Transcriptional Regulation Of Interferon Regulatory Factor-4Binding Protein (IBP) And Its Significance In Breast Cancer Cells

Breast cancer is clearly the most frequent cancer in women. An estimated1.15millionnew cases of breast cancer were identified in2002. In China, breast cancer registries arerecording annual incidence increases of3%to4%. Although the molecular mechanisms andearly diagnosis of breast cancer have been progressed, there are still30%of patients withearly breast cancer recurrent every year. Looking for new target for breast cancer diagnosisand treatment is one of the current problems to be solved. Interferon regulatory factor-4binding protein (IBP) as a guanine nucleotide exchange factor (GEF) has an important rolein the immune system: participating in the immunological synapse formation, associatingwith Th2polarization, and so on. In2009, our group found that IBP is a newly discoveredprotein that is aberrantly expressed in breast cancer. It promotes breast cancer cellsproliferation and migration. It prompted that IBP transcriptional regulation mechanism maybe related to breast cancer development, and in breast cancer IBP may have a moreimportant significance. However, regulation of IBP expression, particularly in breast cancer,still remains unknown. Signal network of IBP and its molecular function in breast cancerstill remain to be further studied.To investigate the mechanism of IBP dysregulation and its possible clinicalsignificance in breast cancer, we did the following experiments:1. Bioinformatics analysis of IBP5’-flanking region and its core promoteridentification.(1) We subcloned5’-deletion mutants of the IBP5’-flanking region into pGL3-basic,assessed the expression of luciferase in HEK293cells. Our results mapped the corepromoter to the-294to-115region.(2) PROMO bioinformatics analysis demonstrated that IBP core promoter contains aputative noncanonical p53-binding site. 2. Wild type p53inhibits the transcriptional activity of the IBP promoter and attenuatesIBP expression(1) Fragment pIV which has the strongest transcriptional activity and harboursp53-binding site, was transiently transfected into HCT116p53-/-or HCT116p53+/+(wild-type p53) cells. pIV exhibited higher luciferase activity in p53knockout HCT116cells(2) pIV exhibited higher luciferase activity in the p53knockdown MCF-7cells.(3) When we treated MCF-7cells with p53activator doxorubicin, pIV luciferaseactivity reduced.(4) Wild-type p53expression plasmid pCMV-p53decreased the luciferase activity ofpIV. pCMV-p53R175H, which expressed a p53mutant, did not affect pIV luciferaseactivity.(5) p53adenovirus infected HCT116the p53-/-cells were transfected with IBPpromoter reporter gene plasmid and the p53binding site mutated reporter plasmid, theresults show that the p53decreases the activity of the IBP promoter through its putativep53-binding site.(6) We detected IBP expression in p53adenovirus, p53RNA interference, and p53inhibitor pifithrin-α, activator Nutlin-3stimulated MCF-7cells respectively, by quantitativePCR and Western Blot. The experiments showed that IBP expression is suppressed by p53in breast cancer cell lines. IBP is a p53responsive gene.3. p53protein binds to IBP core promoter in vivo and in vitro.(1) In vitro, electrophoresis mobility shift assay (EMSA) combined with p21probecompetition and p53antibody supershift experiments showed wild-type p53protein canbind to IBP promoter sequence.(2) Combined with doxorubicin stimulation, chromatin immunoprecipitation assay(ChIP) showed p53binds to IBP promoter region in MCF-7and HCT116p53+/+cells4. IBP is p53dependently suppressed by DNA damaging agents.(1) In MCF-7and ZR-75-1cells (wild-type p53expression cells), IBP was suppressedwith the DNA damaging agent cisplatin, doxorubicin in a dose-dependent andtime-dependent manner.(2) IBP was suppressed was unaffected in HCT116p53-/-cells and MCF-7cells stably expressing p53RNAi, indicating that the suppression of IBP by DNA damaing agents inbreast cancer cells is p53dependent.5. IBP regulates the sensitivity to cisplatin-induced apoptosis in breast cancer cells(1) We first established stable IBP over-expressing and stable IBP-knockdown MCF-7cells. Subsequently, all the cells were exposed to cisplatin, cell growth and IC50weremeasured. Over-expression of IBP increased proliferation and survival of MCF-7cells, andIBP knockdown increased cisplatin sensitivity of MCF-7cells.(2) To confirm that IBP depletion increased cisplatin induced apoptosis in MCF-7cells,we tested PARP and Annexin V-PI expression. These results demonstrate that IBPparticipates in the suppression of cisplatin-induced apoptosis in MCF-7cells.6. The mechanisms of which IBP regulates the sensitivity to cisplatin-inducedapoptosis in breast cancer cells.(1) The basal expression of p53in IBP-knockdown MCF-7cells was markedlyelevated. The p21expression was consistent with p53expression in IBP-knockdown andIBP-over-expressing MCF-7cells. Furthermore, in IBP-over-expressing cells, decreasedlevel of phosphorylated p53could be induced by cisplatin. This data suggests that IBPover-expression in breast cancer cells decreases p53accumulation and activation inresponse to cisplatin.(2) We tested Bcl-2and Bax levels in IBP-over-expressing cells. The levels of Bcl-2were highly elevated in IBP-over-expressing cells, and Bax expression was markedlyreduced. This result shows that IBP regulates Bcl-2family expression, and IBP disrupts p53dependent apoptotic pathway in breast cancer cells.(3) High level of AKT and MDM2phosphorylation was found in IBP-over-expressingMCF-7cells. When we treated IBP-over-expressing cells with AKT inhibitor Ly294002orwortmanin, p53expression was elevated. These results suggest that IBP may negativelyregulate p53activation through AKT in breast cancer cells.(4) In stable IBP-knockdown MCF-7cells, we inhibited p53expression, then cellswere exposed to cisplatin, and cell growth was measured. Inhibition of p53could decreasecisplatin sensitivity in IBP-knockdown MCF-7cells. Moreover, IBP knockdown alsoincreased cisplatin sensitivity of HCT116p53-/-cells. Furthermore, in IBP-over-expressingMCF-7cells, AKT inhibitors Ly294002could attenuate cisplatin resistance and increase cisplatin induced apoptosis. These results suggest that IBP may impair cisplatinchemosensitivity in breast cancer cells partly through AKT/p53pathway.In summary, we mapped IBP core promoter to the-294to-115region, and found thatit contains a noncanonical p53-binding site. IBP expression could be suppressed whenwild-type p53was directly bound to the IBP promoter. Further investigation revealed thatthere is a feedback loop between IBP and p53pathway. IBP over-expression inactivated p53pathway through AKT. In breast cancer cell lines, IBP was down-regulated by the DNAdamage agent in p53dependent way. While high levels of IBP were found to decreasecisplatin-induced growth suppression and apoptotic cell death, which was associated withdecreased p53accumulation, activation and imbalanced Bcl-2family member expression.This study may lay a foundation for further clarification of IBP expression regulationmechanism and clinical significance in breast cancer.