017 ), and aneuploid yeast ( Supplemental Fig. S6 ; Dephoure et al 2014 ).…”

“…We first assessed the difference in protein expression between four published cell lines engineered to have a stable gain of Chromosome 5, normalized to their corresponding euploid controls ( Stingele et al 2012 ). We found that there was a highly significant correlation between the expression difference of proteins encoded on Chromosome 5 among CCLE lines with Chromosome 5 gains and the expression of those same proteins in the engineered cell lines (corr = 0.431, P < 5 × 10 −10 ) ( Supplemental Fig.…”

“…Despite these findings, aneuploidy itself has also been observed to induce substantial tumor-suppressive stresses ( Sheltzer et al 2017 ; Vasudevan et al 2020 , 2021 ). Aneuploid cells have increased metabolic requirements ( Williams et al 2008 ; Tang et al 2011 ; Stingele et al 2012 ; Sheltzer 2013 ; Dephoure et al 2014 ; Schukken et al 2020 ), display high levels of senescence ( Estrada et al 2013 ; Sheltzer et al 2017 ; He et al 2018 ; Macedo et al 2018 ; Giam et al 2020 ), show significant genomic instability ( Sheltzer et al 2011 ; Passerini et al 2016 ), and are sensitive to compounds that interfere with protein folding and turnover ( Torres et al 2010 ; Tang et al 2011 ; Donnelly et al 2014 ; Donnelly and Storchová 2015 ). Aneuploidy-associated stresses may be caused by the deregulation of gene expression, which leads to the imbalanced production of key cellular proteins.…”

“…We have significantly less insight into how aneuploidy shapes the proteome of human cancers. Experiments performed on single cancer cell lines harboring a few chromosome gains have suggested that human cells overexpress most proteins on gained chromosomes, with ribosomes and some protein complex subunits showing dosage compensation ( Stingele et al 2012 ; McShane et al 2016 ; Viganó et al 2018 ; Brennan et al 2019 ; Hwang et al 2021 ). Similarly, studies of subchromosomal amplifications in cancers suggest that up to ∼30% of DNA copy number changes do not result in corresponding changes in protein abundance ( Zhang et al 2014 , 2016 ; Mertins et al 2016 ; Gonçalves et al 2017 ; Sousa et al 2019 ).…”

Proteogenomic analysis of cancer aneuploidy and normal tissues reveals divergent modes of gene regulation across cellular pathways

“…TMT-based proteome quantifications, widely adopted in CPTAC database, suffer from the issue of ratio compression and may underestimate the actual change ( Savitski et al, 2013 ). Nevertheless, previous studies using other techniques such as stable isotope labeling with amino acids in cell culture (SILAC) or MS3-based proteomics found widespread protein compensation for complex genes after DNA gain in yeast and pairs of isogenic diploid and aneuploid cell lines, consistent with our observations ( Dephoure et al, 2014 ; Hwang et al, 2021 ; Stingele et al, 2012 ). To further exclude the impact of ratio compression on our results, we performed the analysis shown in Figure 1 on The Cancer Genome Atlas (TCGA; Weinstein et al, 2013 ) COAD samples for which label-free proteomics data is available ( Zhang et al, 2014 ).…”

“…Next, for each SCNA group (loss, deep loss, gain, high gain), we compared the genes belonging to protein complexes (‘CORUM,’ composed of 3449 protein complex genes) with those that do not (‘NoCORUM,’ non-complex genes, i.e., remaining genes) ( Ruepp et al, 2008 ; Figure 1B–D ). In general, we found that protein complex genes had a stronger protein-level compensation compared to non-complex genes ( Figure 1B , FDR < 0.001, Supplementary file 1D ), consistent with previous studies examining the effect of chromosome gains ( Oromendia et al, 2012 ; Stingele et al, 2012 ; Torres et al, 2010 ). Importantly, this was true not only for gains, but also for losses ( Figure 1B , FDR < 0.001, Supplementary file 1D ) and across tumor types ( Figure 1C , FDR < 0.001, Supplementary file 1E ).…”

“…We found that strong compensation at the protein level is common among the seven tumors studied here, while compensation at the RNA level is less common and showed heterogeneous tissue-specific patterns. The protein-level compensation is stronger for genes in protein complexes than non-complex genes, consistent with previous reports ( Stingele et al, 2012 ; Torres et al, 2010 ). Interestingly, the existence of protein-level compensation and its higher degree for protein complex genes were true not only for DNA gains, but also for DNA losses.…”

“…We calculated the pan-cancer distributions of the correlation coefficients for complex and non-complex genes using the mean correlation coefficient value across the seven tumor types ( Figure 2C , Supplementary file 2A ). As expected, we found that the median of the RNA–protein correlations was significantly lower for protein complex genes than for non-complex genes (FDR < 0.001), indicating that protein complex genes tend to have stronger protein-level regulation compared to non-complex genes ( Stingele et al, 2012 ). Strikingly, the opposite was true for RNA-level regulation, where the median of the DNA–RNA correlations was significantly higher for protein complex genes than for non-complex genes (FDR < 0.001).…”

“…Work done in model organisms and isogenic human cells suggests that RNA levels change proportionally to the DNA levels in aneuploid cells (i.e., there is no RNA-level compensation) while the protein levels do not (i.e., there is protein-level compensation) ( Oromendia et al, 2012 ; Stingele et al, 2012 ; Torres et al, 2010 ). In our pan-cancer analysis ( Figure 1B ), this was generally the case, with a significant protein-level compensation (false discovery rate [FDR] < 0.001) and no significant RNA-level compensation except in the high gain group (see also below; FDR < 0.001; Supplementary file 1B ).…”

