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.

Micronuclei-based model system reveals functional consequences of chromothripsis in human cells

“…The cognate aneuploid cell lines with and without chromothripsis allowed us to determine the functional consequences of chromosomal rearrangements. Gain of a chromosome generally impairs cellular proliferation in human cells and most trisomic human cells proliferate slower than their isogenic parental cell line (Sheltzer et al, 2017; Stingele et al, 2012). Strikingly, we found that the cell lines with a rearranged chromosome formed colonies in soft agar more readily than the cell lines with an identical, but intact chromosome (Figure 5a,b).…”

“…Finally, the micronuclei-mediated rearrangements obtained via MMCT can be stably propagated in cell lines, allowing the analysis of their consequences. A gain of a chromosome is often detrimental for human cells, which is reflected by impaired proliferation (Sheltzer et al, 2017; Stingele et al, 2012). Comparison of proliferation of cell lines carrying intact extra chromosomes with cell lines carrying rearranged chromosomes revealed that the chromosomal rearrangements reduce the negative effects of chromosome gain on cellular proliferation by reducing the amount of extra DNA (Figure 5).…”

“…Cells with the extra chromosome were selected in DMEM with the appropriate antibiotic. For detailed description see Stingele et al (2012). All trisomic and tetrasomic cell lines originate from a single cell.…”

Systematic proteome and proteostasis profiling in human Trisomy 21 fibroblast cells

“…The tight control of protein complex stoichiometry has been reported as a buffering mechanism against aneuploidy in different systems 10 , 11 , 30 , 31 . To analyze the contribution of protein complex stoichiometry to the T21 phenotype, we classified proteins into two groups depending on their inclusion (“complex_in”) or exclusion (“complex_out”; Methods) from known stable heteromeric protein complexes 32 , 33 , and compared their log2 FCs.…”

“…Previous studies have investigated the proteomes of different aneuploidy systems in yeast 11 , fly 30 , human cancer cells 10 , and in artificial microcell fusion-induced human cells carrying different additional chromosomes 7 , 31 . Here we quantified both protein and protein degradation in a unique cohort of T21 primary cells to study the impact of T21 aneuploidy.…”

“…Further, we found that those Chr21 proteins that are members of heteromeric protein complexes were less affected by the increased gene dosage due to aneuploidy, a buffering effect that is mediated by accelerated protein degradation. Although tight stoichiometric control of protein complex subunits was previously reported as a buffering mechanism in aneuploidy 10 , 11 , 30 , 31 , no studies have yet directly quantified the regulation of Chr21-wide protein turnover or in clinically derived samples. It has been a long-standing hypothesis that proteins are stabilized by complex formation and by protein degradation against misfolded proteins 47 .…”

Stable aneuploid tumors cells are more sensitive to TTK inhibition than chromosomally unstable cell lines

“…Since the DNA for sequence analysis was isolated from passage +4 or +5 from the original vials from ATCC, it is likely that these mutations are also present in batches of these cell lines used in other laboratories. Indeed, the two mutations in hTERT-RPE1 were also found in DNA from cell batches of the Max Planck Institute in Martinsried (Germany), who had obtained the cell line from the University of Manchester (U.K.) [ 45 ].…”

Aneuploidy increases resistance to chemotherapeutics by antagonizing cell division

“…1, 2, or 3) (21) enabled us to assess whether paclitaxel resistance was indeed due to trisomy 3. This control was important because, unlike the trisomic MEFs that were obtained from crosses (4), trisomic HCT116 lines were generated by microcell-mediated chromosome transfer, which involves drug selection and a single cell cloning step (22). These procedures could introduce genetic alterations that might confer the observed drug resistance.…”

“…For cisplatin, we tested the response of multiple trisomic mouse embryonic fibroblast (MEF) cell lines and their euploid littermate controls (4). Because MEFs are not very responsive to paclitaxel, we tested the efficacy of paclitaxel against a pseudodiploid colon cancer cell line, HCT116, and four aneuploid derivatives of this cell line (22).…”

