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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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Intracellular CD24 and proliferation of prostate cancer cells.(a) Measurement of cell surface and total cellular CD24 by flow cytometry. The prostate cancer cell lines PC3, DU145 and LNCaP were stained with anti-CD24 with or without permeabilization to measure either cell surface (top) or total cellular CD24 (bottom). (b) Analysis of CD24 in the cytoplasmic and plasma membrane fraction (Cp+Cm), nucleoplasm (Np) and the insoluble fraction that includes chromatin by western blot. The efficiency of the fractionation was monitored by the presence of β-actin (Cp+Cm+Np), Lamin (Np) and H1.5 (chromatin). (c) Confocal microscopy reveals dynamic upregulation and localization of CD24 during mitosis. Note alterations of CD24 distribution and signal intensity at different phases during mitosis. See Supplementary Fig. 6 for single-colour images. (d) Upregulation of CD24 during mitosis as revealed by western blot of cellular lysates prepared from nocodazale-synchronized DU145 cells. (e) Diagram of CD24-GFP and GFP-CD24 constructs. The N-terminus-tagged CD24 was produced by inserting the GFP-coding sequence after the signal peptide, whereas the C-terminus-tagged CD24 was generated by inserting the GFP-coding sequence before the stop codon of CD24. (f) Tagging the C-terminus with GFP prevents cell surface localization of CD24. The images show the distinct distribution of green fluorescence in cells transfected with either CD24-GFP (C) or GFP-CD24 (N). (g) Verification of cell surface versus intracellular CD24 expression by cell surface staining. CD24-GFP (C) was not present on the surface of DU145 cells as it was not detected by an anti-CD24 mAb added to non-permeabilized cells. CD24-GFP (N) was used as positive control for cell surface CD24. (h) Verification of fusion protein expression by western blot using anti-CD24 and anti-GFP antibodies. (i) Intracellular CD24 encoded by CD24-GFP (C) is at least as potent in promoting proliferation of DU145 cells ad GFP-CD24 (N). CD24-silenced DU145 cells were transfected with CD24-GFP (C) or GFP-CD24 (N) plasmids. After drug selection, an equal number of drug-resistant transfectants were plated onto 10-cm plates. The colonies were stained with crystal violet. All images have been reproduced three times.
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f5: Intracellular CD24 and proliferation of prostate cancer cells.(a) Measurement of cell surface and total cellular CD24 by flow cytometry. The prostate cancer cell lines PC3, DU145 and LNCaP were stained with anti-CD24 with or without permeabilization to measure either cell surface (top) or total cellular CD24 (bottom). (b) Analysis of CD24 in the cytoplasmic and plasma membrane fraction (Cp+Cm), nucleoplasm (Np) and the insoluble fraction that includes chromatin by western blot. The efficiency of the fractionation was monitored by the presence of β-actin (Cp+Cm+Np), Lamin (Np) and H1.5 (chromatin). (c) Confocal microscopy reveals dynamic upregulation and localization of CD24 during mitosis. Note alterations of CD24 distribution and signal intensity at different phases during mitosis. See Supplementary Fig. 6 for single-colour images. (d) Upregulation of CD24 during mitosis as revealed by western blot of cellular lysates prepared from nocodazale-synchronized DU145 cells. (e) Diagram of CD24-GFP and GFP-CD24 constructs. The N-terminus-tagged CD24 was produced by inserting the GFP-coding sequence after the signal peptide, whereas the C-terminus-tagged CD24 was generated by inserting the GFP-coding sequence before the stop codon of CD24. (f) Tagging the C-terminus with GFP prevents cell surface localization of CD24. The images show the distinct distribution of green fluorescence in cells transfected with either CD24-GFP (C) or GFP-CD24 (N). (g) Verification of cell surface versus intracellular CD24 expression by cell surface staining. CD24-GFP (C) was not present on the surface of DU145 cells as it was not detected by an anti-CD24 mAb added to non-permeabilized cells. CD24-GFP (N) was used as positive control for cell surface CD24. (h) Verification of fusion protein expression by western blot using anti-CD24 and anti-GFP antibodies. (i) Intracellular CD24 encoded by CD24-GFP (C) is at least as potent in promoting proliferation of DU145 cells ad GFP-CD24 (N). CD24-silenced DU145 cells were transfected with CD24-GFP (C) or GFP-CD24 (N) plasmids. After drug selection, an equal number of drug-resistant transfectants were plated onto 10-cm plates. The colonies were stained with crystal violet. All images have been reproduced three times.

