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

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-null 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.

Keywords: BRCA1; PIG3; breast cancer; p53; prohibitin.

Figure 1. PIG3 and BRCA1 are associated with OS in breast cancer patients

(A) High magnification (200×) regions shown immunohistochemical analysis of PIG3 and BRCA1 high or low expression breast cancer patient tissues. (B) High PIG3 and/or BRCA1 expression was associated with better OS (all P < 0.05).

Figure 2. BRCA1 positively regulates PIG3 expression in a p53-dependent manner

(A and B) Cell viability was measured by MTT assay, and apoptosis was detected by flow cytometry following transfection of plasmid encoding BRCA1 into MCF-7, T47D, SK-BR-3, and MDA-MB-157 cells. (C) BRCA1, p53, and PIG3 protein levels were determined by western blotting following transfection of plasmid encoding BRCA1 into MCF-7, T47D, SK-BR-3, and MDA-MB-157 cells, and normalized to GAPDH expression. (D) BRCA1, p53, and PIG3 mRNA expression levels were determined by RT-PCR following transfection of plasmid encoding BRCA1 into HCT116p53^+/+ and HCT116p53^−/− cells, and normalized to GAPDH expression. (E and F) Localization and expression of p53 and PIG3 were determined by fluorescence microscopy following the same treatment as (D), with DMSO treatment as a control. Nuclei were stained with DAPI. The high magnification (200×) regions were shown above. (G) Relative levels of PIG3 mRNA in 206 breast cancer cell samples with no BRCA1 mutation (WT) and 392 breast cancer cell samples with a BRCA1 mutation (MT). Data analyzed using Oncomine (www.oncomine.org) from original published data (Garnett et al., 2012). Data are the mean of three independent experiments. *P < 0.05, as compared with untreated cells.

Figure 3. BRCA1 induces PIG3 expression independent of PHB

(A) PHB protein levels were determined by western blotting following transfection of plasmid encoding BRCA1 into MCF-7, T47D, SK-BR-3, and MDA-MB-157 cells, and normalized to GAPDH expression. (B) PHB mRNA expression levels were determined by RT-PCR following transfection of plasmid encoding BRCA1 into HCT116p53^+/+ and HCT116p53^−/− cells, and normalized to GAPDH expression. Data are the mean of three independent experiments. *P < 0.05, as compared with untreated cells. (C) Localization and expression of PHB were determined by fluorescence microscopy following the same treatment as (B), with DMSO treatment as a control. Nuclei were stained with DAPI. The high magnification (200×) regions were shown above.

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.

Figure 5. Schematic model depicting the critical role of BRCA1 in the regulation of PIG3-mediated apoptosis