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.
Subject(s)
Adenocarcinoma/genetics , Colorectal Neoplasms/genetics , Cyclin C/genetics , Tumor Suppressor Protein p53/genetics , Adenocarcinoma/drug therapy , Adenocarcinoma/metabolism , Aminopyridines/pharmacology , Benzimidazoles/pharmacology , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , Cyclin C/biosynthesis , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cytochalasin B/analogs & derivatives , Cytochalasin B/pharmacology , Diploidy , Flow Cytometry , Gene Knockdown Techniques , Genes, p53 , HCT116 Cells , Humans , Protein Kinase Inhibitors/pharmacology , Tetraploidy , Tumor Suppressor Protein p53/metabolismABSTRACT
Convenient syntheses of 6beta-tritiated Delta(7)-cholestenol and 3alpha-tritiated Delta(7)-cholestene-3beta,5alpha-diol are described. It was shown that the conversion of 6beta-tritiated Delta(7)-cholestenol into cholesterol is accompanied by the complete retention of label. It was unambiguously established that the overall reaction leading to the introduction of the double bond in the 5,6-position in cholesterol occurs via a cis-elimination involving the 5alpha- and 6alpha-hydrogen atoms and that during this process the 6beta-hydrogen atom remains completely undisturbed. Metabolic studies with 3alpha-tritiated Delta(7)-cholestene-3beta,5alpha-diol revealed that under anaerobic conditions the compound is not converted into cholesterol. This observation, coupled with the previous work of Slaytor & Bloch (1965), is interpreted to exclude a hydroxylation-dehydration mechanism for the origin of the 5,6-double bond in cholesterol. It was also shown that under aerobic conditions 3alpha-tritiated Delta(7)-cholestene-3beta,5alpha-diol is efficiently converted into cholesterol and that this conversion occurs through the intermediacy of 7-dehydrocholesterol. Cumulative experimental evidence presented in this paper and elsewhere is used to suggest that the 5,6-double bond in cholesterol originates through an oxygen-dependent dehydrogenation process and a hypothetical mechanism for this and related reactions is outlined.
