Chromosomal instability, tolerance of mitotic errors and multidrug resistance are promoted by tetraploidization in human cells

Abstract

Up to 80% of human cancers, in particular solid tumors, contain cells with abnormal chromosomal numbers, or aneuploidy, which is often linked with marked chromosomal instability. Whereas in some tumors the aneuploidy occurs by missegregation of one or a few chromosomes, aneuploidy can also arise during proliferation of inherently unstable tetraploid cells generated by whole genome doubling from diploid cells. Recent findings from cancer genome sequencing projects suggest that nearly 40% of tumors underwent whole genome doubling at some point of tumorigenesis, yet its contribution to cancer phenotypes and benefits for malignant growth remain unclear. Here, we investigated the consequences of a whole genome doubling in both cancerous and non-transformed p53 positive human cells. SNP array analysis and multicolor karyotyping revealed that induced whole-genome doubling led to variable aneuploidy. We found that chromosomal instability (CIN) is a frequent, but not a default outcome of whole genome doubling. The CIN phenotypes were accompanied by increased tolerance to mitotic errors that was mediated by suppression of the p53 signaling. Additionally, the expression of pro-apoptotic factors, such as iASPP and cIAP2, was downregulated. Furthermore, we found that whole genome doubling promotes resistance to a broad spectrum of chemotherapeutic drugs and stimulates anchorage-independent growth even in non-transformed p53-positive human cells. Taken together, whole genome doubling provides multifaceted benefits for malignant growth. Our findings provide new insight why genome-doubling promotes tumorigenesis and correlates with poor survival in cancer.

Keywords: CIN; aneuploidy; cancer; drug resistance; p53; tetraploidy; whole genome doubling.

Figure 1.

Posttetraploid progenies (PTs) are chromosomally unstable. (A) Schematic depiction of the experimental strategy. (B, C) SNP array profiles of HCT116, RPE1 and their posttetraploid derivatives. Copy numbers are indicated by colors. The log R represents the copy number; B-allele frequency (BAF) indicates the allele composition: BAF of 0 or 1 represents genotype of AA / A- / BB / B-, respectively; BAF of 0.5 represents AB. (D) Multicolor FISH karyotyping of 2 cells from the HPT2 cell line (number of chromosomes was 72 and 79, respectively). Note the difference in copy number of chromosomes 1, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18 and 20.

Figure 2.

Posttetraploid cells display chromosomal instability and an increased frequency of abnormal mitosis. (A) Fluorescence in situ hybridization (FISH) against centromeric regions of HCT116 and HCT116-derived PTs. Comparison of chromosome number distribution for chromosome 7 in early passages and 36 passages later; mean and SEM of 2 independent FISH experiments. Chromosome 7 – red, chromosome 1- green, DNA was counterstained with DAPI (objective 63x, bar: 10 μm). Percentage of abnormal mitoses evaluated in fixed images of HCT116 (B) and RPE1 (C) and their respective posttetraploid derivatives; mean and SD of 3 independent experiments. AnaphBridge – cells that contain an anaphase bridge; LaggingChr – cells containing a lagging chromosome; AnaphBridge-LaggingChr – cells containing both an anaphase bridge and a lagging chromosome; Multipolar – cells that underwent multipolar anaphase.

Figure 3.

Posttetraploid cells are tolerant to mitotic errors. (A) Frequency of cell cycle arrest/cell death after bipolar mitosis with no apparent defects (normal mitosis) and with visible chromosome segregation defects (abnormal mitosis). Mean of 4 independent experiments and SD is plotted. Unpaired Student t-test was used to test for statistical significance. (B) Examples of p53 accumulation in the nuclei and micronuclei of the micronucleated cells. Yellow and white arrowheads indicate the micronuclei with and without p53 enrichment, respectively. p53-red, DNA was counterstained with DAPI, bar: 10 μm. (C and D) Accumulation of p53 in the nuclei of cells forming micronuclei (MN⁺) in HCT116, RPE1 and their respective posttetraploid derivatives (panels C, D, respectively). Mean of 4 independent experiments and SEM are plotted.

Figure 4.

The p53 pathway is deregulated in posttetraploids. (A, B) Changes in abundance in p53, p38 and p21 and phosphorylation of p53 on Ser15 (p53-p) and phosphorylation of p38-p on Threonine180 and Tyrosine 182 (p38-p) in HCT116, RPE1 and respective posttetraploid cell lines with and without VS83 treatment. Four independent experiments were performed, an example of immunoblotting is shown; a Ponceau S stain was used as a loading control. (C, D) Quantification of the response to the missegregation triggered by release from VS83 treatment. The relative signal levels are presented as fold change of treated-to-untreated cells. Mean of at least 3 independent experiments with SD is shown, * marks statistically significant difference (P < 0.05). (E) Heat map of transcriptional fold changes of 91 significantly altered p53 interactors in posttetraploid cell lines (normalized to the respective parental cell lines).

Figure 5.

Posttetraploid cell lines are resistant to a broad spectrum of chemotherapeutic drugs and transform in vitro. (A) Dose-response curves of compounds showing different sensitivities in proliferation assays with the hTERT-RPE1 cell line and the posttetraploid cell lines RPT1, RPT3 and RPT4. (B) Dose-response curves of compounds showing different sensitivities in proliferation assays with the HCT116 cell line and the posttetraploid cell lines HPT1, HPT2 and HPT4. The posttetraploids lines are resistant to a broad spectrum of anti-cancerous drugs; except HPT1, 2 and 4 that are relatively more sensitive to 6-mercaptopurine. Fitted curve for 2 replicates from one or 2 independent experiments is plotted. Note that no fitted curve was determined for HPT1 upon etoposide treatment. See Material and Methods for details. (C) Phase contrast images of anchorage-independent colony growth in soft agar.