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.

Context is everything: aneuploidy in cancer

“…Systematic introduction of extra chromosomes into yeast genomes revealed that single chromosome gains lead to slower proliferation and various detrimental metabolic and physiological consequences 7 . Studies in mouse and human cell lines reached similar conclusions: single chromosome gains generally impair proliferation, alter metabolism and induce various stress responses 8,16 . Further, oncogene-transformed trisomic cells exhibit reduced tumorigenicity compared to their diploid counterparts 5 .…”

“…The answer to such questions is not straight-forward. Single trisomies are sufficient to significantly affect cellular functions 5,16,28 and are, by the classical definition, aneuploid. However, at the same time, when tumors with single chromosome gains or losses are classified in the "diploid" group, the prognostic value of high degree of aneuploidy becomes stronger 29 .…”

“…Second, as discussed below, aneuploidy can play distinct, often opposite, roles in different contexts. Third, introducing or eliminating specific chromosomes remains technically challenging and laborious, despite tools such as microcell-mediated chromosome transfer 15,16 , Cre-Lox recombination 17 and CRISPR-Cas9 gene editing 9,18,19 . Consequently, we lack the ability to systematically characterize the consequences of aneuploidy across a wide range of chromosomes and cell types.…”

Genome-wide gene expression perturbation induced by loss of C2 chromosome in allotetraploid Brassica napus L.

“…Another finding for both pairs was that the downregulated genes was dominant in the term of “transcription regulator activity” (χ 2 test, P < 0.01), consistent with the fact of a priority of downregulated gene expression. However, in term of “translation regulator activity,” the upregulated genes manifested booming (χ 2 test, P < 0.01), strengthening the idea that some transcript expression profilings would be adjusted at the process of translation (Stingele et al, 2012 ). It seemed likely that the sophisticated mechanisms responding to deficiency of C2 not only functioned at the beginning of transcription but also threaded throughout the process of gene expression.…”

Transcriptome research of human amniocytes identifies hub genes associated with developmental dysplasia in down syndrome

“…This may explain why these genes also directly contribute to embryonic development, including "nervous system development", "heart and skeletal muscle development" and "face morphogenesis", indicating a close correlation between altered cellular processes and dysplastic phenotypes manifested in DS fetuses. In contrast, downregulated genes principally affect "DNA replication and cell cycle", which was revealed as a shared phenotype among multiple aneuploids [36]. Indeed, the downregulated genes may result from the upregulated genes, in other words, the upregulated genes disrupted the developmental program and consequently led to defects in cell proliferation in DS, which was confirmed through proliferation assay using EdU staining (Supplementary Figure 4B, 4C).…”

Effects of aneuploidy on gene expression: implications for cancer

“…Annotations related to inflammatory and stress responses and extracellular regions are upregulated in all mammalian model aneuploid cells, whereas lysosomes, vacuoles and membrane metabolism are consistently upregulated only in human aneuploids. The deregulated pathways determined by transcriptome analysis largely overlap with the proteome analysis performed in model human aneuploid cell lines . These findings suggest a model in which changes in expression throughout the whole genome are largely driven by aneuploidy per se .…”

“…It should be noted that the environmental stress response reflects changes in the cell‐cycle distribution rather than a genuine stress response ; thus, identification of the environmental stress response in aneuploid cells further emphasizes the marked negative effect of de novo aneuploidy on cell‐cycle progression. Aneuploid mammalian cells and amniocytes from trisomic pregnancies also show uniform aneuploidy‐driven pathway deregulation . Gene ontology terms associated with DNA and RNA pathways such as DNA replication, repair, chromosome condensation and segregation, cell‐cycle progression, ribosome biogenesis and RNA processing were consistently downregulated in human and mouse aneuploid cell lines .…”

“…This is true also for aneuploid cells with complex karyotypes, because expression of the subunits of macromolecular complexes encoded on the aneuploid chromosomes was significantly reduced in three of five budding yeast strains with highly altered karyotypes . Other protein classes such as protein kinases are subjected to dosage compensation in human cell lines . The abundance adjustment of subunits of macromolecular complexes and kinases likely contributes to restoring the protein stoichiometry.…”

