Limits...
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.

Show MeSH

Related in: MedlinePlus

CD24 silencing restores transcriptional activity of p53.(a) Effect of CD24 silencing on expression of NPM, ARF, MDM2 and p53. Scrambled (Scr) or CD24-shRNA-silenced DU145 cells were analysed for CD24, NPM, ARF, MDM2 and p53 by western blot. (b) In ARF-deficient U2OS cells, ectopic expression of CD24 had no effect on the levels of MDM2 and p53. Actin levels were used as loading control. (c) Endogenous mutant p53 is responsible for the upregulation of p21 in CD24-silenced DU145 cells. (d) In DU145 cells, silencing CD24 restores transcriptional induction of the p21 promoter. DU145 cells transduced with Scr or shRNA for eitherTP53 or CD24 were transfected with a luciferase reporter containing the p21 promoter. The WT reporter has an intact p53-binding site, whereas the mutant promoter has the site deleted. (e,f) CD24 silencing restores transcriptional activity of multiple TP53 mutants. DU145 cells in which the endogenous TP53 was silenced by shRNA were transduced with Scr or CD24 shRNA lentivirus. The stable cell lines were then transfected with p53 mutants in conjunction with WT or mutant p21 promoter reporter constructs. p53 levels (e) were determined by western blot, while luciferase activity (f) was measured at 72 h after transfection. The mutants used were p53R273H, p53V143A, p53R280T and p53R175H. Numbers on the x axis indicate the amino-acid positions of the p53 mutations. (g) As in e and f, except that the endogenous p21 and MDM2 mRNA were measured by quantitative PCR. Data shown are means and s.d. of triplicate samples. All data have been reproduced two or three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4300525&req=5

f8: CD24 silencing restores transcriptional activity of p53.(a) Effect of CD24 silencing on expression of NPM, ARF, MDM2 and p53. Scrambled (Scr) or CD24-shRNA-silenced DU145 cells were analysed for CD24, NPM, ARF, MDM2 and p53 by western blot. (b) In ARF-deficient U2OS cells, ectopic expression of CD24 had no effect on the levels of MDM2 and p53. Actin levels were used as loading control. (c) Endogenous mutant p53 is responsible for the upregulation of p21 in CD24-silenced DU145 cells. (d) In DU145 cells, silencing CD24 restores transcriptional induction of the p21 promoter. DU145 cells transduced with Scr or shRNA for eitherTP53 or CD24 were transfected with a luciferase reporter containing the p21 promoter. The WT reporter has an intact p53-binding site, whereas the mutant promoter has the site deleted. (e,f) CD24 silencing restores transcriptional activity of multiple TP53 mutants. DU145 cells in which the endogenous TP53 was silenced by shRNA were transduced with Scr or CD24 shRNA lentivirus. The stable cell lines were then transfected with p53 mutants in conjunction with WT or mutant p21 promoter reporter constructs. p53 levels (e) were determined by western blot, while luciferase activity (f) was measured at 72 h after transfection. The mutants used were p53R273H, p53V143A, p53R280T and p53R175H. Numbers on the x axis indicate the amino-acid positions of the p53 mutations. (g) As in e and f, except that the endogenous p21 and MDM2 mRNA were measured by quantitative PCR. Data shown are means and s.d. of triplicate samples. All data have been reproduced two or three times.

Mentions: Given the effect of ARF on MDM2 and its substrate p53, we evaluated if CD24 affected the expression of MDM2 and p53 in an ARF-dependent manner. As shown in Fig. 8a, in ARF-expressing DU145 cells, CD24 silencing increased the levels of ARF and p53 and reduced that of MDM2. Elevated ARF levels contributed to CD24-mediated reduction of MDM2 and p53 as CD24 had no effect on the levels of MDM2 and p53 in ARF-deficient U2OS cells (Fig. 8b). Furthermore, expression of p21 following shRNA silencing of CD24 was due to elevated p53 levels as shRNA silencing of p53 in DU145 cells substantially reduced p21 levels (Fig. 8c).


