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Intracellular CD24 disrupts the ARF-NPM interaction and enables mutational and viral oncogene-mediated p53 inactivation.

Wang L, Liu R, Ye P, Wong C, Chen GY, Zhou P, Sakabe K, Zheng X, Wu W, Zhang P, Jiang T, Bassetti MF, Jube S, Sun Y, Zhang Y, Zheng P, Liu Y - Nat Commun (2015)

Bottom Line: CD24 competitively inhibits ARF binding to NPM, resulting in decreased ARF, increase MDM2 and decrease levels of p53 and the p53 target p21/CDKN1A.CD24 silencing prevents functional inactivation of p53 by both somatic mutation and viral oncogenes, including the SV40 large T antigen and human papilloma virus 16 E6-antigen.In support of the functional interaction between CD24 and p53, in silico analyses reveal that TP53 mutates at a higher rate among glioma and prostate cancer samples with higher CD24 mRNA levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

ABSTRACT
CD24 is overexpressed in nearly 70% human cancers, whereas TP53 is the most frequently mutated tumour-suppressor gene that functions in a context-dependent manner. Here we show that both targeted mutation and short hairpin RNA (shRNA) silencing of CD24 retard the growth, progression and metastasis of prostate cancer. CD24 competitively inhibits ARF binding to NPM, resulting in decreased ARF, increase MDM2 and decrease levels of p53 and the p53 target p21/CDKN1A. CD24 silencing prevents functional inactivation of p53 by both somatic mutation and viral oncogenes, including the SV40 large T antigen and human papilloma virus 16 E6-antigen. In support of the functional interaction between CD24 and p53, in silico analyses reveal that TP53 mutates at a higher rate among glioma and prostate cancer samples with higher CD24 mRNA levels. These data provide a general mechanism for functional inactivation of ARF and reveal an important cellular context for genetic and viral inactivation of TP53.

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CD24 inhibits tumour-suppressor activity of ARF by antagonizing the NPM–ARF interaction.(a) Recombinant CD24 associates with recombinant NPM and blocks the ARF–NPM interaction. Control GST and NPM–GST fusion proteins were incubated with indicated amounts of CD24-Fc and/or ARF. The GST or GST–NPM1-associated proteins were determined by western blot after pull-down with glutathione beads. (b,c) Proximity ligation assay reveals CD24 antagonism of NPM–ARF association. Scrambled or CD24 shRNA-transduced DU145 cells were compared for endogenous NPM–ARF association. Images were obtained using an Olympus BX61 microscope using a × 60 oil objective. Scale bar, 50 μm. Image J software was used to quantify fluorescent intensities. Mean fluorescent intensities were determined from more than 100 cells per slide. This experiment has been repeated three times. Data shown are means and s.d. of three independent repeats (b) and representative images (c). (d) Cd24 reduces Npm-associated p19Arf. Spleen lysates from WT and Cd24−/− mice were precipitated with anti-Npm mAb. The amounts of Npm and p19Arf in the immunoprecipitates were determined by western blot. IgG heavy chain (IgHC) was used as loading controls. (e) Regulation of ARF levels and stability by endogenous CD24. Scrambled or shRNA-silenced DU145 cells were arrested at G2/M phase overnight and then treated with CHX for 0, 2 and 4 h. The levels of CD24 (upper panel) and p14ARF were analysed by western blot. (f) Decay kinetics of p14ARF are shown as % of ARF protein at different times after CHX treatment. (g) ShRNA silencing of CD24 does not affect the levels of the ARF E3 ligase ULF. (h) Ectopic expression of CD24 reduces ARF levels. U2OS cells that ectopically expressed CD24 alone, ARF alone or both were analysed for their CD24 and ARF levels by western blot. (i) CD24 expression alleviates ARF tumour-suppressor activity. Data shown are growth kinetics of U2OS cells transfected with the given plasmids. Data shown are means and s.d. of cell numbers during the 6-day period. All data in this figure have been reproduced two or three times.
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f7: CD24 inhibits tumour-suppressor activity of ARF by antagonizing the NPM–ARF interaction.(a) Recombinant CD24 associates with recombinant NPM and blocks the ARF–NPM interaction. Control GST and NPM–GST fusion proteins were incubated with indicated amounts of CD24-Fc and/or ARF. The GST or GST–NPM1-associated proteins were determined by western blot after pull-down with glutathione beads. (b,c) Proximity ligation assay reveals CD24 antagonism of NPM–ARF association. Scrambled or CD24 shRNA-transduced DU145 cells were compared for endogenous NPM–ARF association. Images were obtained using an Olympus BX61 microscope using a × 60 oil objective. Scale bar, 50 μm. Image J software was used to quantify fluorescent intensities. Mean fluorescent intensities were determined from more than 100 cells per slide. This experiment has been repeated three times. Data shown are means and s.d. of three independent repeats (b) and representative images (c). (d) Cd24 reduces Npm-associated p19Arf. Spleen lysates from WT and Cd24−/− mice were precipitated with anti-Npm mAb. The amounts of Npm and p19Arf in the immunoprecipitates were determined by western blot. IgG heavy chain (IgHC) was used as loading controls. (e) Regulation of ARF levels and stability by endogenous CD24. Scrambled or shRNA-silenced DU145 cells were arrested at G2/M phase overnight and then treated with CHX for 0, 2 and 4 h. The levels of CD24 (upper panel) and p14ARF were analysed by western blot. (f) Decay kinetics of p14ARF are shown as % of ARF protein at different times after CHX treatment. (g) ShRNA silencing of CD24 does not affect the levels of the ARF E3 ligase ULF. (h) Ectopic expression of CD24 reduces ARF levels. U2OS cells that ectopically expressed CD24 alone, ARF alone or both were analysed for their CD24 and ARF levels by western blot. (i) CD24 expression alleviates ARF tumour-suppressor activity. Data shown are growth kinetics of U2OS cells transfected with the given plasmids. Data shown are means and s.d. of cell numbers during the 6-day period. All data in this figure have been reproduced two or three times.