“…Aneuploidy affects the expression not only of the genes located on the supernumerary chromosomes, but also of multiple other genes across the entire genome [7,9,23,26,42]. This global gene deregulation can originate from at least two sources ( Fig.…”

“…Despite these difficulties, the impact of aneuploidy on gene expression in whole chromosome aneuploid model cell lines is evident. The majority of genes encoded on the supernumerary chromosomes are differentially expressed according to the gene copy number changes in yeast , murine and human cell lines , Arabidopsis thaliana and maize . Gene expression also scales with copy number changes in cells from patients with trisomy syndromes and mouse models of Down's syndrome.…”

“…One possible cause might be a specific cellular stress response elicited by the imbalanced protein levels in aneuploid cells, which in turn, feeds back to transcriptional regulation. Indeed, the gain of an extra chromosome impairs protein folding and activates protein degradation pathways in model aneuploids . This negatively affects the delicate balancing act between proteins that need to be folded and the cellular protein folding capacity, thus causing proteotoxic stress .…”

“…Impaired proliferation and delayed progress through phases G 1 and S of the cell cycle have been observed in disomic yeasts, as well as in trisomic and tetrasomic mammalian cell lines . The slower proliferation of aneuploid cells offers another possible explanation for their characteristic transcriptional patterns.…”

“…Protein abundance also scales with changes in the gene copy number, as the expression from the extra chromosome increases up to 1.6–1.9‐fold in disomic yeast strains and up to 1.3‐ and 1.7‐fold in trisomic and tetrasomic mammalian cells, respectively . Genome‐wide comparison revealed that ~ 20% of the proteins encoded on supernumerary chromosomes did not show elevated expression in yeast or in human cell lines and the compensated proteins were enriched for subunits of macromolecular protein complexes . This is true also for aneuploid cells with complex karyotypes, because expression of the subunits of macromolecular complexes encoded on the aneuploid chromosomes was significantly reduced in three of five budding yeast strains with highly altered karyotypes .…”

“…The propagation of cells that have missegregated chromosomes is limited because they often arrest in the G 1 phase directly following the erroneous mitosis . Even if aneuploid cells overcome this burden and continue to divide, their proliferation is often impaired by the detrimental changes caused by aneuploidy . The cause of the altered physiology likely lies in the large‐scale gene copy number alterations and the corresponding changes in gene expression.…”

On the Dependency of Cellular Protein Levels on mRNA Abundance

“…These findings resonate with an earlier study using a fly brain tumor model (Jü schke et al, 2013), which showed that levels of proteins participating in common complexes were more strongly controlled than their matching mRNAs. Consistently, studies in breast cancer cells and yeast cells have shown that stable protein complexes tend to maintain constant protein expression, despite changed gene copy number and mRNA levels (Dephoure et al, 2014;Geiger et al, 2010;Stingele et al, 2012). Thus, molecular requirements such as protein complex stoichiometry seem to constrain protein level variation in somatically mutated cells more strongly than transcript variation.…”

“…Indeed, it seems difficult to imagine a generic (i.e., non-gene-specific) process by which translation rates could be adapted to erroneous mRNA levels (Bader et al, 2015). Thus, adaptation of protein turnover through protein degradation remains as a plausible candidate process (Battle et al, 2015;Stingele et al, 2012). It is well established that the stoichiometry of ribosomal proteins is controlled via degradation of excess proteins that cannot be incorporated into ribosomes (A) Buffering of protein levels against mRNA variation can occur at multiple scales, including intra-and inter-individual genomic variation, as well as interspecies variation.…”

The effect of trisomic chromosomes on spatial genome organization and global transcription in embryonic stem cells

“…This primary dosage effect due to trisomy may cause secondary transcriptional changes through trans-acting factors and proteomic stress response. 77,78 This observation offers an explanation for the shared dysregulated pathways and phenotypes across different trisomic cells. 18,44 Previous imaging studies of trisomic cells reported chromosome compaction in either trisomic chromosomes 34 or disomic chromosomes.…”