“…Another way to investigate gene regulation is to measure how RNA and protein abundances change upon alterations in DNA copy number (somatic copy number alterations [SCNAs]) of a given gene that naturally occur in human cancers or that are experimentally engineered in cells. Previous proteomics analyses of aneuploid yeast and human cells have shown that, while most genes exhibit high correlation between DNA copy number and RNA abundance, a significant fraction of genes (20–30%) do not show protein abundance changes that are proportional to the DNA or RNA changes ( Gonçalves et al, 2017 ; Jovanovic et al, 2015 ; McShane et al, 2016 ; Stingele et al, 2012 ; Torres et al, 2007 ). In other words, the change of protein abundance is less than what it is expected based on its DNA change; this phenomenon is referred to as gene compensation.…”

Convergent network effects along the axis of gene expression during prostate cancer progression

“…S7). This result is consistent with the concept that protein complex stoichiometry contributes to the buffering of mRNA changes at the level of proteins [ 21 , 22 , 33 – 35 ]. Thus, molecular patterns in high-grade PCa are more strongly perturbed at all layers, and the effects of genomic variation are progressively but non-uniformly attenuated along the axis of gene expression.…”

Convergent network effects along the axis of gene expression during prostate cancer progression

“…S6). This result is consistent with the concept that protein complex stoichiometry contributes to the buffering of mRNA changes at the level of proteins [22,[32][33][34].…”

Widespread Post-transcriptional Attenuation of Genomic Copy-Number Variation in Cancer

“…The enrichment analysis revealed that proteins involved in complexes and modules of functionally interacting proteins displayed a significant agreement with the copy-number measurements at the transcript level, but this agreement is generally lost at the protein level ( Figure 2 B). This recapitulates previous findings in models of aneuploidy in yeast ( Dephoure et al., 2014 ) and human cell lines ( Stingele et al., 2012 ), showing that these observations generalize from the aneuploidy models to the hundreds of patient tumor samples studied here. To validate the generality of the set of attenuated proteins, we confirmed that these are also recapitulated in independent proteomic cell line panels of triple-negative breast cancer and ovarian cancer ( Coscia et al., 2016 , Lawrence et al., 2015 ) ( Figure 2 C).…”

“…Since copy-number changes are buffered and not observed at the protein level, these are therefore less likely to be drivers of cancer progression and similarly less likely to explain changes in drug associations. Notably, this attenuation was more pronounced in protein subunits and complexes, in agreement with previous observations ( Dephoure et al., 2014 , Stingele et al., 2012 ). This is likely explained by the fact that the stoichiometry of complexes needs to be preserved, and that proteins over-represented compared with other members of the complex are likely degraded due to increased instability ( McShane et al., 2016 ).…”

“…It has been hypothesized that non-attenuated subunits could act as scaffolding proteins or rate-limiting for the assembly of the complex ( Dephoure et al., 2014 ). However, past studies based on aneuploidy models were conducted on a small number of yeast strains or cell lines ( Dephoure et al., 2014 , Stingele et al., 2012 ). Given the large number of tumor samples analyzed here we reasoned that we could more readily identify such subunits that can act as drivers of complex assembly.…”

“…In females, one of the two X chromosomes is inactivated by a specialized RNA-based silencing mechanism ( Avner and Heard, 2001 , Lyon, 1961 ), but such a mechanism does not exist for gene-dosage imbalances in the autosomal chromosomes. Protein and mRNA abundance measurements in models of aneuploidy in yeast and human cells have shown that most autosomal gene duplications are propagated to the protein level, with the notable exception of protein complex subunits that showed attenuated (i.e., less than expected) changes in protein abundance ( Dephoure et al., 2014 , Stingele et al., 2012 ). In yeast aneuploid strains, the discrepancy between gene copy-number and protein abundance has been shown to be mostly due to control of protein abundance by degradation ( Dephoure et al., 2014 ).…”

Post-Translational Dosage Compensation Buffers Genetic Perturbations to Stoichiometry of Protein Complexes

“…Given that the biological function of dosage compensation is to maintain subunit stoichiometry, this result explains our previous observation that cellular systems are very fragile to subunit gene overexpression [ 17 ]. This is also consistent with previous observations that the stoichiometric imbalance caused by aneuploidy strongly correlates with impaired cell growth [ 18 , 33 ]. Similarly, our data support a classical hypothesis called the balance hypothesis that predicts deleterious effects due to imbalanced subunit stoichiometry [ 34 ].…”

“…The robustness in cellular systems to gene copy number changes has been investigated mainly using two approaches: generating aneuploidy of specific chromosomes [ 12 , 18 ] and introducing a plasmid carrying an individual target gene [ 17 ]. The generation of aneuploid cells containing one extra chromosome doubles the number of genes in the additional chromosome.…”

“…(iii) The degradation of subunits via an N-terminal degradation signal at the nascent chain level has been supported by experimental evidence [ 38 ]. In addition, autophagy might be included because higher expression of autophagy-related proteins has been detected in aneuploid mammalian cells [ 15 , 18 ].…”

“…The generation of aneuploid cells containing one extra chromosome doubles the number of genes in the additional chromosome. Several recent studies using aneuploid yeast and mammalian cells have revealed fragility of cellular systems against gene copy number increase in a genome-wide manner [ 12 , 18 ]. The use of a multicopy plasmid carrying an individual target gene dramatically increases its copy number.…”

“…Systematic investigations of the robustness in cellular systems have been performed by focusing on the effects of manipulating gene copy number on cell growth [ 12 , 16 – 18 ]. We previously measured cell robustness to gene overexpression using a genetic technique termed genetic tug-of-war (gTOW), by which fragility to protein overproduction is indirectly and quantitatively assessed as an upper limit of gene copy number in Saccharomyces cerevisiae [ 17 , 19 , 20 ].…”