Mentions: To determine whether CD24 competes with ARF for interaction with NPM, we co-incubated recombinant ARF with either control glutathione S-transferase (GST) or NPM–GST in the presence of increasing amounts of CD24-Fc or control IgG-Fc. The proteins that associated with the GST-fusion proteins were pulled down using glutathione beads and identified by western blot using anti-NPM, anti-CD24 or anti-ARF. As shown in Fig. 7a, CD24-Fc, but not control IgG-Fc, blocked the ARF–NPM association in a dose-dependent manner. To determine whether CD24 inhibits the ARF–NPM association in cancer cells, we used a proximity ligation assay to assess the association between NPM and ARF in DU145 cells with or without CD24 silencing. A summary of three experiments is shown in Fig. 7b, whereas representative images from one experiment are shown in Fig. 7c. These data demonstrate that shRNA silencing of CD24 increased the NPM–ARF association in tumour cells. To test if CD24 also affects the ARF–NPM interaction in normal cells, we compared the amounts of Npm-associated Arf in WT and Cd24−/− thymocytes. As shown in Fig. 7d, a substantial increase in Npm-associated Arf was observed in Cd24−/− thymocytes. These results demonstrate that CD24 inhibited ARF association with NPM.


Intracellular CD24 disrupts the ARF-NPM interaction and enables mutational and viral oncogene-mediated p53 inactivation.

Wang L, Liu R, Ye P, Wong C, Chen GY, Zhou P, Sakabe K, Zheng X, Wu W, Zhang P, Jiang T, Bassetti MF, Jube S, Sun Y, Zhang Y, Zheng P, Liu Y - Nat Commun (2015)

