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
In non-alcoholic fatty liver disease (NAFLD) and insulin resistance, hepatic de novo lipogenesis is often elevated, but the underlying mechanisms remain poorly understood. Recently, we show that CDK8 functions to suppress de novo lipogenesis. Here, we identify the mammalian target of rapamycin complex 1 (mTORC1) as a critical regulator of CDK8 and its activating partner CycC. Using pharmacologic and genetic approaches, we show that increased mTORC1 activation causes the reduction of the CDK8-CycC complex in vitro and in mouse liver in vivo. In addition, mTORC1 is more active in three mouse models of NAFLD, correlated with the lower abundance of the CDK8-CycC complex. Consistent with the inhibitory role of CDK8 on de novo lipogenesis, nuclear SREBP-1c proteins and lipogenic enzymes are accumulated in NAFLD models. Thus, our results suggest that mTORC1 activation in NAFLD and insulin resistance results in down-regulation of the CDK8-CycC complex and elevation of lipogenic protein expression.
Subject(s)
Cyclin C/biosynthesis , Cyclin-Dependent Kinase 8/biosynthesis , Down-Regulation , Gene Expression Regulation, Enzymologic , Multiprotein Complexes/metabolism , TOR Serine-Threonine Kinases/metabolism , Animals , Cyclin C/genetics , Cyclin-Dependent Kinase 8/genetics , HEK293 Cells , Humans , Lipogenesis/genetics , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Knockout , Mice, Obese , Multiprotein Complexes/genetics , Non-alcoholic Fatty Liver Disease/genetics , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Sterol Regulatory Element Binding Protein 1/genetics , Sterol Regulatory Element Binding Protein 1/metabolism , TOR Serine-Threonine Kinases/geneticsABSTRACT
Steroid receptor coactivator 2 (SRC-2) is a coactivator that regulates nuclear receptor activity. We previously reported that SRC-2 protein is degraded through the action of cAMP-dependent protein kinase A (PKA) and cAMP response element binding protein (CREB). In the study presented here, we aimed to identify proteins that interact with and thereby regulate SRC-2. We isolated cyclin C (CCNC) as an interacting partner with the SRC-2 degradation domain aa 347-758 in a yeast two-hybrid assay and confirmed direct interaction in an in vitro assay. The protein level of SRC-2 was increased with CCNC overexpression in COS-1 cells and decreased with CCNC silencing in COS-1 and MCF-7 cells. In a pulse-chase assay, we further show that silencing of CCNC resulted in a different SRC-2 degradation pattern during the first 6 h after the pulse. Finally, we provide evidence that CCNC regulates expression of cell cycle genes upregulated by SRC-2. In conclusion, our results suggest that CCNC temporarily protects SRC-2 against degradation and this event is involved in the transcriptional regulation of SRC-2 cell cycle target genes.
