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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 is required for viral oncogene-mediated inactivation of p53 function.(a) By increasing the overall levels of p53 protein, Cd24 silencing increases the proportion of SV40 T antigen-unbound p53. SV40 T antigen was tagged with Flag and introduced into the mouse prostate cancer cell line C3 with or without transduction of CD24 shRNA. The total cellular (left panel) or SV40 T antigen-associated or non-associated p53 (right panel) were determined by either western blot or anti-Flag immunoprecipitation followed by western blot. Note that CD24 was not co-precipitated. S: supernatant; P: pellet. (b) Cd24 silencing reveals p53 function in SV40 large T antigen (SV40 Tag)-transduced (top) and human papilloma viral protein E6 (HPV E6)-transduced C3 cells (bottom). SV40 Tag- or HPV E6-transduced C3 cells were transfected with WT or p53-binding site mutant p21 promoter linked with a luciferase reporter. The function of the endogenous Cd24 and Tp53 locus was confirmed by shRNA silencing of either genes. Data shown are means and s.d. of relative luciferase activity. (c) In the Tp53+/+ TRAMP model, targeted mutation of Cd24 increased the levels of p53 and p21. Data shown are photographs of immunohistochemical analysis of p21 and p53 levels from either Cd24+/+ or Cd24−/− prostate cancer samples. (d) Cd24 silencing increases the proportion of HPV E6-unbound p53, as in a, except that HPV E6 was used instead of SV40 TAg. All data in this figure have been reproduced two or three times. Co-IP, co-immunoprecipitation; Mut, mutant; WB, western blot.
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f9: CD24 is required for viral oncogene-mediated inactivation of p53 function.(a) By increasing the overall levels of p53 protein, Cd24 silencing increases the proportion of SV40 T antigen-unbound p53. SV40 T antigen was tagged with Flag and introduced into the mouse prostate cancer cell line C3 with or without transduction of CD24 shRNA. The total cellular (left panel) or SV40 T antigen-associated or non-associated p53 (right panel) were determined by either western blot or anti-Flag immunoprecipitation followed by western blot. Note that CD24 was not co-precipitated. S: supernatant; P: pellet. (b) Cd24 silencing reveals p53 function in SV40 large T antigen (SV40 Tag)-transduced (top) and human papilloma viral protein E6 (HPV E6)-transduced C3 cells (bottom). SV40 Tag- or HPV E6-transduced C3 cells were transfected with WT or p53-binding site mutant p21 promoter linked with a luciferase reporter. The function of the endogenous Cd24 and Tp53 locus was confirmed by shRNA silencing of either genes. Data shown are means and s.d. of relative luciferase activity. (c) In the Tp53+/+ TRAMP model, targeted mutation of Cd24 increased the levels of p53 and p21. Data shown are photographs of immunohistochemical analysis of p21 and p53 levels from either Cd24+/+ or Cd24−/− prostate cancer samples. (d) Cd24 silencing increases the proportion of HPV E6-unbound p53, as in a, except that HPV E6 was used instead of SV40 TAg. All data in this figure have been reproduced two or three times. Co-IP, co-immunoprecipitation; Mut, mutant; WB, western blot.

Mentions: p53 protein is inactivated by SV40 T antigen and this inactivation requires interaction between p53 and SV40 T antigen57. To determine whether Cd24 abrogates p53 function in SV40 T antigen-induced cancer cells, we compared the SV40 TAg-transduced mouse prostate cancer cell line C3 with or without endogenous Cd24 expression. As shown in Fig. 9a, shRNA silencing of Cd24 resulted in increased p53 levels, as expected. To determine whether the increased p53 was subjected to inactivation by SV40 T antigen, we assessed the relative amount of SV40 T antigen-associated versus non-associated p53 in Cd24-expressing and non-expressing C3 cells. As shown in Fig. 9a, right panels, Cd24 shRNA silencing resulted in a massive increase of SV40 T antigen-free p53, although a small increase in SV40 T antigen-associated p53 was also observed. As CD24 was not co-precipitated with SV40 T antigen, it is likely that the primary function of CD24 is to decrease p53 levels. Consistent with inactivation of p53 by large T antigen in CD24-expressing C3 cells, the p21 promoter was largely inactive in the scrambled shRNA-transduced C3 cells (Fig. 9b, top panel). Importantly, shRNA silencing of Cd24 caused a major increase in p21 promoter activity (Fig. 9b, top panel). This increase was fully attributable to p53 as it required a p53-binding site on the p21 promoter and was abrogated by shRNA silencing of TP53 (Fig. 9b, top panel). We compared the levels of p21 and p53 in prostate cancer from Cd24+/+, Cd24+/− and Cd24−/− TRAMP mice by immunohistochemistry (IHC). The tumour cells in this model have WT TP53 (ref. 58). As shown in Fig. 9c, Cd24+/− and Cd24−/− prostate cancers showed considerably higher levels of p53 and p21 than the WT prostate cancer.


