Limits...
BRCA1 regulates PIG3-mediated apoptosis in a p53-dependent manner.

Zhang W, Luo J, Chen F, Yang F, Song W, Zhu A, Guan X - Oncotarget (2015)

Bottom Line: Moreover, we reveal that overexpression of BRCA1 significantly increased expression of PIG3 in cells with intact p53, whereas no increase in PIG3 was observed in p53- MDA-MB-157 cells and p53-depleted HCT116p53-/- cells.Meanwhile, ectopic expression of BRCA1 could not lead to an increase expression level of prohibitin (PHB), which we have previously identified to induce PIG3-mediated apoptosis.Finally, ChIP analysis revealed that PHB can bind to the PIG3 promoter and activate PIG3 transcription independent of p53, although p53 presence did enhance this process.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China.

ABSTRACT
BRCA1 plays a key role in the regulation of p53-dependent target gene transcription activation. Meanwhile, the p53 inducible gene 3 (PIG3) is a downstream target of p53 and is involved in p53-initiated apoptosis. However, little is known about whether BRCA1 can regulate PIG3-mediated apoptosis. Using a tissue microarray containing 149 breast cancer patient samples, we found that BRCA1 and PIG3 expression status were significantly positively correlated (r = 0.678, P < 0.001) and identified a significant positive correlation between high expression of BRCA1 and/or PIG3 and overall survival (OS). Moreover, we reveal that overexpression of BRCA1 significantly increased expression of PIG3 in cells with intact p53, whereas no increase in PIG3 was observed in p53- MDA-MB-157 cells and p53-depleted HCT116p53-/- cells. Meanwhile, ectopic expression of BRCA1 could not lead to an increase expression level of prohibitin (PHB), which we have previously identified to induce PIG3-mediated apoptosis. Finally, ChIP analysis revealed that PHB can bind to the PIG3 promoter and activate PIG3 transcription independent of p53, although p53 presence did enhance this process. Taken together, our findings suggest that BRCA1 regulates PIG3-mediated apoptosis in a p53-dependent manner, and that PIG3 expression is associated with a better OS in breast cancer patients.

No MeSH data available.


Related in: MedlinePlus

PHB regulates PIG3-mediated apoptosis in a p53-depentent or -independent manner(A and B) PIG3 and PHB protein levels were determined by western blotting and analyzed by grayscale software following transfection of plasmids encoding PHB or siPHB, into HCT116p53+/+ and HCT116p53−/− cells in the presence or absence of 10 nM camptothecin. (C) ChIP assay for p53 and PHB binding of the PIG3 (TGYCC)15 motif in HCT116p53+/+ and HCT116p53−/− cell lines in the presence or absence of camptothecin. (D) Plasmids encoding PIG3 were co-transfected into HCT116p53+/+ and HCT116p53−/− cells with or without PHB. At 24 h post-transfection, the cells were subjected to luciferase assay. For comparison, the luciferase activity of the ncRNA-transfected cells was set as 1. Data are the mean of three independent experiments. *P < 0.05, as compared with untreated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4480703&req=5

Figure 4: PHB regulates PIG3-mediated apoptosis in a p53-depentent or -independent manner(A and B) PIG3 and PHB protein levels were determined by western blotting and analyzed by grayscale software following transfection of plasmids encoding PHB or siPHB, into HCT116p53+/+ and HCT116p53−/− cells in the presence or absence of 10 nM camptothecin. (C) ChIP assay for p53 and PHB binding of the PIG3 (TGYCC)15 motif in HCT116p53+/+ and HCT116p53−/− cell lines in the presence or absence of camptothecin. (D) Plasmids encoding PIG3 were co-transfected into HCT116p53+/+ and HCT116p53−/− cells with or without PHB. At 24 h post-transfection, the cells were subjected to luciferase assay. For comparison, the luciferase activity of the ncRNA-transfected cells was set as 1. Data are the mean of three independent experiments. *P < 0.05, as compared with untreated cells.

Mentions: Our previous studies have reported that the PIG3 promoter motif (TGYCC)15 is required for effective transcriptional activity of the promoter, and that PHB could contribute to PIG3-mediated apoptosis [18, 19]. To further investigate the roles of PHB and p53 in PIG3-mediated apoptosis, we examined the expression of PIG3 and PHB in HCT116p53+/+ and HCT116p53−/− cells overexpressing PHB in the presence or absence of camptothecin (CPT), a topoisomerase 1 inhibitor and an effective apoptotic stressor [20]. Protein expression of PIG3 was elevated in the HCT116p53+/+ cells compared with HCT116p53−/− cells, regardless of CPT treatment (Figure 4A). Following PHB overexpression, expression of PIG3 was increased in HCT116p53+/+ cells, while PIG3 was also significantly increased in the absence of p53 in HCT116p53−/− cells transfected with PHB. To investigate whether PHB could regulate the expression of PIG3 in a p53-independent manner, we also examined the effect of PHB depletion by RNA interference on the expression of PIG3. Transfection of HCT116p53−/− cells with PHB-targeting shRNA constructs efficiently suppressed PHB expression and inhibited expression of PIG3 (Figure 4B). Similarly, when the PHB-depleted cells were exposed to 10 nM camptothecin, PIG3 down-regulation was observed (Figure 4B).


