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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 interacts with NPM during mitosis.(a,b) Dynamic association between CD24 and NPM in DU145 cells. (a) Merged images displaying DNA (stained with DAPI, blue), NPM (green) and CD24 (red). Cells at prophase, metaphase and anaphase were identified by their chromatin distribution. The representative image was obtained from a Meta laser-scanning confocal microscope. (b) Overlay between CD24 (red) and NPM (green) signals of cells at the three indicated phases during mitosis. The intensity (y axis) of signals was simultaneously obtained from the laser-scanning cross-section with the major diameter and overlap of the red and the green signals was analysed using LSM image browser. (c) Reciprocal co-precipitation between CD24 and NPM in DU145 cells. The cells lysates were prepared after 2 h of release from nocodazale arrest. The cell lysates were precipitated with antibodies specific for either NPM or CD24 or IgG control. The precipitates were probed by western blot with indicated antibodies. (d) Localizing the CD24-binding domain in NPM to the regions of AA196-294 based on co-IP of Myc-tagged NPM fragments and HA-tagged CD24 in HA-CD24 and NPM-Myc co-transfected HEK293 cells. The association was confirmed by western blot with anti-HA and anti-Myc mAbs. All images have been reproduced three times. DAPI, 4',6-diamidino-2-phenylindole. Co-IP, co-immunoprecipitation; IgLC, immunoglobulin light chain; IP, immunoprecipitation; IB, immunoblot.
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f6: CD24 interacts with NPM during mitosis.(a,b) Dynamic association between CD24 and NPM in DU145 cells. (a) Merged images displaying DNA (stained with DAPI, blue), NPM (green) and CD24 (red). Cells at prophase, metaphase and anaphase were identified by their chromatin distribution. The representative image was obtained from a Meta laser-scanning confocal microscope. (b) Overlay between CD24 (red) and NPM (green) signals of cells at the three indicated phases during mitosis. The intensity (y axis) of signals was simultaneously obtained from the laser-scanning cross-section with the major diameter and overlap of the red and the green signals was analysed using LSM image browser. (c) Reciprocal co-precipitation between CD24 and NPM in DU145 cells. The cells lysates were prepared after 2 h of release from nocodazale arrest. The cell lysates were precipitated with antibodies specific for either NPM or CD24 or IgG control. The precipitates were probed by western blot with indicated antibodies. (d) Localizing the CD24-binding domain in NPM to the regions of AA196-294 based on co-IP of Myc-tagged NPM fragments and HA-tagged CD24 in HA-CD24 and NPM-Myc co-transfected HEK293 cells. The association was confirmed by western blot with anti-HA and anti-Myc mAbs. All images have been reproduced three times. DAPI, 4',6-diamidino-2-phenylindole. Co-IP, co-immunoprecipitation; IgLC, immunoglobulin light chain; IP, immunoprecipitation; IB, immunoblot.

Mentions: In order to understand how CD24 encourages cancer cell growth, we identified CD24-associated proteins by mass spectrometric analysis of trypsin-digested anti-CD24 immunoprecipitates from WT but not CD24-deficient spleen cells48. We identified 12 unique NPM peptides in immunoprecipitates from Cd24+/+ spleen cells and none from Cd24−/− spleen cells (Supplementary Table 2). These data suggest that CD24 may interact with NPM. NPM is a nucleolar protein and forms a punctate structure in the nucleus that dispersed during mitosis. Interestingly, the CD24–NPM association occurred transiently during mitosis. CD24 was more broadly distributed than NPM at the interphase (Fig. 6a), although some overlap between the two signals can also be observed (Fig. 6b). However, at metaphase and anaphase, CD24 and NPM were largely co-localized (Fig. 6a,b; see also Supplementary Fig. 8 for single-colour images). To confirm this observation in human tumour cells, we immunoprecipitated CD24 from DU145 cells and probed them with an anti-NPM monoclonal antibody. As shown in Fig. 6c, anti-CD24 co-precipitated NPM. Conversely, anti-NPM co-precipitated CD24. To map the region in NPM that interacts with CD24, we used a series of Myc-tagged deletion mutants of NPM and HA-tagged CD24. As shown in Fig. 6d, full-length (1–294) and all but one (AA1-177) truncation mutant co-precipitated CD24. As a small NPM C-terminal only fragment encompassing AA196-294 co-precipitated CD24, CD24 interacts with the C-terminus of NPM. However, as the AA196-294 fragment showed reduced association compared with the AA117-294 fragment, a contribution of the NPM core region to the interaction cannot be ruled out.


