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HDACi inhibits liposarcoma via targeting of the MDM2-p53 signaling axis and PTEN, irrespective of p53 mutational status.

Ou WB, Zhu J, Eilers G, Li X, Kuang Y, Liu L, Mariño-Enríquez A, Yan Z, Li H, Meng F, Zhou H, Sheng Q, Fletcher JA - Oncotarget (2015)

Bottom Line: We demonstrated that simultaneous knockdown of MDM2 and p53 in p53-mutant LPS lines resulted in increased apoptosis, anti-proliferative effects, and cell cycle arrest, as compared to either intervention alone.HDACi treatment resulted in the dephosphorylation and depletion of MDM2 and p53 without affecting CDK4 and JUN expression, irrespective of p53 mutational status in MDM2-amplified LPS.Moreover, the pro-apoptotic and anti-proliferative effect of HDACi warrants further evaluation as a therapeutic strategy in MDM2-amplified LPS.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.

ABSTRACT
The MDM2-p53 pathway plays a prominent role in well-differentiated liposarcoma (LPS) pathogenesis. Here, we explore the importance of MDM2 amplification and p53 mutation in LPS independently, to determine whether HDACi are therapeutically useful in LPS. We demonstrated that simultaneous knockdown of MDM2 and p53 in p53-mutant LPS lines resulted in increased apoptosis, anti-proliferative effects, and cell cycle arrest, as compared to either intervention alone. HDACi treatment resulted in the dephosphorylation and depletion of MDM2 and p53 without affecting CDK4 and JUN expression, irrespective of p53 mutational status in MDM2-amplified LPS. In control mesothelioma cell lines, HDACi treatment resulted in down-regulation of p53 in the p53 mutant cell line JMN1B, but resulted in no changes of MDM2 and p53 in two mesothelioma lines with normal MDM2 and wild-type p53. HDACi treatment substantially decreased LPS and mesothelioma proliferation and survival, and was associated with upregulation of PTEN and p21, and inactivation of AKT. Our findings indicate that wild-type p53 depletion by HDACi is MDM2 amplification-dependent. These findings underscore the importance of targeting both MDM2 and p53 in LPS and other cancers harboring p53 mutations. Moreover, the pro-apoptotic and anti-proliferative effect of HDACi warrants further evaluation as a therapeutic strategy in MDM2-amplified LPS.

No MeSH data available.


Related in: MedlinePlus

Additive effects were observed through coordinated knockdowns of MDM2 and TP53 as demonstrated by immunoblotting (A) cell culture appearance (B) cell viability (C) and cell cycle analyses (D) showing that MDM2 and TP53 knockdown had greater anti-proliferative effects and cell cycle arrest, compared to either intervention alone in MDM2-amplified and p53 mutant liposarcoma cell lines(A) MDM2, p53, and p21 were evaluated by immunoblotting at 10 days post-infection with stable MDM2 and/or TP53 shRNA expression. Actin staining is a loading control. (B) Cell culture appearance in LPS141/239 and LPS510 at 10 days after infection by lentiviral MDM2 and/or TP53 shRNA constructs, showing greater growth inhibition compared to either intervention alone. (C) Cell viability evaluated by a Cell-titer Glo® ATP-based luminescence assay in liposarcoma cell lines (LPS141, LPS141/239, LPS141/266, and LPS510), at 72 hours following stable MDM2 and/or TP53 shRNA expression for 10 days. Data were normalized to the empty lentivirus infections or DMSO, and represent the mean values (± s.d.) from quadruplicate cultures. Statistically significant differences between empty vector control and target gene shRNAs are presented as *p < 0.05, **p < 0.01, ***p < 0.001. (D) Cell cycle analyses performed at 72 hours following stable lentiviral MDM2 and/or TP53 shRNA expression for 10 days.
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Figure 4: Additive effects were observed through coordinated knockdowns of MDM2 and TP53 as demonstrated by immunoblotting (A) cell culture appearance (B) cell viability (C) and cell cycle analyses (D) showing that MDM2 and TP53 knockdown had greater anti-proliferative effects and cell cycle arrest, compared to either intervention alone in MDM2-amplified and p53 mutant liposarcoma cell lines(A) MDM2, p53, and p21 were evaluated by immunoblotting at 10 days post-infection with stable MDM2 and/or TP53 shRNA expression. Actin staining is a loading control. (B) Cell culture appearance in LPS141/239 and LPS510 at 10 days after infection by lentiviral MDM2 and/or TP53 shRNA constructs, showing greater growth inhibition compared to either intervention alone. (C) Cell viability evaluated by a Cell-titer Glo® ATP-based luminescence assay in liposarcoma cell lines (LPS141, LPS141/239, LPS141/266, and LPS510), at 72 hours following stable MDM2 and/or TP53 shRNA expression for 10 days. Data were normalized to the empty lentivirus infections or DMSO, and represent the mean values (± s.d.) from quadruplicate cultures. Statistically significant differences between empty vector control and target gene shRNAs are presented as *p < 0.05, **p < 0.01, ***p < 0.001. (D) Cell cycle analyses performed at 72 hours following stable lentiviral MDM2 and/or TP53 shRNA expression for 10 days.