“…Consistent with previous studies, 73 we observed the transcriptional upregulation of trisomic chromosomes. This primary dosage effect due to trisomy may cause secondary transcriptional changes through trans‐acting factors and proteomic stress response 77,78 . This observation offers an explanation for the shared dysregulated pathways and phenotypes across different trisomic cells 18,44 .…”

Living in CIN: Mitotic Infidelity and Its Consequences for Tumor Promotion and Suppression

“…Proteomic analysis of HCT116 and RPE cells with one or two additional chromosomes revealed that, while transcription reflects gene copy number, protein levels for ~25% of genes on the extra chromosomes are lower than what would be predicted based on gene copy number and are instead similar to the levels in diploid cells (Stingele et al, 2012). Proteins whose expression was downregulated post-transcriptionally included those that function in DNA replication and repair, which is consistent with the observed growth defect during interphase, as well as with a delay in DNA synthesis (Passerini et al, 2016).…”

“…Consistent with this, trisomy 21 patients have reduced stature and head circumference (Van Gameren-Oosterom et al, 2012). HCT116 cells with extra copies of chromosome 3 or 5 also showed marked growth delay under standard conditions (Stingele et al, 2012). Therefore in yeast, mice, and human, chromosome gains confer a growth disadvantage under conditions optimized for euploid cell growth.…”

“…In principle, aneuploid karyotypes can remain stable over many generations, or they can exhibit CIN and continue to evolve. It has long been proposed that aneuploidy causes CIN due to an imbalance of genes and protein complexes involved in chromosome segregation during mitosis (Duesberg et al, 1998), and aneuploidy has been shown to cause profound changes in the transcriptome and proteome, some of which are conserved among distinct aneuploid karyotypes (Pavelka et al, 2010; Stingele et al, 2012; Torres et al, 2007b; Upender et al, 2004). Meiosis in yeast strains with an uneven ploidy (3n or 5n) results in the production of both stably aneuploid and CIN yeast strains, with CIN yeast strains predominating (Pavelka et al, 2010; Sheltzer et al, 2011; Zhu et al, 2012).…”

“…Chromosome gains generally lead to concomitant increases in transcription that mirror chromosome copy numbers in yeast and human cells (Pavelka et al, 2010; Stingele et al, 2012; Torres et al, 2007a). Proteomic analysis of HCT116 and RPE cells with one or two additional chromosomes revealed that, while transcription reflects gene copy number, protein levels for ~25% of genes on the extra chromosomes are lower than what would be predicted based on gene copy number and are instead similar to the levels in diploid cells (Stingele et al, 2012).…”

“…Proteins whose expression was downregulated post-transcriptionally included those that function in DNA replication and repair, which is consistent with the observed growth defect during interphase, as well as with a delay in DNA synthesis (Passerini et al, 2016). One mechanism contributing to the detrimental effects of chromosome gains is the induction of a stress response, in part due to additional genetic copies of individual subunits of multiprotein complexes (Dephoure et al, 2014; Stingele et al, 2012). …”

Understanding How Genetic Mutations Collaborate with Genomic Instability in Cancer

“…Conversely, genes involved in carbohydrate metabolism displayed decreased expression. A similar but not identical response was found in human cells when comparing DNA, mRNA, and protein levels of euploid and aneuploid human cell lines [6]. For this purpose, multiple human tri-and tetrasomic cell lines were transcriptionally profiled and compared to aneuploid human cancer cell lines.…”

“…Finally, autophagy, a process that removes surplus and damaged proteins and organelles, is upregulated in aneuploid cells [6]. Stable aneuploid human colon cancer cells display increased numbers of LC3 foci, an autophagy marker [43], and upregulated p62-dependent autophagy.…”

“…As aneuploidy leads to increased gene expression and thus increased protein production from the aneuploid chromosomes (in case of chromosome gains) [39], aneuploidy disrupts the protein complex stoichiometry [6,40]. The resulting excess of 'aneuploid' proteins causes proteotoxic stress, such as impaired protein folding [41], unscheduled protein degradation [42,43] and increased protein aggregation [44].…”