“…G1 Cell Cycle Delay Causes Chemotherapeutic Resistance. Aneuploidy causes cell cycle delays, especially in G1 and S phase (20,22,23). Many frontline chemotherapeutics target specific phases of the cell cycle and were discovered based on their ability to kill rapidly growing cancers or cancer cell lines.…”

“…These aneuploidy-associated stresses lead to cell cycle changes and slow proliferation (4,21). Specifically, aneuploidy causes G1 delays in the yeast, Saccharomyces cerevisiae, and lengthens G1 and S phase in mammalian cells under most circumstances (20,22,23).…”

Circular RNA ZNF609/CKAP5 mRNA interaction regulates microtubule dynamics and tumorigenicity

“…Mitotic abnormalities, such as aneuploidy because of aberrant chromosome segregation, can lead to the accumulation of DNA damage ( Ganem and Pellman, 2012 ; Stingele et al., 2012 ; Williams et al., 2008 ).…”

Allele-specific genomic data elucidate the role of somatic gain and copy-number neutral loss of heterozygosity in cancer

“…In line with previous work showing that TP53 mutational status is linked to ploidy and that aneuploid mammalian cells activate p53 ( Hinchcliffe et al., 2016 ) ( Li et al., 2010 ; Soto et al., 2017 ; Thompson and Compton, 2010 ), we observed that TP53 SNVs are enriched in high asP samples (chi-square test p value = 4.7e−21, Table S7 ). As TP53 is involved in cell-cycle regulation and, based on recent data, in aneuploidy-mediated activation of proteotoxic stress response in cells ( Santaguida et al., 2015 ), in the impact of SCNA on the amount of proteins ( Stingele et al., 2012 ), and in proteasome regulation ( Walerych et al., 2016 ), we tested the activation of a set of selected proliferative ( STAR Methods ; Table S18 ) ( Sheltzer, 2013 ) and proteasome-related pathways ( Levin et al., 2018 ; Wang et al., 2017 ) in high asP samples compared with diploid samples ( Figure S10 ). The results suggest the activation of opposite transcriptional programs upon the presence of high asP in different tumor types ( Table S16 ) possibly related to different genetic and tissue-specific transcriptional backgrounds and their interactions with TP53 status, affecting proliferation and global protein homeostasis.…”

ROCK2 Knockout Improves Proliferation Rate in a Cellular Model of Down Syndrome

“…1B), indicating a successful transfection. Note that the RPE1-21/3-Cas9 cells carry a histone H3:GFP fusion (Stingele S, et al 2012) and thus both the cytoplasm and nuclei of transfected cells appear green in the aneuploid cells (Fig. 1B, lower panel).…”

“…H-TERT immortalized RPE-1 H2B-GFP 21/3 cells were originally produced by microcell mediated chromosome transfer by the Storchova lab (Stingele, et al 2012; Passerini, et al 2016) and generously shared with us. This cell line and the parental H-TERT immortalized euploid RPE-1 cell line were infected to express Cas9 using LentiCas9-Blast (Sanjana NE, et al 2014), and subsequently used in the whole genome CRISPR screen as described (Replogle JM, et al m anuscript in preparation ).…”

“…Many different altered phenotypes are associated with human trisomy 21 or Down Syndrome (Antonarakis SE, et al 2020) including hypocellularity (Guidi S, et al 2011). Analysis of aneuploid cells carrying a third copy of chromosome 21 reveal that a reduction in cell proliferation rate versus euploid counterparts is one measure of decreased cell fitness (Contestabile A, et al 2009; Hwang S, et al 2019; Hwang S et al 2021; Inoue M, et al 2019; Gimeno A, et al 2014; Guidi S, et al 2011; Meharena HS, et al 2022; Soppa U, et al 2014; Stingele S, et al 2012; Williams BR, et al 2008). A whole genome CRISPR screen was undertaken in order to identify genes and pathways that would improve trisomy 21 cell proliferation and that could be targets for therapeutic intervention (Replogle JM, et al m anuscript in preparation ).…”

