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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

MDM2 is regulated post-translationally(A) LS8817 (left) and LS8107 (right) cells were treated with 1 μM PD0332991 for forty eight hours, and 75 μg/ml cycloheximide was added for the indicated times before proteins were extracted and amount of MDM2 measured by immunoblot. Tubulin is a loading control. Representative autoradiograms are shown below the graphs. (B) LS8817 cells were transduced with either a FLAG-tagged MDM2 or a FLAG-tagged C464A mutant of MDM2 as indicated and selected for five days. PD0332991 and cycloheximide were added as described in the legend to panel A. A representative immunoblot is shown and the graph was compiled from two independent experiments (mean + S.E.M.). (C) The indicated cell lines were treated with 1 μM PD0332991 for two days and 5μM MG132 added 2 hours prior to protein extraction. MDM2 was immunoprecipitated and HAUSP and MDM2 measured in the immunoprecipitate by immunoblot. IgG was used as a non-specific antibody control for the immunoprecipitation. (D) Proteins were extracted from asynchronously growing or cells treated with 1 μM PD0332991 for 2 days and the expression of HAUSP measured by immunoblot. Tubulin is a loading control. (E, F) The indicated cells were transduced with two different lentiviruses expressing shRNA targeting HAUSP or a scrambled control and selected for 10 days. All cells had exited the mitotic cycle. (E) Extracts were prepared and the accumulation of proteins detected by immunoblot. For comparison the effect of the MDM2 shM380 lentiviral knockdown is shown. (F) The accumulation of SA-β-gal and HP1γ positive cells was measured as described in other figures. This experiment was done twice with similar results.
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Figure 6: MDM2 is regulated post-translationally(A) LS8817 (left) and LS8107 (right) cells were treated with 1 μM PD0332991 for forty eight hours, and 75 μg/ml cycloheximide was added for the indicated times before proteins were extracted and amount of MDM2 measured by immunoblot. Tubulin is a loading control. Representative autoradiograms are shown below the graphs. (B) LS8817 cells were transduced with either a FLAG-tagged MDM2 or a FLAG-tagged C464A mutant of MDM2 as indicated and selected for five days. PD0332991 and cycloheximide were added as described in the legend to panel A. A representative immunoblot is shown and the graph was compiled from two independent experiments (mean + S.E.M.). (C) The indicated cell lines were treated with 1 μM PD0332991 for two days and 5μM MG132 added 2 hours prior to protein extraction. MDM2 was immunoprecipitated and HAUSP and MDM2 measured in the immunoprecipitate by immunoblot. IgG was used as a non-specific antibody control for the immunoprecipitation. (D) Proteins were extracted from asynchronously growing or cells treated with 1 μM PD0332991 for 2 days and the expression of HAUSP measured by immunoblot. Tubulin is a loading control. (E, F) The indicated cells were transduced with two different lentiviruses expressing shRNA targeting HAUSP or a scrambled control and selected for 10 days. All cells had exited the mitotic cycle. (E) Extracts were prepared and the accumulation of proteins detected by immunoblot. For comparison the effect of the MDM2 shM380 lentiviral knockdown is shown. (F) The accumulation of SA-β-gal and HP1γ positive cells was measured as described in other figures. This experiment was done twice with similar results.

Mentions: We next wanted to gain some insight into the regulation of MDM2. In all the cell lines MDM2 transcripts were modestly reduced by PD0332991, possibly reflecting the CDK4 inhibitor induced loss of p53 (Supplementary Figure 6). In contrast, MDM2 turnover was accelerated in LS8817, LS141 and LS0082 responder cells, but not in the LS8107, LS7785-1 and LS7785-10 non-responder cells (Figure 6A and Supplementary Figure 7). Adding the proteasome inhibitor MG132 to PD0332991-treated LS8817 or LS141 cells prevented the decrease in MDM2 (Supplementary Figure 7). Thus, the PD0332991-triggered loss of MDM2 was at least partially due to increased post-translational proteasome-dependent turnover. We did not look at turnover in LS8313 non-responder cells.


