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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 reduces levels of p53 and p21 and promotes tumour cell proliferation.(a) Heat-map depiction of alterations in gene expression in DU145 cells following CD24 gene silencing. RNA isolated from five independent cultures of Scr, three cultures of Sh1 and three cultures of Sh2 were compared using Affymeatrix Human U133 Plus 2.0 microarrays. Only those genes that met the statistical threshold of P<0.001 and mean alterations that reached the cutoff of >150% or<66% of Scr levels are displayed. The genes are listed in Supplementary Data set and the pathway analysis summary of affected genes is provided in Supplementary Table 1. (b) CD24 silencing increases p21/CDKN1A mRNA. Error bars, s.d. n=3. The data have been reproduced three times. (c) CD24 silencing increased levels of p53 and p21 proteins, but not p27 protein. Representative images of western blots are shown and the data have been reproduced three times. (d) CD24 silencing reduces cell cycle re-entry after starvation. Scr, Sh1 and Sh2 clones were starved for 48 h in serum-free medium and then shifted into serum containing medium. The cells were harvested and analysed for cell cycle progression based on DNA content. These data have been reproduced three times. (e) Ectopic expression of CD24 in LNCaP cell line decreases levels of p53 and p21, but not p27. Representative images of western blots are shown and the data have been reproduced two times. (f) CD24 transduction increases proliferation of LNCaP cells. Data shown are triplicate means and s.d. of triplicates and have been reproduced twice. LSD, least significant difference.
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f4: CD24 reduces levels of p53 and p21 and promotes tumour cell proliferation.(a) Heat-map depiction of alterations in gene expression in DU145 cells following CD24 gene silencing. RNA isolated from five independent cultures of Scr, three cultures of Sh1 and three cultures of Sh2 were compared using Affymeatrix Human U133 Plus 2.0 microarrays. Only those genes that met the statistical threshold of P<0.001 and mean alterations that reached the cutoff of >150% or<66% of Scr levels are displayed. The genes are listed in Supplementary Data set and the pathway analysis summary of affected genes is provided in Supplementary Table 1. (b) CD24 silencing increases p21/CDKN1A mRNA. Error bars, s.d. n=3. The data have been reproduced three times. (c) CD24 silencing increased levels of p53 and p21 proteins, but not p27 protein. Representative images of western blots are shown and the data have been reproduced three times. (d) CD24 silencing reduces cell cycle re-entry after starvation. Scr, Sh1 and Sh2 clones were starved for 48 h in serum-free medium and then shifted into serum containing medium. The cells were harvested and analysed for cell cycle progression based on DNA content. These data have been reproduced three times. (e) Ectopic expression of CD24 in LNCaP cell line decreases levels of p53 and p21, but not p27. Representative images of western blots are shown and the data have been reproduced two times. (f) CD24 transduction increases proliferation of LNCaP cells. Data shown are triplicate means and s.d. of triplicates and have been reproduced twice. LSD, least significant difference.

Mentions: To understand the molecular mechanism by which CD24 silencing causes growth suppression, we compared the gene expression profiles of scrambled, non-silenced control (Scr) versus CD24 shRNA-silenced DU145 cells by gene array analysis. Using P<0.001 and less than 66% or greater than 150% of control levels as a cutoff, we found that as a result of CD24 gene silencing, 408 genes were downregulated, whereas 618 genes were upregulated (Fig. 4a and Supplementary Data set 1). The top ten pathways affected are listed in Supplementary Table 1. Among them, the most significantly affected pathway was cell cycle regulation (P=2 × 10−5). For example, the CDKN1A mRNA, which encodes p21, was significantly elevated (Fig. 4b). This increase in p21 corresponded with elevated levels of p53 protein (Fig. 4c). Given the function of p21/CDKN1A in G1 arrest, we evaluated the role of CD24 in cell entry into S phase and beyond. We starved DU145 cells under serum-free conditions for 48 h, which arrested the cells at the G0-G1 phase. After 24 h of serum stimulation, more than 70% of the scrambled shRNA-transduced DU145 cells were at either S or G2-M phases (Fig. 4d). In contrast, only 39–49% of the CD24 shRNA-transduced cells entered S or G2-M phases (Fig. 4d). Therefore, CD24 contributes to cell re-entry into cell cycle following starvation. To determine whether CD24 overexpression is sufficient to promote tumour cell growth, we ectopically expressed CD24 in the LNCaP cell line that does not express CD24. As shown in Fig. 4e, CD24 expression reduced p53 and p21 without marked effect on p27. Consistent with reduced expression of tumour-suppressor genes, CD24 increased LNCaP cell proliferation (Fig. 4f).