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 interacts with NPM during mitosis.(a,b) Dynamic association between CD24 and NPM in DU145 cells. (a) Merged images displaying DNA (stained with DAPI, blue), NPM (green) and CD24 (red). Cells at prophase, metaphase and anaphase were identified by their chromatin distribution. The representative image was obtained from a Meta laser-scanning confocal microscope. (b) Overlay between CD24 (red) and NPM (green) signals of cells at the three indicated phases during mitosis. The intensity (y axis) of signals was simultaneously obtained from the laser-scanning cross-section with the major diameter and overlap of the red and the green signals was analysed using LSM image browser. (c) Reciprocal co-precipitation between CD24 and NPM in DU145 cells. The cells lysates were prepared after 2 h of release from nocodazale arrest. The cell lysates were precipitated with antibodies specific for either NPM or CD24 or IgG control. The precipitates were probed by western blot with indicated antibodies. (d) Localizing the CD24-binding domain in NPM to the regions of AA196-294 based on co-IP of Myc-tagged NPM fragments and HA-tagged CD24 in HA-CD24 and NPM-Myc co-transfected HEK293 cells. The association was confirmed by western blot with anti-HA and anti-Myc mAbs. All images have been reproduced three times. DAPI, 4',6-diamidino-2-phenylindole. Co-IP, co-immunoprecipitation; IgLC, immunoglobulin light chain; IP, immunoprecipitation; IB, immunoblot.
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Show All Figures
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f6: CD24 interacts with NPM during mitosis.(a,b) Dynamic association between CD24 and NPM in DU145 cells. (a) Merged images displaying DNA (stained with DAPI, blue), NPM (green) and CD24 (red). Cells at prophase, metaphase and anaphase were identified by their chromatin distribution. The representative image was obtained from a Meta laser-scanning confocal microscope. (b) Overlay between CD24 (red) and NPM (green) signals of cells at the three indicated phases during mitosis. The intensity (y axis) of signals was simultaneously obtained from the laser-scanning cross-section with the major diameter and overlap of the red and the green signals was analysed using LSM image browser. (c) Reciprocal co-precipitation between CD24 and NPM in DU145 cells. The cells lysates were prepared after 2 h of release from nocodazale arrest. The cell lysates were precipitated with antibodies specific for either NPM or CD24 or IgG control. The precipitates were probed by western blot with indicated antibodies. (d) Localizing the CD24-binding domain in NPM to the regions of AA196-294 based on co-IP of Myc-tagged NPM fragments and HA-tagged CD24 in HA-CD24 and NPM-Myc co-transfected HEK293 cells. The association was confirmed by western blot with anti-HA and anti-Myc mAbs. All images have been reproduced three times. DAPI, 4',6-diamidino-2-phenylindole. Co-IP, co-immunoprecipitation; IgLC, immunoglobulin light chain; IP, immunoprecipitation; IB, immunoblot.
Mentions: In order to understand how CD24 encourages cancer cell growth, we identified CD24-associated proteins by mass spectrometric analysis of trypsin-digested anti-CD24 immunoprecipitates from WT but not CD24-deficient spleen cells48. We identified 12 unique NPM peptides in immunoprecipitates from Cd24+/+ spleen cells and none from Cd24−/− spleen cells (Supplementary Table 2). These data suggest that CD24 may interact with NPM. NPM is a nucleolar protein and forms a punctate structure in the nucleus that dispersed during mitosis. Interestingly, the CD24–NPM association occurred transiently during mitosis. CD24 was more broadly distributed than NPM at the interphase (Fig. 6a), although some overlap between the two signals can also be observed (Fig. 6b). However, at metaphase and anaphase, CD24 and NPM were largely co-localized (Fig. 6a,b; see also Supplementary Fig. 8 for single-colour images). To confirm this observation in human tumour cells, we immunoprecipitated CD24 from DU145 cells and probed them with an anti-NPM monoclonal antibody. As shown in Fig. 6c, anti-CD24 co-precipitated NPM. Conversely, anti-NPM co-precipitated CD24. To map the region in NPM that interacts with CD24, we used a series of Myc-tagged deletion mutants of NPM and HA-tagged CD24. As shown in Fig. 6d, full-length (1–294) and all but one (AA1-177) truncation mutant co-precipitated CD24. As a small NPM C-terminal only fragment encompassing AA196-294 co-precipitated CD24, CD24 interacts with the C-terminus of NPM. However, as the AA196-294 fragment showed reduced association compared with the AA117-294 fragment, a contribution of the NPM core region to the interaction cannot be ruled out.

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