Mentions: MDM2 and TP53 gene expression was stably silenced by lentivirus-mediated shRNAs (Figure 4), and knockdown was evaluated by immunoblotting, in LPS141, LPS141/239, LPS141/266, and LPS510, and MESO924, at 10 days after transduction (Figure 4A). MESO924 has normal MDM2 and wild-type p53. We achieved greater than 60% knockdown of the targets with at least one construct. MDM2 knockdown increased p53 and p21 expression, irrespective of p53 mutational status, in p53 wild-type LPS141 and MESO924. p53 knockdown had little effect on MDM2 and p21 in LPS cell lines but decreased MDM2 and p21 expression in MESO924 (Figure 4A). MDM2 or p53 knockdown alone dramatically reduced LPS510 and LPS141/239 cell growth. The combination of MDM2 or p53 knockdown had an additive effect on cell growth, compared to either intervention alone (Figure 4B). MDM2 knockdown, in p53 wild-type LPS141, and p53-mutant LPS141/239 and LPS510, resulted in ~40% and ~60–70% inhibition of cell viability, respectively, at 10 days after MDM2 silencing, compared to the empty vector control. MDM2 knockdown resulted in a mild reduction in viability for a p53 mutant LPS141/266 (Figure 4C). p53 knockdown with at least one construct resulted in 20–70% reduction in viability of three mutant p53 LPS cell lines (LPS141/266, LPS141/239, and LPS510), but had little effect on viability of p53 wild-type LPS141. Combination of MDM2 and p53 knockdown resulted in 40–80% reduction in cell viability and showed a greater effect on cell growth, in three mutant p53 LPS cell lines (LPS141/266, LPS141/239, and LPS510), compared to either intervention alone (Figure 4B).


HDACi inhibits liposarcoma via targeting of the MDM2-p53 signaling axis and PTEN, irrespective of p53 mutational status.

Ou WB, Zhu J, Eilers G, Li X, Kuang Y, Liu L, Mariño-Enríquez A, Yan Z, Li H, Meng F, Zhou H, Sheng Q, Fletcher JA - Oncotarget (2015)

Additive effects were observed through coordinated knockdowns of MDM2 and TP53 as demonstrated by immunoblotting (A) cell culture appearance (B) cell viability (C) and cell cycle analyses (D) showing that MDM2 and TP53 knockdown had greater anti-proliferative effects and cell cycle arrest, compared to either intervention alone in MDM2-amplified and p53 mutant liposarcoma cell lines(A) MDM2, p53, and p21 were evaluated by immunoblotting at 10 days post-infection with stable MDM2 and/or TP53 shRNA expression. Actin staining is a loading control. (B) Cell culture appearance in LPS141/239 and LPS510 at 10 days after infection by lentiviral MDM2 and/or TP53 shRNA constructs, showing greater growth inhibition compared to either intervention alone. (C) Cell viability evaluated by a Cell-titer Glo® ATP-based luminescence assay in liposarcoma cell lines (LPS141, LPS141/239, LPS141/266, and LPS510), at 72 hours following stable MDM2 and/or TP53 shRNA expression for 10 days. Data were normalized to the empty lentivirus infections or DMSO, and represent the mean values (± s.d.) from quadruplicate cultures. Statistically significant differences between empty vector control and target gene shRNAs are presented as *p < 0.05, **p < 0.01, ***p < 0.001. (D) Cell cycle analyses performed at 72 hours following stable lentiviral MDM2 and/or TP53 shRNA expression for 10 days.
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Related In: Results  -  Collection