Mentions: Interestingly, cell surface staining by flow cytometry revealed that only DU145 cells expressed cell surface CD24 (Fig. 5a). To determine whether CD24 is expressed intracellularly, we fixed the cells with 4% paraformaldehyde and permeabilized them with detergent to allow antibody access to intracellular CD24. As shown in Fig. 5a, permeabilization increased the levels of CD24 detected in a cell by an average of fivefold in DU145 cells. Thus, the majority of CD24 must reside intracellularly. To determine whether CD24 resides in the cytoplasm or in the nucleus, we compared the amount of CD24 in 0.1% NP-40 soluble (cytoplasmic and plasma membrane) and insoluble (putatively nuclear) fractions. The 0.1% NP-40 insoluble fraction was incubated in 3 mM EDTA and 0.2 mM EGTA to isolate the nucleoplasm. CD24 was detected in both cytoplasmic and nuclear fractions (Fig. 5b). As membrane-bound GPI-anchored molecules may be insoluble because of its association with caveolae47, we used confocal microscopy to confirm the intracellular distribution of CD24 relative to that of microtubules and DNA. We observed increased CD24 levels and alterations in CD24 distribution during mitosis. As shown in Fig. 5c and Supplementary Fig. 6, compared with interphase, increased levels of CD24 were found in prophase and metaphase. Also during metaphase, CD24 seemed to distribute itself more evenly with less staining of CD24 in the membrane and more staining in the cytoplasm. This shift was reversed as the cells entered telophase. To confirm the increase in CD24 levels, we arrested DU145 cells at G2 using nocodazole and assessed CD24 protein levels at different time points following nocodazole removal. Concomitant with an increase in Cyclin B1, a marked increase of CD24 was observed as early as 30 min after removal of nocodazole. The increase was transient and largely disappeared at 2 h after nocodazole removal (Fig. 5d).


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)

Intracellular CD24 and proliferation of prostate cancer cells.(a) Measurement of cell surface and total cellular CD24 by flow cytometry. The prostate cancer cell lines PC3, DU145 and LNCaP were stained with anti-CD24 with or without permeabilization to measure either cell surface (top) or total cellular CD24 (bottom). (b) Analysis of CD24 in the cytoplasmic and plasma membrane fraction (Cp+Cm), nucleoplasm (Np) and the insoluble fraction that includes chromatin by western blot. The efficiency of the fractionation was monitored by the presence of β-actin (Cp+Cm+Np), Lamin (Np) and H1.5 (chromatin). (c) Confocal microscopy reveals dynamic upregulation and localization of CD24 during mitosis. Note alterations of CD24 distribution and signal intensity at different phases during mitosis. See Supplementary Fig. 6 for single-colour images. (d) Upregulation of CD24 during mitosis as revealed by western blot of cellular lysates prepared from nocodazale-synchronized DU145 cells. (e) Diagram of CD24-GFP and GFP-CD24 constructs. The N-terminus-tagged CD24 was produced by inserting the GFP-coding sequence after the signal peptide, whereas the C-terminus-tagged CD24 was generated by inserting the GFP-coding sequence before the stop codon of CD24. (f) Tagging the C-terminus with GFP prevents cell surface localization of CD24. The images show the distinct distribution of green fluorescence in cells transfected with either CD24-GFP (C) or GFP-CD24 (N). (g) Verification of cell surface versus intracellular CD24 expression by cell surface staining. CD24-GFP (C) was not present on the surface of DU145 cells as it was not detected by an anti-CD24 mAb added to non-permeabilized cells. CD24-GFP (N) was used as positive control for cell surface CD24. (h) Verification of fusion protein expression by western blot using anti-CD24 and anti-GFP antibodies. (i) Intracellular CD24 encoded by CD24-GFP (C) is at least as potent in promoting proliferation of DU145 cells ad GFP-CD24 (N). CD24-silenced DU145 cells were transfected with CD24-GFP (C) or GFP-CD24 (N) plasmids. After drug selection, an equal number of drug-resistant transfectants were plated onto 10-cm plates. The colonies were stained with crystal violet. All images have been reproduced three times.
