CDK4/6 inhibitors induce repication stress to cause long-term cell cycle withdrawal
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Epithelial Cell
SUBMITTER: 
Tony Ly
LAB HEAD: Tony Ly
PROVIDER: PXD023435 | Pride | 2021-12-20
REPOSITORIES: Pride
Similar Datasets
Project description:A long-term goal in cancer research has been to inhibit the cell cycle in tumour cells without causing toxicity in proliferative healthy tissues. The best evidence that this is achievable is provided by CDK4/6 inhibitors, which arrest the cell cycle in G1, are well-tolerated in patients, and are effective in treating ER+/HER2- breast cancer. CDK4/6 inhibitors are effective because they arrest tumour cells more efficiently than some healthy cell types and, in addition, they affect the tumour microenvironment to enhance anti-tumour immunity. We demonstrate here another reason to explain their efficacy. Tumour cells are specifically vulnerable to CDK4/6 inhibition because during the G1 arrest, oncogenic signals drive toxic cell overgrowth. This overgrowth causes permanent cell cycle withdrawal by either preventing progression from G1 or by inducing replication stress and genotoxic damage during the subsequent S-phase and mitosis. Inhibiting or reverting oncogenic signals that converge onto mTOR can rescue this excessive growth, DNA damage and cell cycle exit in cancer cells. Conversely, inducing oncogenic signals in non-transformed cells can drive these toxic phenotypes and sensitize cells to CDK4/6 inhibition. Together, this demonstrates how oncogenic signals that have evolved to stimulate constitutive tumour growth and proliferation driven subverted to cause toxic cell growth and irreversible cell cycle exit when proliferation is halted in G1.
2023-11-17 | PXD043792 | Pride
Project description:Cell size and the cell cycle are intrinsically coupled and abnormal increases in cell size are associated with senescence and permanent cell cycle arrest. The mechanism by which overgrowth primes cells to withdraw from the cell cycle remains unknown. We investigate this here using CDK4/6 inhibitors that arrest cell cycle progression during G0/G1 and are used in the clinic to treat ER+/HER2- metastatic breast cancer. We demonstrate that CDK4/6 inhibition promotes cellular overgrowth during G0/G1, causing p38MAPK-p53-p21-dependent cell cycle withdrawal. We find that cell cycle withdrawal is triggered by two waves of p21 induction. First, overgrowth during a long-term G0/G1 arrest induces an osmotic stress response. This stress response produces the first wave of p21 induction. Second, when CDK4/6 inhibitors are removed, a fraction of cells escape long term G0/G1 arrest and enter S-phase where overgrowth-driven replication stress results in a second wave of p21 induction that causes cell cycle withdrawal from G2, or the subsequent G1. We propose a model whereby both waves of p21 induction contribute to promote permanent cell cycle arrest. This model could explain why cellular hypertrophy is associated with senescence and why CDK4/6 inhibitors have long-lasting effects in patients.
2023-11-27 | PXD036519 | Pride
Project description:The p53 tumour suppressor is a transcription factor that can regulate the expression of numerous genes encoding either proteins or microRNAs (miRNAs). The predominant outcomes of a typical p53 response are the initiation of apoptotic cascades and the activation of cell cycle checkpoints. HT29-tsp53 cells express a temperature sensitive variant of p53 and in the absence of exogenous DNA damage, these cells preferentially undergo G1 phase cell cycle arrest at the permissive temperature that correlates with increased expression of the cyclin-dependent kinase inhibitor p21WAF1. Recent evidence also suggests that a variety of miRNAs can induce G1 arrest by inhibiting the expression of proteins like CDK4 and CDK6. Here we used oligonucleotide microarrays to identify p53-regulated miRNAs that are induced in these cells undergoing G1 arrest. At the permissive temperature, the expression of several miRNAs was increased through a combination of either transcriptional or post-transcriptional regulation. In particular, miR-34a-5p, miR-143-3p and miR-145-5p were strongly induced and they reached levels comparable to that of reference miRNAs (miR-191 and miR-103). Importantly, miR-34a-5p and miR-145-5p are known to silence the Cdk4 and/or Cdk6 G1 cyclin-dependent kinases (cdks). Surprisingly, there was no p53-dependent decrease in the expression of either of these G1 cdks. To search for other potential targets of p53-regulated miRNAs, p53-downregulated mRNAs were identified through parallel microarray analysis of mRNA expression. Once again, there was no clear effect of p53 on the repression of mRNAs under these conditions despite a remarkable increase in p53-induced mRNA expression. Therefore, despite a strong p53 transcriptional response, there was no clear evidence that p53-responsive miRNA contributed to gene silencing. Taken together, the changes in cell cycle distribution in this cell line at the permissive temperature is likely attributable to transcriptional upregulation of the CDKN1A mRNA and p21WAF1 protein and not to the down regulation of CDK4 or CDK6 by p53-regulated miRNAs. Two independent experiments were performed with 2 samples in each experiment (1 control and 1 treatment condition). In the control sample, RNA was isolated cells maintained at the restrictive temperature (37ËC). The treatment treated sample, was incubated for 16 hours at the permissive temperature (32ËC).