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)

MDM2 is regulated post-translationally(A) LS8817 (left) and LS8107 (right) cells were treated with 1 μM PD0332991 for forty eight hours, and 75 μg/ml cycloheximide was added for the indicated times before proteins were extracted and amount of MDM2 measured by immunoblot. Tubulin is a loading control. Representative autoradiograms are shown below the graphs. (B) LS8817 cells were transduced with either a FLAG-tagged MDM2 or a FLAG-tagged C464A mutant of MDM2 as indicated and selected for five days. PD0332991 and cycloheximide were added as described in the legend to panel A. A representative immunoblot is shown and the graph was compiled from two independent experiments (mean + S.E.M.). (C) The indicated cell lines were treated with 1 μM PD0332991 for two days and 5μM MG132 added 2 hours prior to protein extraction. MDM2 was immunoprecipitated and HAUSP and MDM2 measured in the immunoprecipitate by immunoblot. IgG was used as a non-specific antibody control for the immunoprecipitation. (D) Proteins were extracted from asynchronously growing or cells treated with 1 μM PD0332991 for 2 days and the expression of HAUSP measured by immunoblot. Tubulin is a loading control. (E, F) The indicated cells were transduced with two different lentiviruses expressing shRNA targeting HAUSP or a scrambled control and selected for 10 days. All cells had exited the mitotic cycle. (E) Extracts were prepared and the accumulation of proteins detected by immunoblot. For comparison the effect of the MDM2 shM380 lentiviral knockdown is shown. (F) The accumulation of SA-β-gal and HP1γ positive cells was measured as described in other figures. This experiment was done twice with similar results.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4480747&req=5

Figure 6: MDM2 is regulated post-translationally(A) LS8817 (left) and LS8107 (right) cells were treated with 1 μM PD0332991 for forty eight hours, and 75 μg/ml cycloheximide was added for the indicated times before proteins were extracted and amount of MDM2 measured by immunoblot. Tubulin is a loading control. Representative autoradiograms are shown below the graphs. (B) LS8817 cells were transduced with either a FLAG-tagged MDM2 or a FLAG-tagged C464A mutant of MDM2 as indicated and selected for five days. PD0332991 and cycloheximide were added as described in the legend to panel A. A representative immunoblot is shown and the graph was compiled from two independent experiments (mean + S.E.M.). (C) The indicated cell lines were treated with 1 μM PD0332991 for two days and 5μM MG132 added 2 hours prior to protein extraction. MDM2 was immunoprecipitated and HAUSP and MDM2 measured in the immunoprecipitate by immunoblot. IgG was used as a non-specific antibody control for the immunoprecipitation. (D) Proteins were extracted from asynchronously growing or cells treated with 1 μM PD0332991 for 2 days and the expression of HAUSP measured by immunoblot. Tubulin is a loading control. (E, F) The indicated cells were transduced with two different lentiviruses expressing shRNA targeting HAUSP or a scrambled control and selected for 10 days. All cells had exited the mitotic cycle. (E) Extracts were prepared and the accumulation of proteins detected by immunoblot. For comparison the effect of the MDM2 shM380 lentiviral knockdown is shown. (F) The accumulation of SA-β-gal and HP1γ positive cells was measured as described in other figures. This experiment was done twice with similar results.
Mentions: We next wanted to gain some insight into the regulation of MDM2. In all the cell lines MDM2 transcripts were modestly reduced by PD0332991, possibly reflecting the CDK4 inhibitor induced loss of p53 (Supplementary Figure 6). In contrast, MDM2 turnover was accelerated in LS8817, LS141 and LS0082 responder cells, but not in the LS8107, LS7785-1 and LS7785-10 non-responder cells (Figure 6A and Supplementary Figure 7). Adding the proteasome inhibitor MG132 to PD0332991-treated LS8817 or LS141 cells prevented the decrease in MDM2 (Supplementary Figure 7). Thus, the PD0332991-triggered loss of MDM2 was at least partially due to increased post-translational proteasome-dependent turnover. We did not look at turnover in LS8313 non-responder cells.

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