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Figure 4: Additive effects were observed through coordinated knockdowns of MDM2 and TP53 as demonstrated by immunoblotting (A) cell culture appearance (B) cell viability (C) and cell cycle analyses (D) showing that MDM2 and TP53 knockdown had greater anti-proliferative effects and cell cycle arrest, compared to either intervention alone in MDM2-amplified and p53 mutant liposarcoma cell lines(A) MDM2, p53, and p21 were evaluated by immunoblotting at 10 days post-infection with stable MDM2 and/or TP53 shRNA expression. Actin staining is a loading control. (B) Cell culture appearance in LPS141/239 and LPS510 at 10 days after infection by lentiviral MDM2 and/or TP53 shRNA constructs, showing greater growth inhibition compared to either intervention alone. (C) Cell viability evaluated by a Cell-titer Glo® ATP-based luminescence assay in liposarcoma cell lines (LPS141, LPS141/239, LPS141/266, and LPS510), at 72 hours following stable MDM2 and/or TP53 shRNA expression for 10 days. Data were normalized to the empty lentivirus infections or DMSO, and represent the mean values (± s.d.) from quadruplicate cultures. Statistically significant differences between empty vector control and target gene shRNAs are presented as *p < 0.05, **p < 0.01, ***p < 0.001. (D) Cell cycle analyses performed at 72 hours following stable lentiviral MDM2 and/or TP53 shRNA expression for 10 days.
Mentions: MDM2 and TP53 gene expression was stably silenced by lentivirus-mediated shRNAs (Figure 4), and knockdown was evaluated by immunoblotting, in LPS141, LPS141/239, LPS141/266, and LPS510, and MESO924, at 10 days after transduction (Figure 4A). MESO924 has normal MDM2 and wild-type p53. We achieved greater than 60% knockdown of the targets with at least one construct. MDM2 knockdown increased p53 and p21 expression, irrespective of p53 mutational status, in p53 wild-type LPS141 and MESO924. p53 knockdown had little effect on MDM2 and p21 in LPS cell lines but decreased MDM2 and p21 expression in MESO924 (Figure 4A). MDM2 or p53 knockdown alone dramatically reduced LPS510 and LPS141/239 cell growth. The combination of MDM2 or p53 knockdown had an additive effect on cell growth, compared to either intervention alone (Figure 4B). MDM2 knockdown, in p53 wild-type LPS141, and p53-mutant LPS141/239 and LPS510, resulted in ~40% and ~60–70% inhibition of cell viability, respectively, at 10 days after MDM2 silencing, compared to the empty vector control. MDM2 knockdown resulted in a mild reduction in viability for a p53 mutant LPS141/266 (Figure 4C). p53 knockdown with at least one construct resulted in 20–70% reduction in viability of three mutant p53 LPS cell lines (LPS141/266, LPS141/239, and LPS510), but had little effect on viability of p53 wild-type LPS141. Combination of MDM2 and p53 knockdown resulted in 40–80% reduction in cell viability and showed a greater effect on cell growth, in three mutant p53 LPS cell lines (LPS141/266, LPS141/239, and LPS510), compared to either intervention alone (Figure 4B).

Bottom Line: We demonstrated that simultaneous knockdown of MDM2 and p53 in p53-mutant LPS lines resulted in increased apoptosis, anti-proliferative effects, and cell cycle arrest, as compared to either intervention alone.HDACi treatment resulted in the dephosphorylation and depletion of MDM2 and p53 without affecting CDK4 and JUN expression, irrespective of p53 mutational status in MDM2-amplified LPS.Moreover, the pro-apoptotic and anti-proliferative effect of HDACi warrants further evaluation as a therapeutic strategy in MDM2-amplified LPS.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.

ABSTRACT
The MDM2-p53 pathway plays a prominent role in well-differentiated liposarcoma (LPS) pathogenesis. Here, we explore the importance of MDM2 amplification and p53 mutation in LPS independently, to determine whether HDACi are therapeutically useful in LPS. We demonstrated that simultaneous knockdown of MDM2 and p53 in p53-mutant LPS lines resulted in increased apoptosis, anti-proliferative effects, and cell cycle arrest, as compared to either intervention alone. HDACi treatment resulted in the dephosphorylation and depletion of MDM2 and p53 without affecting CDK4 and JUN expression, irrespective of p53 mutational status in MDM2-amplified LPS. In control mesothelioma cell lines, HDACi treatment resulted in down-regulation of p53 in the p53 mutant cell line JMN1B, but resulted in no changes of MDM2 and p53 in two mesothelioma lines with normal MDM2 and wild-type p53. HDACi treatment substantially decreased LPS and mesothelioma proliferation and survival, and was associated with upregulation of PTEN and p21, and inactivation of AKT. Our findings indicate that wild-type p53 depletion by HDACi is MDM2 amplification-dependent. These findings underscore the importance of targeting both MDM2 and p53 in LPS and other cancers harboring p53 mutations. Moreover, the pro-apoptotic and anti-proliferative effect of HDACi warrants further evaluation as a therapeutic strategy in MDM2-amplified LPS.

No MeSH data available.


Related in: MedlinePlus