Limits...
MDM2 turnover and expression of ATRX determine the choice between quiescence and senescence in response to CDK4 inhibition.

Kovatcheva M, Liu DD, Dickson MA, Klein ME, O'Connor R, Wilder FO, Socci ND, Tap WD, Schwartz GK, Singer S, Crago AM, Koff A - Oncotarget (2015)

Bottom Line: Failure to reduce MDM2 does not prevent CDK4i-induced withdrawal from the cell cycle but the cells remain in a reversible quiescent state.CDK4i-induced senescence associated with loss of MDM2 is also observed in some breast cancer, lung cancer and glioma cell lines indicating that this is not limited to WD/DDLS cells in which MDM2 is overexpressed or in cells that contain wild type p53.Interestingly, in seven patients the changes in MDM2 expression were correlated with outcome.

View Article: PubMed Central - PubMed

Affiliation: The Louis V. Gerstner Graduate School of Biomedical Sciences, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, USA.

ABSTRACT
CDK4 inhibitors (CDK4i) earned Breakthrough Therapy Designation from the FDA last year and are entering phase III clinical trials in several cancers. However, not all tumors respond favorably to these drugs. CDK4 activity is critical for progression through G1 phase and into the mitotic cell cycle. Inhibiting this kinase induces Rb-positive cells to exit the cell cycle into either a quiescent or senescent state. In this report, using well-differentiated and dedifferentiated liposarcoma (WD/DDLS) cell lines, we show that the proteolytic turnover of MDM2 is required for CDK4i-induced senescence. Failure to reduce MDM2 does not prevent CDK4i-induced withdrawal from the cell cycle but the cells remain in a reversible quiescent state. Reducing MDM2 in these cells drives them into the more stable senescent state. CDK4i-induced senescence associated with loss of MDM2 is also observed in some breast cancer, lung cancer and glioma cell lines indicating that this is not limited to WD/DDLS cells in which MDM2 is overexpressed or in cells that contain wild type p53. MDM2 turnover depends on its E3 ligase activity and expression of ATRX. Interestingly, in seven patients the changes in MDM2 expression were correlated with outcome. These insights identify MDM2 and ATRX as new regulators controlling geroconversion, the process by which quiescent cells become senescent, and this insight may be exploited to improve the activity of CDK4i in cancer therapy.

No MeSH data available.


Related in: MedlinePlus

Inhibition of CDK4 triggers either senescence or quiescence in WD/DDLS(A) Copy number alterations in WD/DDLS cell lines. Amplification (red) and deletions (blue) were identified using the RAE algorithm [81]. (B) Cells were grown in the presence (white) or absence (black) of 1 μM PD0332991 for 2 days and labeled with BrdU for the last two hours before fixation and immunofluorescence. The percentage (mean and standard deviation) of cells that incorporated BrdU into the nuclear DNA was determined and plotted (*p<0.05). (C) Cells staining for SA-β-gal seven days after 1 μM PD0332991 treatment (white) or in untreated asynchronously growing cultures (black) were quantitated in three or more independent experiments and the mean and standard deviation plotted. (*p<0.05). Representative phase contrast micrographs for LS8817 and LS8107 are shown. (D) This panel is arranged as described in panel C but we determined the percentage of cells in which for HP1γ foci accumulated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4480747&req=5

Figure 1: Inhibition of CDK4 triggers either senescence or quiescence in WD/DDLS(A) Copy number alterations in WD/DDLS cell lines. Amplification (red) and deletions (blue) were identified using the RAE algorithm [81]. (B) Cells were grown in the presence (white) or absence (black) of 1 μM PD0332991 for 2 days and labeled with BrdU for the last two hours before fixation and immunofluorescence. The percentage (mean and standard deviation) of cells that incorporated BrdU into the nuclear DNA was determined and plotted (*p<0.05). (C) Cells staining for SA-β-gal seven days after 1 μM PD0332991 treatment (white) or in untreated asynchronously growing cultures (black) were quantitated in three or more independent experiments and the mean and standard deviation plotted. (*p<0.05). Representative phase contrast micrographs for LS8817 and LS8107 are shown. (D) This panel is arranged as described in panel C but we determined the percentage of cells in which for HP1γ foci accumulated.

Mentions: We looked at the response of a panel of seven Rb-positive patient derived WD/DDLS cell lines. These cell lines had common amplifications of MDM2 and CDK4 and a heterogenous assortment of copy number alterations as identified by array CGH (Figure 1A). As expected, within 48 hours PD0332991 induced the accumulation of G0/G1 cells in all the cell lines with significantly reduced phosphorylated Rb (Supplementary Figure 1). Why total Rb decreased in some cells but not others is not clear. Bromodeoxyuridine (BrdU) incorporation was also dramatically reduced in all the cells (Figure 1B). However, the accumulation of perinuclear senescence associated β-galactosidase (SA-β-gal, Figure 1C) and focal HP1γ, a marker of senescence associated heterochromatic foci (SAHF, Figure 1D), increased only in LS8817, LS141 and LS0082 cells. Similar results were seen at a range of doses as low as 100nM and as high as 10 μM. The failure of LS7785-1, LS7785-10, LS8107 and LS8313 to undergo senescence was not associated with increased apoptosis or adipocytic differentiation. Thus, we defined LS8817, LS141 and LS0082 cells as responders: cells that undergo senescence when treated with PD0332991. The other four cell lines were defined as non-responders, which undergo quiescence when treated with the drug.