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 reduces levels of p53 and p21 and promotes tumour cell proliferation.(a) Heat-map depiction of alterations in gene expression in DU145 cells following CD24 gene silencing. RNA isolated from five independent cultures of Scr, three cultures of Sh1 and three cultures of Sh2 were compared using Affymeatrix Human U133 Plus 2.0 microarrays. Only those genes that met the statistical threshold of P<0.001 and mean alterations that reached the cutoff of >150% or<66% of Scr levels are displayed. The genes are listed in Supplementary Data set and the pathway analysis summary of affected genes is provided in Supplementary Table 1. (b) CD24 silencing increases p21/CDKN1A mRNA. Error bars, s.d. n=3. The data have been reproduced three times. (c) CD24 silencing increased levels of p53 and p21 proteins, but not p27 protein. Representative images of western blots are shown and the data have been reproduced three times. (d) CD24 silencing reduces cell cycle re-entry after starvation. Scr, Sh1 and Sh2 clones were starved for 48 h in serum-free medium and then shifted into serum containing medium. The cells were harvested and analysed for cell cycle progression based on DNA content. These data have been reproduced three times. (e) Ectopic expression of CD24 in LNCaP cell line decreases levels of p53 and p21, but not p27. Representative images of western blots are shown and the data have been reproduced two times. (f) CD24 transduction increases proliferation of LNCaP cells. Data shown are triplicate means and s.d. of triplicates and have been reproduced twice. LSD, least significant difference.
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f4: CD24 reduces levels of p53 and p21 and promotes tumour cell proliferation.(a) Heat-map depiction of alterations in gene expression in DU145 cells following CD24 gene silencing. RNA isolated from five independent cultures of Scr, three cultures of Sh1 and three cultures of Sh2 were compared using Affymeatrix Human U133 Plus 2.0 microarrays. Only those genes that met the statistical threshold of P<0.001 and mean alterations that reached the cutoff of >150% or<66% of Scr levels are displayed. The genes are listed in Supplementary Data set and the pathway analysis summary of affected genes is provided in Supplementary Table 1. (b) CD24 silencing increases p21/CDKN1A mRNA. Error bars, s.d. n=3. The data have been reproduced three times. (c) CD24 silencing increased levels of p53 and p21 proteins, but not p27 protein. Representative images of western blots are shown and the data have been reproduced three times. (d) CD24 silencing reduces cell cycle re-entry after starvation. Scr, Sh1 and Sh2 clones were starved for 48 h in serum-free medium and then shifted into serum containing medium. The cells were harvested and analysed for cell cycle progression based on DNA content. These data have been reproduced three times. (e) Ectopic expression of CD24 in LNCaP cell line decreases levels of p53 and p21, but not p27. Representative images of western blots are shown and the data have been reproduced two times. (f) CD24 transduction increases proliferation of LNCaP cells. Data shown are triplicate means and s.d. of triplicates and have been reproduced twice. LSD, least significant difference.
Mentions: To understand the molecular mechanism by which CD24 silencing causes growth suppression, we compared the gene expression profiles of scrambled, non-silenced control (Scr) versus CD24 shRNA-silenced DU145 cells by gene array analysis. Using P<0.001 and less than 66% or greater than 150% of control levels as a cutoff, we found that as a result of CD24 gene silencing, 408 genes were downregulated, whereas 618 genes were upregulated (Fig. 4a and Supplementary Data set 1). The top ten pathways affected are listed in Supplementary Table 1. Among them, the most significantly affected pathway was cell cycle regulation (P=2 × 10−5). For example, the CDKN1A mRNA, which encodes p21, was significantly elevated (Fig. 4b). This increase in p21 corresponded with elevated levels of p53 protein (Fig. 4c). Given the function of p21/CDKN1A in G1 arrest, we evaluated the role of CD24 in cell entry into S phase and beyond. We starved DU145 cells under serum-free conditions for 48 h, which arrested the cells at the G0-G1 phase. After 24 h of serum stimulation, more than 70% of the scrambled shRNA-transduced DU145 cells were at either S or G2-M phases (Fig. 4d). In contrast, only 39–49% of the CD24 shRNA-transduced cells entered S or G2-M phases (Fig. 4d). Therefore, CD24 contributes to cell re-entry into cell cycle following starvation. To determine whether CD24 overexpression is sufficient to promote tumour cell growth, we ectopically expressed CD24 in the LNCaP cell line that does not express CD24. As shown in Fig. 4e, CD24 expression reduced p53 and p21 without marked effect on p27. Consistent with reduced expression of tumour-suppressor genes, CD24 increased LNCaP cell proliferation (Fig. 4f).

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