CD24 inhibits tumour-suppressor activity of ARF by antagonizing the NPM–ARF interaction.(a) Recombinant CD24 associates with recombinant NPM and blocks the ARF–NPM interaction. Control GST and NPM–GST fusion proteins were incubated with indicated amounts of CD24-Fc and/or ARF. The GST or GST–NPM1-associated proteins were determined by western blot after pull-down with glutathione beads. (b,c) Proximity ligation assay reveals CD24 antagonism of NPM–ARF association. Scrambled or CD24 shRNA-transduced DU145 cells were compared for endogenous NPM–ARF association. Images were obtained using an Olympus BX61 microscope using a × 60 oil objective. Scale bar, 50 μm. Image J software was used to quantify fluorescent intensities. Mean fluorescent intensities were determined from more than 100 cells per slide. This experiment has been repeated three times. Data shown are means and s.d. of three independent repeats (b) and representative images (c). (d) Cd24 reduces Npm-associated p19Arf. Spleen lysates from WT and Cd24−/− mice were precipitated with anti-Npm mAb. The amounts of Npm and p19Arf in the immunoprecipitates were determined by western blot. IgG heavy chain (IgHC) was used as loading controls. (e) Regulation of ARF levels and stability by endogenous CD24. Scrambled or shRNA-silenced DU145 cells were arrested at G2/M phase overnight and then treated with CHX for 0, 2 and 4 h. The levels of CD24 (upper panel) and p14ARF were analysed by western blot. (f) Decay kinetics of p14ARF are shown as % of ARF protein at different times after CHX treatment. (g) ShRNA silencing of CD24 does not affect the levels of the ARF E3 ligase ULF. (h) Ectopic expression of CD24 reduces ARF levels. U2OS cells that ectopically expressed CD24 alone, ARF alone or both were analysed for their CD24 and ARF levels by western blot. (i) CD24 expression alleviates ARF tumour-suppressor activity. Data shown are growth kinetics of U2OS cells transfected with the given plasmids. Data shown are means and s.d. of cell numbers during the 6-day period. All data in this figure have been reproduced two or three times.
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f7: CD24 inhibits tumour-suppressor activity of ARF by antagonizing the NPM–ARF interaction.(a) Recombinant CD24 associates with recombinant NPM and blocks the ARF–NPM interaction. Control GST and NPM–GST fusion proteins were incubated with indicated amounts of CD24-Fc and/or ARF. The GST or GST–NPM1-associated proteins were determined by western blot after pull-down with glutathione beads. (b,c) Proximity ligation assay reveals CD24 antagonism of NPM–ARF association. Scrambled or CD24 shRNA-transduced DU145 cells were compared for endogenous NPM–ARF association. Images were obtained using an Olympus BX61 microscope using a × 60 oil objective. Scale bar, 50 μm. Image J software was used to quantify fluorescent intensities. Mean fluorescent intensities were determined from more than 100 cells per slide. This experiment has been repeated three times. Data shown are means and s.d. of three independent repeats (b) and representative images (c). (d) Cd24 reduces Npm-associated p19Arf. Spleen lysates from WT and Cd24−/− mice were precipitated with anti-Npm mAb. The amounts of Npm and p19Arf in the immunoprecipitates were determined by western blot. IgG heavy chain (IgHC) was used as loading controls. (e) Regulation of ARF levels and stability by endogenous CD24. Scrambled or shRNA-silenced DU145 cells were arrested at G2/M phase overnight and then treated with CHX for 0, 2 and 4 h. The levels of CD24 (upper panel) and p14ARF were analysed by western blot. (f) Decay kinetics of p14ARF are shown as % of ARF protein at different times after CHX treatment. (g) ShRNA silencing of CD24 does not affect the levels of the ARF E3 ligase ULF. (h) Ectopic expression of CD24 reduces ARF levels. U2OS cells that ectopically expressed CD24 alone, ARF alone or both were analysed for their CD24 and ARF levels by western blot. (i) CD24 expression alleviates ARF tumour-suppressor activity. Data shown are growth kinetics of U2OS cells transfected with the given plasmids. Data shown are means and s.d. of cell numbers during the 6-day period. All data in this figure have been reproduced two or three times.
Mentions: To determine whether CD24 competes with ARF for interaction with NPM, we co-incubated recombinant ARF with either control glutathione S-transferase (GST) or NPM–GST in the presence of increasing amounts of CD24-Fc or control IgG-Fc. The proteins that associated with the GST-fusion proteins were pulled down using glutathione beads and identified by western blot using anti-NPM, anti-CD24 or anti-ARF. As shown in Fig. 7a, CD24-Fc, but not control IgG-Fc, blocked the ARF–NPM association in a dose-dependent manner. To determine whether CD24 inhibits the ARF–NPM association in cancer cells, we used a proximity ligation assay to assess the association between NPM and ARF in DU145 cells with or without CD24 silencing. A summary of three experiments is shown in Fig. 7b, whereas representative images from one experiment are shown in Fig. 7c. These data demonstrate that shRNA silencing of CD24 increased the NPM–ARF association in tumour cells. To test if CD24 also affects the ARF–NPM interaction in normal cells, we compared the amounts of Npm-associated Arf in WT and Cd24−/− thymocytes. As shown in Fig. 7d, a substantial increase in Npm-associated Arf was observed in Cd24−/− thymocytes. These results demonstrate that CD24 inhibited ARF association with NPM.

Bottom Line: CD24 competitively inhibits ARF binding to NPM, resulting in decreased ARF, increase MDM2 and decrease levels of p53 and the p53 target p21/CDKN1A.CD24 silencing prevents functional inactivation of p53 by both somatic mutation and viral oncogenes, including the SV40 large T antigen and human papilloma virus 16 E6-antigen.In support of the functional interaction between CD24 and p53, in silico analyses reveal that TP53 mutates at a higher rate among glioma and prostate cancer samples with higher CD24 mRNA levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

ABSTRACT
CD24 is overexpressed in nearly 70% human cancers, whereas TP53 is the most frequently mutated tumour-suppressor gene that functions in a context-dependent manner. Here we show that both targeted mutation and short hairpin RNA (shRNA) silencing of CD24 retard the growth, progression and metastasis of prostate cancer. CD24 competitively inhibits ARF binding to NPM, resulting in decreased ARF, increase MDM2 and decrease levels of p53 and the p53 target p21/CDKN1A. CD24 silencing prevents functional inactivation of p53 by both somatic mutation and viral oncogenes, including the SV40 large T antigen and human papilloma virus 16 E6-antigen. In support of the functional interaction between CD24 and p53, in silico analyses reveal that TP53 mutates at a higher rate among glioma and prostate cancer samples with higher CD24 mRNA levels. These data provide a general mechanism for functional inactivation of ARF and reveal an important cellular context for genetic and viral inactivation of TP53.

Show MeSH
Related in: MedlinePlus