Cyclin D mediates tolerance of genome-doubling in cancers with functional p53

Abstract

Background: Aneuploidy and chromosomal instability (CIN) are common features of human malignancy that fuel genetic heterogeneity. Although tolerance to tetraploidization, an intermediate state that further exacerbates CIN, is frequently mediated by TP53 dysfunction, we find that some genome-doubled tumours retain wild-type TP53. We sought to understand how tetraploid cells with a functional p53/p21-axis tolerate genome-doubling events.

Methods: We performed quantitative proteomics in a diploid/tetraploid pair within a system of multiple independently derived TP53 wild-type tetraploid clones arising spontaneously from a diploid progenitor. We characterized adapted and acute tetraploidization in a variety of flow cytometry and biochemical assays and tested our findings against human tumours through bioinformatics analysis of the TCGA dataset.

Results: Cyclin D1 was found to be specifically overexpressed in early but not late passage tetraploid clones, and this overexpression was sufficient to promote tolerance to spontaneous and pharmacologically induced tetraploidy. We provide evidence that this role extends to D-type cyclins and their overexpression confers specific proliferative advantage to tetraploid cells. We demonstrate that tetraploid clones exhibit elevated levels of functional p53 and p21 but override the p53/p21 checkpoint by elevated expression of cyclin D1, via a stoichiometry-dependent and CDK activity-independent mechanism. Tetraploid cells do not exhibit increased sensitivity to abemaciclib, suggesting that cyclin D-overexpressing tumours might not be specifically amenable to treatment with CDK4/6 inhibitors.

Conclusions: Our study suggests that D-type cyclin overexpression is an acute event, permissive for rapid adaptation to a genome-doubled state in TP53 wild-type tumours and that its overexpression is dispensable in later stages of tumour progression.

Figure 1.

Tetraploidy tolerance in a TP53 WT background. (A) The proportion of genome-doubled (GD) versus non-genome-doubled (nGD) tumours is indicated for each cancer type. (B) Schematic representation of major genes involved in the G1/S pathway and p53 response pathways. (C) SILAC correlation plot displaying two inversely labelled replicate experiments. Early TC13 cells were labelled ‘heavy’ (H) on the horizontal axis and ‘light’ (L) on the vertical axis, while early DC14 cells were labelled inversely. In either case, ‘heavy’ species were divided by ‘light’ and ratios representing log2 fold differences between clones were plotted. Box plots showing the median (stripe), the 25th–75th percentile (box) and outliers (open circles) for both replicate experiments. Cyclin D1 is indicated. (D) Immunoblot (IB) of all major cyclins in diploid (DC) and tetraploid clones (TC). (E) Immunoblot of cyclin D1 levels in diploid and tetraploid, early and late clones, as indicated by the passage number.

Figure 2.

D-type cyclin-overexpression confers tetraploidy tolerance. (A) DNA ploidy profiles showing the percentage of 8N RPE-FUCCI, control or cyclin D1-overexpressing cells, post-DCB treatment (2 μM, 18 h). The basal levels of tetraploidization are expressed as percentages of the total number of cells analysed. (B) Quantification of the mean percentage of 8N cells from triplicates of three independent experiments. (C) DCB-treated, RPE-FUCCI control or cyclin D1-overexpressing tetraploid (4N) cells were plated for clonogenic assays and stained. Untreated (UNT) cells were plated for plating efficiency (PE). (D) Quantification of colony forming assays, presented as a fold change relative to RPE-FUCCI control cells. (E) Colony forming units from triplicates of two independent experiments were counted and presented as a fold change relative to RPE-FUCCI control cells.

Figure 3.

Mechanistic insights into the cyclin D-mediated tetraploidy tolerance. (A) Immunoblot (IB) of Serine 15 (S15) phosphorylated p53, total p53 and p21 protein levels in diploid (DC) and tetraploid (TC) HCT116 clones, after 5-FU treatment (5 μM; 16 h). (B) Graph demonstrating the median intensity of p21 in arbitrary units (a.u) in all phases of the cell cycle across the diploid and tetraploid clones. (C) Subcellular fractionation of HCT116 isogenic cells immunoblotted for p53 and p21 protein levels in diploid and tetraploid clones. H2B was used as a purity control and actin as a loading control. (D) Cell lysates from diploid and tetraploid clones were subjected to five rounds of immunodepletion with an anti-p21 antibody (IP1-5) and subsequently immunoprecipitated with anti-cyclin D1 antibody. Arrows indicate specific bands, asterisks indicate immunoglobulins cross-reacting with secondary antibodies. (E) Colony forming fraction of diploid and tetraploid clones after treatment with increasing doses of abemaciclib. Data shown as the average of three experiments, with three diploid and four tetraploid clones per data point in triplicate (errors bars are ±SEM).

Figure 4.

D-type cyclin expression in tumours. (A) Correlation of gene expression of D-type cyclins (CCND1-3) and components of the G1/S, p53-dependent checkpoint (CDKN1A, CDKN2A, TP53) with TP53 mutant (red) versus TP53 wild-type (blue) tumours in colorectal adenocarcinomas (COAD). Gene expression was normalized to TBP expression. (B) Spearman rank correlation analysis of gene expression of D-type cyclins and components of the G1/S p53-depenent checkpoint in TP53 wild-type colorectal adenocarcinoma. The colour range indicated reflects the correlation values and the size of circles indicates P values, with larger circles representing smaller P values.