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

Loss of MDM2 can trigger senescence in WD/DDLS(A) The indicated cells were transduced with two different MDM2 knockdown lentiviral vectors (M376 or M380) or a scrambled non-specific vector (scr) and selected in puromycin for five days prior to extraction of proteins for immunoblotting. (B) The percentage of cells staining for SA-β-gal 10 days after knockdown were quantitated as described in the legend to figure 1C. (*p<0.05). (C) This is arranged as in panel B but the percentage of cells staining for HP1γ foci were quantitated (*p<0.05). (D) LS8107 cells were first arrested in PD0332991 for 2 days and MDM2 was subsequently knocked down with shM380 and cells selected as described in panel A. The effect on the accumulation of SA-β-gal positive cells and expression of MDM2 and p53 is shown. Tubulin is a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Loss of MDM2 can trigger senescence in WD/DDLS(A) The indicated cells were transduced with two different MDM2 knockdown lentiviral vectors (M376 or M380) or a scrambled non-specific vector (scr) and selected in puromycin for five days prior to extraction of proteins for immunoblotting. (B) The percentage of cells staining for SA-β-gal 10 days after knockdown were quantitated as described in the legend to figure 1C. (*p<0.05). (C) This is arranged as in panel B but the percentage of cells staining for HP1γ foci were quantitated (*p<0.05). (D) LS8107 cells were first arrested in PD0332991 for 2 days and MDM2 was subsequently knocked down with shM380 and cells selected as described in panel A. The effect on the accumulation of SA-β-gal positive cells and expression of MDM2 and p53 is shown. Tubulin is a loading control.

Mentions: We then asked if knocking down MDM2 would be sufficient to induce senescence in the non-responder cells. To accomplish this, we generated two independent targeting vectors and transduced LS8107 and LS7785-1 non-responder cells with these or a scrambled control. Rb phosphorylation and the amount of cyclin A were reduced in the cells in which MDM2 was knocked down, indicative of growth arrest, even in the absence of PD0332991 (Figure 4A). Arf and p16 levels did not increase but there was a modest increase in p21, which is consistent with an increase in p53 activity when knocking down MDM2. p53 is wild type and functional in all of these WD/DDLS cells as demonstrated by the fact that nutlin-3 induced inhibition of MDM2-p53 interaction triggers apoptosis [35].


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)

Loss of MDM2 can trigger senescence in WD/DDLS(A) The indicated cells were transduced with two different MDM2 knockdown lentiviral vectors (M376 or M380) or a scrambled non-specific vector (scr) and selected in puromycin for five days prior to extraction of proteins for immunoblotting. (B) The percentage of cells staining for SA-β-gal 10 days after knockdown were quantitated as described in the legend to figure 1C. (*p<0.05). (C) This is arranged as in panel B but the percentage of cells staining for HP1γ foci were quantitated (*p<0.05). (D) LS8107 cells were first arrested in PD0332991 for 2 days and MDM2 was subsequently knocked down with shM380 and cells selected as described in panel A. The effect on the accumulation of SA-β-gal positive cells and expression of MDM2 and p53 is shown. Tubulin is a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Loss of MDM2 can trigger senescence in WD/DDLS(A) The indicated cells were transduced with two different MDM2 knockdown lentiviral vectors (M376 or M380) or a scrambled non-specific vector (scr) and selected in puromycin for five days prior to extraction of proteins for immunoblotting. (B) The percentage of cells staining for SA-β-gal 10 days after knockdown were quantitated as described in the legend to figure 1C. (*p<0.05). (C) This is arranged as in panel B but the percentage of cells staining for HP1γ foci were quantitated (*p<0.05). (D) LS8107 cells were first arrested in PD0332991 for 2 days and MDM2 was subsequently knocked down with shM380 and cells selected as described in panel A. The effect on the accumulation of SA-β-gal positive cells and expression of MDM2 and p53 is shown. Tubulin is a loading control.
Mentions: We then asked if knocking down MDM2 would be sufficient to induce senescence in the non-responder cells. To accomplish this, we generated two independent targeting vectors and transduced LS8107 and LS7785-1 non-responder cells with these or a scrambled control. Rb phosphorylation and the amount of cyclin A were reduced in the cells in which MDM2 was knocked down, indicative of growth arrest, even in the absence of PD0332991 (Figure 4A). Arf and p16 levels did not increase but there was a modest increase in p21, which is consistent with an increase in p53 activity when knocking down MDM2. p53 is wild type and functional in all of these WD/DDLS cells as demonstrated by the fact that nutlin-3 induced inhibition of MDM2-p53 interaction triggers apoptosis [35].

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