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f5: Intracellular CD24 and proliferation of prostate cancer cells.(a) Measurement of cell surface and total cellular CD24 by flow cytometry. The prostate cancer cell lines PC3, DU145 and LNCaP were stained with anti-CD24 with or without permeabilization to measure either cell surface (top) or total cellular CD24 (bottom). (b) Analysis of CD24 in the cytoplasmic and plasma membrane fraction (Cp+Cm), nucleoplasm (Np) and the insoluble fraction that includes chromatin by western blot. The efficiency of the fractionation was monitored by the presence of β-actin (Cp+Cm+Np), Lamin (Np) and H1.5 (chromatin). (c) Confocal microscopy reveals dynamic upregulation and localization of CD24 during mitosis. Note alterations of CD24 distribution and signal intensity at different phases during mitosis. See Supplementary Fig. 6 for single-colour images. (d) Upregulation of CD24 during mitosis as revealed by western blot of cellular lysates prepared from nocodazale-synchronized DU145 cells. (e) Diagram of CD24-GFP and GFP-CD24 constructs. The N-terminus-tagged CD24 was produced by inserting the GFP-coding sequence after the signal peptide, whereas the C-terminus-tagged CD24 was generated by inserting the GFP-coding sequence before the stop codon of CD24. (f) Tagging the C-terminus with GFP prevents cell surface localization of CD24. The images show the distinct distribution of green fluorescence in cells transfected with either CD24-GFP (C) or GFP-CD24 (N). (g) Verification of cell surface versus intracellular CD24 expression by cell surface staining. CD24-GFP (C) was not present on the surface of DU145 cells as it was not detected by an anti-CD24 mAb added to non-permeabilized cells. CD24-GFP (N) was used as positive control for cell surface CD24. (h) Verification of fusion protein expression by western blot using anti-CD24 and anti-GFP antibodies. (i) Intracellular CD24 encoded by CD24-GFP (C) is at least as potent in promoting proliferation of DU145 cells ad GFP-CD24 (N). CD24-silenced DU145 cells were transfected with CD24-GFP (C) or GFP-CD24 (N) plasmids. After drug selection, an equal number of drug-resistant transfectants were plated onto 10-cm plates. The colonies were stained with crystal violet. All images have been reproduced three times.
Mentions: Interestingly, cell surface staining by flow cytometry revealed that only DU145 cells expressed cell surface CD24 (Fig. 5a). To determine whether CD24 is expressed intracellularly, we fixed the cells with 4% paraformaldehyde and permeabilized them with detergent to allow antibody access to intracellular CD24. As shown in Fig. 5a, permeabilization increased the levels of CD24 detected in a cell by an average of fivefold in DU145 cells. Thus, the majority of CD24 must reside intracellularly. To determine whether CD24 resides in the cytoplasm or in the nucleus, we compared the amount of CD24 in 0.1% NP-40 soluble (cytoplasmic and plasma membrane) and insoluble (putatively nuclear) fractions. The 0.1% NP-40 insoluble fraction was incubated in 3 mM EDTA and 0.2 mM EGTA to isolate the nucleoplasm. CD24 was detected in both cytoplasmic and nuclear fractions (Fig. 5b). As membrane-bound GPI-anchored molecules may be insoluble because of its association with caveolae47, we used confocal microscopy to confirm the intracellular distribution of CD24 relative to that of microtubules and DNA. We observed increased CD24 levels and alterations in CD24 distribution during mitosis. As shown in Fig. 5c and Supplementary Fig. 6, compared with interphase, increased levels of CD24 were found in prophase and metaphase. Also during metaphase, CD24 seemed to distribute itself more evenly with less staining of CD24 in the membrane and more staining in the cytoplasm. This shift was reversed as the cells entered telophase. To confirm the increase in CD24 levels, we arrested DU145 cells at G2 using nocodazole and assessed CD24 protein levels at different time points following nocodazole removal. Concomitant with an increase in Cyclin B1, a marked increase of CD24 was observed as early as 30 min after removal of nocodazole. The increase was transient and largely disappeared at 2 h after nocodazole removal (Fig. 5d).

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