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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

PD0332991 and MDM2 knockdown induce senescence is p53-mutant SNB19 cells(A) SNB19 glioma cell lines were treated with 1 μM PD0332991 and protein expression measured by immunoblot two days later. Tubulin is a loading control. (B) The accumulation of SA-β-gal and HP1γ foci was measured seven days after drug treatment as described in the legend to figures 1C and 1D. (C) Wild type and mutant MDM2 proteins were expressed in SNB19 glioma cells and analyzed as described in the legend to figure 3D. (D) MDM2 was knocked down in SNB19 cells with shM380 as described in the legend to figure 4A and the effect on accumulation of SA-β-gal positive cells (left) and p53 and p21 are shown (right).
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Figure 5: PD0332991 and MDM2 knockdown induce senescence is p53-mutant SNB19 cells(A) SNB19 glioma cell lines were treated with 1 μM PD0332991 and protein expression measured by immunoblot two days later. Tubulin is a loading control. (B) The accumulation of SA-β-gal and HP1γ foci was measured seven days after drug treatment as described in the legend to figures 1C and 1D. (C) Wild type and mutant MDM2 proteins were expressed in SNB19 glioma cells and analyzed as described in the legend to figure 3D. (D) MDM2 was knocked down in SNB19 cells with shM380 as described in the legend to figure 4A and the effect on accumulation of SA-β-gal positive cells (left) and p53 and p21 are shown (right).

Mentions: We then performed another test of p53-independence. PD0332991 had already been shown to induce senescence in a collection of glioma cell lines [5] and one of these, SNB19, expresses a transcriptionally inactive mutant of p53 (R273H) that does not bind to DNA. We found that MDM2 levels decreased in these cells within 48 hours of drug treatment (Figure 5A) and confirmed that SA-β-gal and SAHF accumulated in seven days after treatment (Figure 5B). Clonogenic growth was also reduced after drug treatment (Supplementary Figure 2B). Similar to what we saw in WD/DDLS cells, enforced MDM2 expression did not affect PD0332991-induced cell cycle exit, but did prevent the accumulation of SA-β-gal (Figure 5C). This was E3 ligase dependent because mutation of the RING abrogated MDM2's ability to suppress senescence; but, it was unrelated to the ability of MDM2 to bind to p53 because mutation of the p53 binding domain did not affect its ability to prevent senescence (Figure 5C). Finally, knocking down MDM2 was able to induce accumulation of SA-β-gal positive cells (Figure 5D). Consequently, MDM2 loss can trigger a p53-independent senescence program.


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)

PD0332991 and MDM2 knockdown induce senescence is p53-mutant SNB19 cells(A) SNB19 glioma cell lines were treated with 1 μM PD0332991 and protein expression measured by immunoblot two days later. Tubulin is a loading control. (B) The accumulation of SA-β-gal and HP1γ foci was measured seven days after drug treatment as described in the legend to figures 1C and 1D. (C) Wild type and mutant MDM2 proteins were expressed in SNB19 glioma cells and analyzed as described in the legend to figure 3D. (D) MDM2 was knocked down in SNB19 cells with shM380 as described in the legend to figure 4A and the effect on accumulation of SA-β-gal positive cells (left) and p53 and p21 are shown (right).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: PD0332991 and MDM2 knockdown induce senescence is p53-mutant SNB19 cells(A) SNB19 glioma cell lines were treated with 1 μM PD0332991 and protein expression measured by immunoblot two days later. Tubulin is a loading control. (B) The accumulation of SA-β-gal and HP1γ foci was measured seven days after drug treatment as described in the legend to figures 1C and 1D. (C) Wild type and mutant MDM2 proteins were expressed in SNB19 glioma cells and analyzed as described in the legend to figure 3D. (D) MDM2 was knocked down in SNB19 cells with shM380 as described in the legend to figure 4A and the effect on accumulation of SA-β-gal positive cells (left) and p53 and p21 are shown (right).
Mentions: We then performed another test of p53-independence. PD0332991 had already been shown to induce senescence in a collection of glioma cell lines [5] and one of these, SNB19, expresses a transcriptionally inactive mutant of p53 (R273H) that does not bind to DNA. We found that MDM2 levels decreased in these cells within 48 hours of drug treatment (Figure 5A) and confirmed that SA-β-gal and SAHF accumulated in seven days after treatment (Figure 5B). Clonogenic growth was also reduced after drug treatment (Supplementary Figure 2B). Similar to what we saw in WD/DDLS cells, enforced MDM2 expression did not affect PD0332991-induced cell cycle exit, but did prevent the accumulation of SA-β-gal (Figure 5C). This was E3 ligase dependent because mutation of the RING abrogated MDM2's ability to suppress senescence; but, it was unrelated to the ability of MDM2 to bind to p53 because mutation of the p53 binding domain did not affect its ability to prevent senescence (Figure 5C). Finally, knocking down MDM2 was able to induce accumulation of SA-β-gal positive cells (Figure 5D). Consequently, MDM2 loss can trigger a p53-independent senescence program.

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