2016-01-07 | E-GEOD-76576 | biostudies-arrayexpress
Project description:The p53 tumour suppressor is a transcription factor that can regulate the expression of numerous genes encoding either proteins or microRNAs (miRNAs). The predominant outcomes of a typical p53 response are the initiation of apoptotic cascades and the activation of cell cycle checkpoints. HT29-tsp53 cells express a temperature sensitive variant of p53 and in the absence of exogenous DNA damage, these cells preferentially undergo G1 phase cell cycle arrest at the permissive temperature that correlates with increased expression of the cyclin-dependent kinase inhibitor p21WAF1. Recent evidence also suggests that a variety of miRNAs can induce G1 arrest by inhibiting the expression of proteins like CDK4 and CDK6. Here we used oligonucleotide microarrays to identify p53-regulated miRNAs that are induced in these cells undergoing G1 arrest. At the permissive temperature, the expression of several miRNAs was increased through a combination of either transcriptional or post-transcriptional regulation. In particular, miR-34a-5p, miR-143-3p and miR-145-5p were strongly induced and they reached levels comparable to that of reference miRNAs (miR-191 and miR-103). Importantly, miR-34a-5p and miR-145-5p are known to silence the Cdk4 and/or Cdk6 G1 cyclin-dependent kinases (cdks). Surprisingly, there was no p53-dependent decrease in the expression of either of these G1 cdks. To search for other potential targets of p53-regulated miRNAs, p53-downregulated mRNAs were identified through parallel microarray analysis of mRNA expression. Once again, there was no clear effect of p53 on the repression of mRNAs under these conditions despite a remarkable increase in p53-induced mRNA expression. Therefore, despite a strong p53 transcriptional response, there was no clear evidence that p53-responsive miRNA contributed to gene silencing. Taken together, the changes in cell cycle distribution in this cell line at the permissive temperature is likely attributable to transcriptional upregulation of the CDKN1A mRNA and p21WAF1 protein and not to the down regulation of CDK4 or CDK6 by p53-regulated miRNAs.
2016-01-07 | GSE76576 | GEO
Project description:By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. Induction of prolonged early-G1 arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G1 and prevents expression of genes programmed for other cell cycle phases. S-phase synchronization upon removal of the early-G1 block (pG1-S) fails to completely restore scheduled gene expression. Consequently, coordinate loss of IRF4 and gain of Bim and Noxa expression sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 and more profoundly in pG1-S in vitro. Induction of pG1 and pG1-S by CDK4/CDK6 inhibition augments tumor-specific bortezomib killing in myeloma xenografts. Inhibition of CDK4/CDK6 in combination therapy thus represents a novel mechanism-based cancer therapy. PD 0332991 (PD) is the only known specific and reversible CDK4/CDK6 inhibitor. Gene expression was measured in myeloma MM1.S cells treated with PD (0.25 uM) in triplicate for 12, 24 or 36 h, or in cells released from G1, induced by 24hPD, for 4 or 18 h.
2012-04-06 | E-GEOD-35728 | biostudies-arrayexpress
Project description:By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. Induction of prolonged early-G1 arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G1 and prevents expression of genes programmed for other cell cycle phases. S-phase synchronization upon removal of the early-G1 block (pG1-S) fails to completely restore scheduled gene expression. Consequently, coordinate loss of IRF4 and gain of Bim and Noxa expression sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 and more profoundly in pG1-S in vitro. Induction of pG1 and pG1-S by CDK4/CDK6 inhibition augments tumor-specific bortezomib killing in myeloma xenografts. Inhibition of CDK4/CDK6 in combination therapy thus represents a novel mechanism-based cancer therapy.
2012-04-07 | GSE35728 | GEO
Project description:Genomic aberrations of Cyclin D1 (CCND1) and CDK4 in neuroblastoma indicate that dysregulation of the G1 entry checkpoint is an important cell cycle aberration in this pediatric tumor. Here we report that analysis of Affymetrix expression data of primary neuroblastic tumors shows an extensive over-expression of Cyclin D1 and CDK4 which correlates with histological subgroups and prognosis respectively. Immunohistochemical analysis demonstrated an over-expression of Cyclin D1 in neuroblasts and a low Cyclin D1 expression in all cell types in ganglioneuroma. This suggests an involvement of G1 regulating genes in neuronal differentiation processes which we further evaluated using RNA interference against Cyclin D1 and its kinase partner CDK4 in several neuroblastoma cell lines. This resulted in pRb pathway inhibition as shown by an almost complete disappearance of CDK4 specific pRb phosphorylation; reduction of E2F transcriptional activity and a decrease of Cyclin A protein levels. The Cyclin D1 and CDK4 knock-down resulted in a significant reduction in cell proliferation, a G1 specific cell cycle arrest and moreover an extensive neuronal differentiation. Affymetrix microarray profiling of siRNA treated cells revealed a shift in expression profile towards a neuronal phenotype. Several new potential downstream players are identified. We conclude that neuroblastoma functionally depend on over-expression of G1 regulating genes to maintain their undifferentiated phenotype. Experiment Overall Design: The Cell line IMR-32 at time point 0 and transiently transfected with siRNA against GFP, Cyclin D1 and CDK4 at time point 48 hours. All experiments are biological triplicates.