“…An important first finding from stable aneuploidy model systems is that aneuploid cells have a significant proliferative disadvantage compared to their euploid counterparts [3,4,6,13]. Furthermore, all aneuploid models showed strong metabolic changes and induction of stress responses, indicating that aneuploidy decreases cellular fitness, and thus is expected to suppress tumorigenesis.…”

“…Recipient cells with the extra human chromosome were selected for using a selection cassette. This technique yielded various stable aneuploid human cell lines including defined trisomic and even tetrasomic RPE1, HE35, and colorectal cancer HCT116 and DLD1 cell lines, which allowed for the studying of effects of aneuploidy using isogenic aneuploid and euploid cell lines [6,13].…”

“…Aneuploidy is highly detrimental during development, which is reflected by the fact that it is the leading cause of spontaneous abortion and mental retardation in humans [2]. When induced experimentally, aneuploidy negatively affects cellular fitness by reducing cell growth and inducing metabolic and proteotoxic stress [3][4][5][6]. However, aneuploidy is a hallmark of cancer [7,8], a disease characterized by uncontrolled proliferation.…”

AMPK-dependent autophagy upregulation serves as a survival mechanism in response to Tumor Treating Fields (TTFields)

“…It has been proposed that cells respond to aneuploidy by engaging proteotoxic stress response, which includes upregulation of pathways leading to the degradation of cellular constituents and protein folding 30 . Specifically, upregulation of autophagy was observed consistently in aneuploid cells 28 . We exploited single-cell analysis to show for the first time that TTFields application specifically triggers autophagy in the progeny cells that divided during treatment.…”

“…Abnormal mitosis following TTFields application results in different cell fates including the formation of aneuploid daughter cells 7 , 8 . Aneuploidy is associated with the activation of regulators of autophagic and lysosomal gene expression 27 , 28 . To explore whether autophagy was more prominent in cells that divided during TTFields application, we analyzed autophagosome dynamics within single cells using U-87 MG cell line stably expressing LC3 protein fused with green fluorescent protein (GFP).…”

Aneuploidy and chromosomal instability: a vicious cycle driving cellular evolution and cancer genome chaos

“…There was no overlap among genes deregulated in each of these trisomies, possibly consistent with the trans-effect seen in aneuploid yeast strains. More recent microarray-based measurements of gene expression in trisomic MEFs [32] and trisomic HCT116 cells [31] also agree that the level for most transcripts expressed from aneuploid chromosomes varies proportionally to the copy number of that chromosome. Nevertheless, as in DLD1 cells [30], few deregulated genes located on various chromosomes were also found.…”

“…However, about 25% of proteins encoded on aneuploid chromosomes were found in lower amounts than expected based on the chromosome copy number. Protein kinases and components of large macromolecular complexes were over-represented among the latter category [31]. This observation suggests that there may be mechanisms that correct for the altered component stoichiometry of protein complex.…”

“…This would be in line with findings in budding yeast, where chromosome instability is associated with certain genome level and chromosome-specific determinants. However, in the case of trisomy 3 and 21, their effect on karyotype stability was not the same in different studies [20],[28,31]. …”

“…For instance, trisomy 3, 7 and 13 created in near-diploid colon carcinoma DLD1 cells by microcell mediated chromosome transfer, and trisomy 3 created in diploid human hTERT mammary cells, have stable chromosome numbers as assayed by spectral karyotyping (SKY) and array-based comparative genomic hybridization (aCGH) [30]. Similarly, HCT116 cells made tri- and tetrasomic for chromosomes 3 and 5, and RPE1 cells trisomic for chromosomes 5, 12 and 21, were not reported to be unstable as assayed by aCGH and chromosome-specific FISH probes [31]. Mouse embryonic fibroblast cell lines trisomic for chromosomes 1, 13, 16 and 19 also were not found to be unstable before immortalization (examined by SKY and aCGH) [32].…”