“…Here we present evidence that in RPE-1 (retinal pigment epithelial-1) trisomy 21 cells, both ROCK2 CRISPR knockout and ROCK drug inhibition result in a modest but significant increase in the proliferation rate. CRISPR was used to knockout ROCK2 in hTERT immortalized aneuploid RPE-1 cells, which were created by microcell mediated chromosome transfer and generously shared by the Storchova lab (Stingele S, et al 2012), and further modified by infection with a Cas9 expressing viral vector (Replogle JM, et al m anuscript in preparation ). Plasmids expressing the two most enriched sgRNAs for ROCK2 identified in the whole genome screen were transfected into the cells to knockout the gene.…”

Chromosomal instability in aneuploid acute lymphoblastic leukemia associates with disease progression

“…Chromosome mis-segregation leads to a myriad of cellular and metabolic stresses which result in a strong negative selection when occurring in diploid cell types, but which are tolerated in aneuploid cancer cells (Chunduri and Storchova, 2019 ; Crasta et al, 2012 ; Donnelly et al, 2014 ; Stingele et al, 2012 ; Torres et al, 2007 ). Cancer cells undergo adaptive resistance rooted from variable cellular signaling networks that involve feedback-dependent homeostatic control.…”

“…The ability to cope with the ongoing genetic imbalances caused by CIN is a fundamental difference between malignant and non-malignant cells. These imbalances result in several aneuploidy-associated stressors that impair overall cellular fitness such as metabolic changes affecting the protein turnover machinery, replication stress affecting genome integrity, and alterations in the mitotic regulatory machinery which fuel chromosome mis-segregation (Cohen-Sharir et al, 2021 ; Crasta et al, 2012 , Donnelly et al, 2014; Garribba et al, 2023 ; Ohashi et al, 2015 ; Oromendia and Amon, 2014 ; Stingele et al, 2012 ; Torres et al, 2007 ). This suggests that CIN must be maintained under a tolerable threshold to preserve cancer cell homeostasis, which may represent a key vulnerability of aneuploid tumors.…”

Complex aneuploidy triggers autophagy and p53-mediated apoptosis and impairs the second lineage segregation in human preimplantation embryos

“…3g). We investigated whether proteotoxic stress and autophagy (Oromendia et al ., 2012; Stingele et al ., 2012; Dürrbaum et al ., 2014; Ohashi et al ., 2015; Singla et al ., 2020) were also present in aneuploid human embryos. The number of puncta per cell of autophagy markers LC3B (19 euploid, 22 aneuploid and 8 reversine-treated embryos stained) and p62 (6 euploid and 9 aneuploid embryos stained) and the intensity levels of proteotoxic stress marker HSP70 (13 euploid, 8 aneuploid and 8 reversine-treated embryos stained) were significantly increased in both aneuploid and reversine-treated embryos (Fig.…”

“…First, aneuploidy results in gene-dosage defects, leading to an unbalanced protein pool and proteotoxic stress (Oromendia et al ., 2012; Ohashi et al ., 2015). If this remains unresolved by activation of autophagy, CASP8/LC3B/p62 will interact to activate the apoptosis cascade (Pan et al ., 2011; Stingele et al ., 2012; Dürrbaum et al ., 2014). Second, aneuploidy results in replication stress that can lead to DNA-damage, resulting in p53 activation and ultimately to apoptosis (Zhu et al ., 2018; Regin et al ., 2022).…”

PTENandARID1Ahaploinsufficiency equip colonic epithelium for oncogenic transformation

“…It has been previously suggested that while the transcriptome reflects copy number alterations, the proteome does not, tending to revert to a diploid state (Stingele et al ., 2012). However, the link between gene dosage and protein abundance remains unclear.…”