BRCA1 regulates PIG3-mediated apoptosis in a p53-dependent manner.

Zhang W, Luo J, Chen F, Yang F, Song W, Zhu A, Guan X - Oncotarget (2015)

PHB regulates PIG3-mediated apoptosis in a p53-depentent or -independent manner(A and B) PIG3 and PHB protein levels were determined by western blotting and analyzed by grayscale software following transfection of plasmids encoding PHB or siPHB, into HCT116p53+/+ and HCT116p53−/− cells in the presence or absence of 10 nM camptothecin. (C) ChIP assay for p53 and PHB binding of the PIG3 (TGYCC)15 motif in HCT116p53+/+ and HCT116p53−/− cell lines in the presence or absence of camptothecin. (D) Plasmids encoding PIG3 were co-transfected into HCT116p53+/+ and HCT116p53−/− cells with or without PHB. At 24 h post-transfection, the cells were subjected to luciferase assay. For comparison, the luciferase activity of the ncRNA-transfected cells was set as 1. Data are the mean of three independent experiments. *P < 0.05, as compared with untreated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480703&req=5

Figure 4: PHB regulates PIG3-mediated apoptosis in a p53-depentent or -independent manner(A and B) PIG3 and PHB protein levels were determined by western blotting and analyzed by grayscale software following transfection of plasmids encoding PHB or siPHB, into HCT116p53+/+ and HCT116p53−/− cells in the presence or absence of 10 nM camptothecin. (C) ChIP assay for p53 and PHB binding of the PIG3 (TGYCC)15 motif in HCT116p53+/+ and HCT116p53−/− cell lines in the presence or absence of camptothecin. (D) Plasmids encoding PIG3 were co-transfected into HCT116p53+/+ and HCT116p53−/− cells with or without PHB. At 24 h post-transfection, the cells were subjected to luciferase assay. For comparison, the luciferase activity of the ncRNA-transfected cells was set as 1. Data are the mean of three independent experiments. *P < 0.05, as compared with untreated cells.
Mentions: Our previous studies have reported that the PIG3 promoter motif (TGYCC)15 is required for effective transcriptional activity of the promoter, and that PHB could contribute to PIG3-mediated apoptosis [18, 19]. To further investigate the roles of PHB and p53 in PIG3-mediated apoptosis, we examined the expression of PIG3 and PHB in HCT116p53+/+ and HCT116p53−/− cells overexpressing PHB in the presence or absence of camptothecin (CPT), a topoisomerase 1 inhibitor and an effective apoptotic stressor [20]. Protein expression of PIG3 was elevated in the HCT116p53+/+ cells compared with HCT116p53−/− cells, regardless of CPT treatment (Figure 4A). Following PHB overexpression, expression of PIG3 was increased in HCT116p53+/+ cells, while PIG3 was also significantly increased in the absence of p53 in HCT116p53−/− cells transfected with PHB. To investigate whether PHB could regulate the expression of PIG3 in a p53-independent manner, we also examined the effect of PHB depletion by RNA interference on the expression of PIG3. Transfection of HCT116p53−/− cells with PHB-targeting shRNA constructs efficiently suppressed PHB expression and inhibited expression of PIG3 (Figure 4B). Similarly, when the PHB-depleted cells were exposed to 10 nM camptothecin, PIG3 down-regulation was observed (Figure 4B).

Bottom Line: Moreover, we reveal that overexpression of BRCA1 significantly increased expression of PIG3 in cells with intact p53, whereas no increase in PIG3 was observed in p53- MDA-MB-157 cells and p53-depleted HCT116p53-/- cells.Meanwhile, ectopic expression of BRCA1 could not lead to an increase expression level of prohibitin (PHB), which we have previously identified to induce PIG3-mediated apoptosis.Finally, ChIP analysis revealed that PHB can bind to the PIG3 promoter and activate PIG3 transcription independent of p53, although p53 presence did enhance this process.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China.

ABSTRACT
BRCA1 plays a key role in the regulation of p53-dependent target gene transcription activation. Meanwhile, the p53 inducible gene 3 (PIG3) is a downstream target of p53 and is involved in p53-initiated apoptosis. However, little is known about whether BRCA1 can regulate PIG3-mediated apoptosis. Using a tissue microarray containing 149 breast cancer patient samples, we found that BRCA1 and PIG3 expression status were significantly positively correlated (r = 0.678, P < 0.001) and identified a significant positive correlation between high expression of BRCA1 and/or PIG3 and overall survival (OS). Moreover, we reveal that overexpression of BRCA1 significantly increased expression of PIG3 in cells with intact p53, whereas no increase in PIG3 was observed in p53- MDA-MB-157 cells and p53-depleted HCT116p53-/- cells. Meanwhile, ectopic expression of BRCA1 could not lead to an increase expression level of prohibitin (PHB), which we have previously identified to induce PIG3-mediated apoptosis. Finally, ChIP analysis revealed that PHB can bind to the PIG3 promoter and activate PIG3 transcription independent of p53, although p53 presence did enhance this process. Taken together, our findings suggest that BRCA1 regulates PIG3-mediated apoptosis in a p53-dependent manner, and that PIG3 expression is associated with a better OS in breast cancer patients.

No MeSH data available.


Related in: MedlinePlus