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 is required for viral oncogene-mediated inactivation of p53 function.(a) By increasing the overall levels of p53 protein, Cd24 silencing increases the proportion of SV40 T antigen-unbound p53. SV40 T antigen was tagged with Flag and introduced into the mouse prostate cancer cell line C3 with or without transduction of CD24 shRNA. The total cellular (left panel) or SV40 T antigen-associated or non-associated p53 (right panel) were determined by either western blot or anti-Flag immunoprecipitation followed by western blot. Note that CD24 was not co-precipitated. S: supernatant; P: pellet. (b) Cd24 silencing reveals p53 function in SV40 large T antigen (SV40 Tag)-transduced (top) and human papilloma viral protein E6 (HPV E6)-transduced C3 cells (bottom). SV40 Tag- or HPV E6-transduced C3 cells were transfected with WT or p53-binding site mutant p21 promoter linked with a luciferase reporter. The function of the endogenous Cd24 and Tp53 locus was confirmed by shRNA silencing of either genes. Data shown are means and s.d. of relative luciferase activity. (c) In the Tp53+/+ TRAMP model, targeted mutation of Cd24 increased the levels of p53 and p21. Data shown are photographs of immunohistochemical analysis of p21 and p53 levels from either Cd24+/+ or Cd24−/− prostate cancer samples. (d) Cd24 silencing increases the proportion of HPV E6-unbound p53, as in a, except that HPV E6 was used instead of SV40 TAg. All data in this figure have been reproduced two or three times. Co-IP, co-immunoprecipitation; Mut, mutant; WB, western blot.
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f9: CD24 is required for viral oncogene-mediated inactivation of p53 function.(a) By increasing the overall levels of p53 protein, Cd24 silencing increases the proportion of SV40 T antigen-unbound p53. SV40 T antigen was tagged with Flag and introduced into the mouse prostate cancer cell line C3 with or without transduction of CD24 shRNA. The total cellular (left panel) or SV40 T antigen-associated or non-associated p53 (right panel) were determined by either western blot or anti-Flag immunoprecipitation followed by western blot. Note that CD24 was not co-precipitated. S: supernatant; P: pellet. (b) Cd24 silencing reveals p53 function in SV40 large T antigen (SV40 Tag)-transduced (top) and human papilloma viral protein E6 (HPV E6)-transduced C3 cells (bottom). SV40 Tag- or HPV E6-transduced C3 cells were transfected with WT or p53-binding site mutant p21 promoter linked with a luciferase reporter. The function of the endogenous Cd24 and Tp53 locus was confirmed by shRNA silencing of either genes. Data shown are means and s.d. of relative luciferase activity. (c) In the Tp53+/+ TRAMP model, targeted mutation of Cd24 increased the levels of p53 and p21. Data shown are photographs of immunohistochemical analysis of p21 and p53 levels from either Cd24+/+ or Cd24−/− prostate cancer samples. (d) Cd24 silencing increases the proportion of HPV E6-unbound p53, as in a, except that HPV E6 was used instead of SV40 TAg. All data in this figure have been reproduced two or three times. Co-IP, co-immunoprecipitation; Mut, mutant; WB, western blot.
Mentions: p53 protein is inactivated by SV40 T antigen and this inactivation requires interaction between p53 and SV40 T antigen57. To determine whether Cd24 abrogates p53 function in SV40 T antigen-induced cancer cells, we compared the SV40 TAg-transduced mouse prostate cancer cell line C3 with or without endogenous Cd24 expression. As shown in Fig. 9a, shRNA silencing of Cd24 resulted in increased p53 levels, as expected. To determine whether the increased p53 was subjected to inactivation by SV40 T antigen, we assessed the relative amount of SV40 T antigen-associated versus non-associated p53 in Cd24-expressing and non-expressing C3 cells. As shown in Fig. 9a, right panels, Cd24 shRNA silencing resulted in a massive increase of SV40 T antigen-free p53, although a small increase in SV40 T antigen-associated p53 was also observed. As CD24 was not co-precipitated with SV40 T antigen, it is likely that the primary function of CD24 is to decrease p53 levels. Consistent with inactivation of p53 by large T antigen in CD24-expressing C3 cells, the p21 promoter was largely inactive in the scrambled shRNA-transduced C3 cells (Fig. 9b, top panel). Importantly, shRNA silencing of Cd24 caused a major increase in p21 promoter activity (Fig. 9b, top panel). This increase was fully attributable to p53 as it required a p53-binding site on the p21 promoter and was abrogated by shRNA silencing of TP53 (Fig. 9b, top panel). We compared the levels of p21 and p53 in prostate cancer from Cd24+/+, Cd24+/− and Cd24−/− TRAMP mice by immunohistochemistry (IHC). The tumour cells in this model have WT TP53 (ref. 58). As shown in Fig. 9c, Cd24+/− and Cd24−/− prostate cancers showed considerably higher levels of p53 and p21 than the WT prostate cancer.

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