MDM2 turnover and expression of ATRX determine the choice between quiescence and senescence in response to CDK4 inhibition.

Kovatcheva M, Liu DD, Dickson MA, Klein ME, O'Connor R, Wilder FO, Socci ND, Tap WD, Schwartz GK, Singer S, Crago AM, Koff A - Oncotarget (2015)

Inhibition of CDK4 triggers either senescence or quiescence in WD/DDLS(A) Copy number alterations in WD/DDLS cell lines. Amplification (red) and deletions (blue) were identified using the RAE algorithm [81]. (B) Cells were grown in the presence (white) or absence (black) of 1 μM PD0332991 for 2 days and labeled with BrdU for the last two hours before fixation and immunofluorescence. The percentage (mean and standard deviation) of cells that incorporated BrdU into the nuclear DNA was determined and plotted (*p<0.05). (C) Cells staining for SA-β-gal seven days after 1 μM PD0332991 treatment (white) or in untreated asynchronously growing cultures (black) were quantitated in three or more independent experiments and the mean and standard deviation plotted. (*p<0.05). Representative phase contrast micrographs for LS8817 and LS8107 are shown. (D) This panel is arranged as described in panel C but we determined the percentage of cells in which for HP1γ foci accumulated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480747&req=5

Figure 1: Inhibition of CDK4 triggers either senescence or quiescence in WD/DDLS(A) Copy number alterations in WD/DDLS cell lines. Amplification (red) and deletions (blue) were identified using the RAE algorithm [81]. (B) Cells were grown in the presence (white) or absence (black) of 1 μM PD0332991 for 2 days and labeled with BrdU for the last two hours before fixation and immunofluorescence. The percentage (mean and standard deviation) of cells that incorporated BrdU into the nuclear DNA was determined and plotted (*p<0.05). (C) Cells staining for SA-β-gal seven days after 1 μM PD0332991 treatment (white) or in untreated asynchronously growing cultures (black) were quantitated in three or more independent experiments and the mean and standard deviation plotted. (*p<0.05). Representative phase contrast micrographs for LS8817 and LS8107 are shown. (D) This panel is arranged as described in panel C but we determined the percentage of cells in which for HP1γ foci accumulated.
Mentions: We looked at the response of a panel of seven Rb-positive patient derived WD/DDLS cell lines. These cell lines had common amplifications of MDM2 and CDK4 and a heterogenous assortment of copy number alterations as identified by array CGH (Figure 1A). As expected, within 48 hours PD0332991 induced the accumulation of G0/G1 cells in all the cell lines with significantly reduced phosphorylated Rb (Supplementary Figure 1). Why total Rb decreased in some cells but not others is not clear. Bromodeoxyuridine (BrdU) incorporation was also dramatically reduced in all the cells (Figure 1B). However, the accumulation of perinuclear senescence associated β-galactosidase (SA-β-gal, Figure 1C) and focal HP1γ, a marker of senescence associated heterochromatic foci (SAHF, Figure 1D), increased only in LS8817, LS141 and LS0082 cells. Similar results were seen at a range of doses as low as 100nM and as high as 10 μM. The failure of LS7785-1, LS7785-10, LS8107 and LS8313 to undergo senescence was not associated with increased apoptosis or adipocytic differentiation. Thus, we defined LS8817, LS141 and LS0082 cells as responders: cells that undergo senescence when treated with PD0332991. The other four cell lines were defined as non-responders, which undergo quiescence when treated with the drug.

Bottom Line: Failure to reduce MDM2 does not prevent CDK4i-induced withdrawal from the cell cycle but the cells remain in a reversible quiescent state.CDK4i-induced senescence associated with loss of MDM2 is also observed in some breast cancer, lung cancer and glioma cell lines indicating that this is not limited to WD/DDLS cells in which MDM2 is overexpressed or in cells that contain wild type p53.Interestingly, in seven patients the changes in MDM2 expression were correlated with outcome.

View Article: PubMed Central - PubMed

Affiliation: The Louis V. Gerstner Graduate School of Biomedical Sciences, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, USA.

ABSTRACT
CDK4 inhibitors (CDK4i) earned Breakthrough Therapy Designation from the FDA last year and are entering phase III clinical trials in several cancers. However, not all tumors respond favorably to these drugs. CDK4 activity is critical for progression through G1 phase and into the mitotic cell cycle. Inhibiting this kinase induces Rb-positive cells to exit the cell cycle into either a quiescent or senescent state. In this report, using well-differentiated and dedifferentiated liposarcoma (WD/DDLS) cell lines, we show that the proteolytic turnover of MDM2 is required for CDK4i-induced senescence. Failure to reduce MDM2 does not prevent CDK4i-induced withdrawal from the cell cycle but the cells remain in a reversible quiescent state. Reducing MDM2 in these cells drives them into the more stable senescent state. CDK4i-induced senescence associated with loss of MDM2 is also observed in some breast cancer, lung cancer and glioma cell lines indicating that this is not limited to WD/DDLS cells in which MDM2 is overexpressed or in cells that contain wild type p53. MDM2 turnover depends on its E3 ligase activity and expression of ATRX. Interestingly, in seven patients the changes in MDM2 expression were correlated with outcome. These insights identify MDM2 and ATRX as new regulators controlling geroconversion, the process by which quiescent cells become senescent, and this insight may be exploited to improve the activity of CDK4i in cancer therapy.

No MeSH data available.


Related in: MedlinePlus