2008-10-25 | E-GEOD-8866 | biostudies-arrayexpress
Project description:Barr2017 - Dynamics of p21 in hTert-RPE1
cells
This deteministic model reveals that a
bistable switch created by Cdt2, promotes irreversible S-phase
entry by keeping p21 levels low, prevents premature S-phase exit
upon DNA damage
This model is described in the article:
DNA damage during S-phase
mediates the proliferation-quiescence decision in the
subsequent G1 via p21 expression.
Barr AR, Cooper S, Heldt FS, Butera
F, Stoy H, Mansfeld J, Novák B, Bakal C.
Nat Commun 2017 Mar; 8: 14728
Abstract:
Following DNA damage caused by exogenous sources, such as
ionizing radiation, the tumour suppressor p53 mediates cell
cycle arrest via expression of the CDK inhibitor, p21. However,
the role of p21 in maintaining genomic stability in the absence
of exogenous DNA-damaging agents is unclear. Here, using live
single-cell measurements of p21 protein in proliferating
cultures, we show that naturally occurring DNA damage incurred
over S-phase causes p53-dependent accumulation of p21 during
mother G2- and daughter G1-phases. High p21 levels mediate G1
arrest via CDK inhibition, yet lower levels have no impact on
G1 progression, and the ubiquitin ligases CRL4Cdt2 and SCFSkp2
couple to degrade p21 prior to the G1/S transition.
Mathematical modelling reveals that a bistable switch, created
by CRL4Cdt2, promotes irreversible S-phase entry by keeping p21
levels low, preventing premature S-phase exit upon DNA damage.
Thus, we characterize how p21 regulates the
proliferation-quiescence decision to maintain genomic
stability.
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2016-06-09 | BIOMD0000000660 | BioModels
Project description:Genomic aberrations of Cyclin D1 (CCND1) and CDK4 in neuroblastoma indicate that dysregulation of the G1 entry checkpoint is an important cell cycle aberration in this pediatric tumor. Here we report that analysis of Affymetrix expression data of primary neuroblastic tumors shows an extensive over-expression of Cyclin D1 and CDK4 which correlates with histological subgroups and prognosis respectively. Immunohistochemical analysis demonstrated an over-expression of Cyclin D1 in neuroblasts and a low Cyclin D1 expression in all cell types in ganglioneuroma. This suggests an involvement of G1 regulating genes in neuronal differentiation processes which we further evaluated using RNA interference against Cyclin D1 and its kinase partner CDK4 in several neuroblastoma cell lines. This resulted in pRb pathway inhibition as shown by an almost complete disappearance of CDK4 specific pRb phosphorylation; reduction of E2F transcriptional activity and a decrease of Cyclin A protein levels. The Cyclin D1 and CDK4 knock-down resulted in a significant reduction in cell proliferation, a G1 specific cell cycle arrest and moreover an extensive neuronal differentiation. Affymetrix microarray profiling of siRNA treated cells revealed a shift in expression profile towards a neuronal phenotype. Several new potential downstream players are identified. We conclude that neuroblastoma functionally depend on over-expression of G1 regulating genes to maintain their undifferentiated phenotype. Keywords: Neuroblastoma, CCND1, Cyclin D1, CDK4
2008-08-20 | GSE8866 | GEO
Project description:Immuno-histochemistry for RelA in breast tumors revealed a range of staining intensity and negative correlation between RelA levels and proliferation-index in estrogen receptor-positive tumors. Conditional expression of RelA arrested proliferation in primary mammary and fallopian tube epithelial cells. RelA dependent CDK4 downregulation was responsible for activating the G1/S checkpoint and cell cycle arrest. RelA target genes, including Interferon Response Factors (IRF), were up-regulated in the arrested cells. Among the IRFs, IRF1 expression correlates with RelA expression. Suppressing IRF1 restored CDK4 levels and rescued RelA-dependent proliferation arrest analogous to abrogating the G1/S checkpoint or restoring CDK4 levels. Apart from cyclins, regulation of CDK4 by the RelA-IRF1 transcriptional network controls proliferation of breast tumors and predicts sensitivity to a CDK4/6 inhibitor.
2015-09-26 | GSE65040 | GEO