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 silencing restores transcriptional activity of p53.(a) Effect of CD24 silencing on expression of NPM, ARF, MDM2 and p53. Scrambled (Scr) or CD24-shRNA-silenced DU145 cells were analysed for CD24, NPM, ARF, MDM2 and p53 by western blot. (b) In ARF-deficient U2OS cells, ectopic expression of CD24 had no effect on the levels of MDM2 and p53. Actin levels were used as loading control. (c) Endogenous mutant p53 is responsible for the upregulation of p21 in CD24-silenced DU145 cells. (d) In DU145 cells, silencing CD24 restores transcriptional induction of the p21 promoter. DU145 cells transduced with Scr or shRNA for eitherTP53 or CD24 were transfected with a luciferase reporter containing the p21 promoter. The WT reporter has an intact p53-binding site, whereas the mutant promoter has the site deleted. (e,f) CD24 silencing restores transcriptional activity of multiple TP53 mutants. DU145 cells in which the endogenous TP53 was silenced by shRNA were transduced with Scr or CD24 shRNA lentivirus. The stable cell lines were then transfected with p53 mutants in conjunction with WT or mutant p21 promoter reporter constructs. p53 levels (e) were determined by western blot, while luciferase activity (f) was measured at 72 h after transfection. The mutants used were p53R273H, p53V143A, p53R280T and p53R175H. Numbers on the x axis indicate the amino-acid positions of the p53 mutations. (g) As in e and f, except that the endogenous p21 and MDM2 mRNA were measured by quantitative PCR. Data shown are means and s.d. of triplicate samples. All data have been reproduced two or three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300525&req=5

f8: CD24 silencing restores transcriptional activity of p53.(a) Effect of CD24 silencing on expression of NPM, ARF, MDM2 and p53. Scrambled (Scr) or CD24-shRNA-silenced DU145 cells were analysed for CD24, NPM, ARF, MDM2 and p53 by western blot. (b) In ARF-deficient U2OS cells, ectopic expression of CD24 had no effect on the levels of MDM2 and p53. Actin levels were used as loading control. (c) Endogenous mutant p53 is responsible for the upregulation of p21 in CD24-silenced DU145 cells. (d) In DU145 cells, silencing CD24 restores transcriptional induction of the p21 promoter. DU145 cells transduced with Scr or shRNA for eitherTP53 or CD24 were transfected with a luciferase reporter containing the p21 promoter. The WT reporter has an intact p53-binding site, whereas the mutant promoter has the site deleted. (e,f) CD24 silencing restores transcriptional activity of multiple TP53 mutants. DU145 cells in which the endogenous TP53 was silenced by shRNA were transduced with Scr or CD24 shRNA lentivirus. The stable cell lines were then transfected with p53 mutants in conjunction with WT or mutant p21 promoter reporter constructs. p53 levels (e) were determined by western blot, while luciferase activity (f) was measured at 72 h after transfection. The mutants used were p53R273H, p53V143A, p53R280T and p53R175H. Numbers on the x axis indicate the amino-acid positions of the p53 mutations. (g) As in e and f, except that the endogenous p21 and MDM2 mRNA were measured by quantitative PCR. Data shown are means and s.d. of triplicate samples. All data have been reproduced two or three times.
Mentions: Given the effect of ARF on MDM2 and its substrate p53, we evaluated if CD24 affected the expression of MDM2 and p53 in an ARF-dependent manner. As shown in Fig. 8a, in ARF-expressing DU145 cells, CD24 silencing increased the levels of ARF and p53 and reduced that of MDM2. Elevated ARF levels contributed to CD24-mediated reduction of MDM2 and p53 as CD24 had no effect on the levels of MDM2 and p53 in ARF-deficient U2OS cells (Fig. 8b). Furthermore, expression of p21 following shRNA silencing of CD24 was due to elevated p53 levels as shRNA silencing of p53 in DU145 cells substantially reduced p21 levels (Fig. 8c).

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