The Mechanism Of Breast Cancer Resistance Protein-Mediated Multidrug Resistance Modulated By Progesterone In Breast Cancer

[Background and objective]Breast cancer is the most prevalent type of cancer in women, and the greatest obstacle in chemotherapy treatment is multidrug resistance (MDR). One of the causes of MDR is that a wide range of structurally and functionally unrelated drugs are pumped out of the cancer cells by the ATP-binding cassette (ABC) transporter proteins. P-glycoprotein (P-gp), Breast Cancer Resistance Protein (BCRP) and Multidrug Resistance Associated Protein (MRP) are three primary types of ABC transporters in human relating to the MDR phenomenon and they act as an efflux pump, which leads to diminished intracellular accumulation of the anticancer drugs and reduced cytotoxic effects.As an ABC transporter protein, BCRP was firstly identified from drug selected human breast cancer cells (MCF-7) and was found to express in many normal tissue and cancer cells isolated from various solid tumors. Many studies have shown that BCRP mediates resistance to adjuvant chemotherapeutic agents, such as mitoxantrone, topotecan and SN-38, by pumping them out of the cell.BCRP is localized to4q22in chromosome and made up by16exons and15introns encoding a72kDa,655-amino acid protein. The BCRP promoter is localized from-266to-36bp. BCRP is designated as a half transporter. In previous studies, the positive BCRP expression in solid tumors were dertermined by positively cytoplasmic staining. However, this approach was not appropriate to reflect the functional status of the protein.Breast cancer is a kind of hormone-dependent systematic disease and may be regulated by steroid hormones, such as estrogen, progesterone and testosterone via classical pathways, but the data have some conflict. Estrogen plays an important role in progesterone action since the level of PR in both mammary gland and breast cancer is regulated by estrogen. It was believed that PR was only a marker of the active function of ER. However, there are still5%of breast cancer patients immunohistochemically with ER-/PR+and it often occurs in young or middle-aged women. The ER-/PR+tumors are larger in size and more aggressive compared with the group of ER+/PR+. Therefore, in those ER negative breast cancer cells, the PR expression level should be greatly compromised, which implies the role of PR remains enigma.Our group have firstly revealed that BCRP expression is down-regulated by toremifen by reversing BCRP-mediated MDR through TOR-ER complexes, which binds to the estrogen response element (ERE) in the BCRP promoter and inhibits the transcription of BCRP. Recent research revealed that progesterone response element (PRE) in BCRP promoter is just the ERE, but it is still unknown whether this novel element plays a role in the BCRP-mediated MDR pathway in breast tumor cells.In this study, we first examined expression of BCRP, ERa, PR and Her-2in95cases of primary invasive ductal carcinoma by immunohistochemistry. Furthermore, two constructs encoding full length BCRP driven by endogenous BCRP promoter or constitutive CMV promoter were transfected into PR-positive T47D cells, PR-positive MCF-7cells and PR-negative MDA-MB-231breast cancer cells, respectively. After PROG or RU-486treatment, the role of PRE in BCRP-mediated MDR was determined by qRT-PCR, RT-PCR, western blotting, flow cytometric analysis and cytometric analysis, and the possible mechanism of MDR mediated by BCRP in human breast cancers was elaborated after the treatment of progesterone.[Methods]1.Immunohistochemistry:We examined the membranous expression of BCRP, ERa, PR and human epidermal growth factor receptor-2(Her-2) in95women breast cancer samples by2-step plus Poly-HRP Anti-Mouse/Rabbit IgG Detection System (PV-9000). Then, we analyzed the relationship between BCRP and ERa, PR or Her-2, respectively, as well as the correlation between BCRP and the clinical pathological characters in invasive ductal carcinoma.2.Plasmids and cloning:3) The construction of plasmids:The pcDNA3.1/BCRP cDNA (stored in our lab) was digested with Apa I/BamH I to get full length BCRP cDNA. Then, BCRP cDNA was inserted into pEGFP-C1including Enhanced Green Fluorescence Protein (EGFP) and stable selective marker, which was named as pEGFP-C1/C-BCRP;4) To generate pEGFP/P-BCRP construct, BCRP promoter was artificially synthesized and cloned into pMD20-T vector. Then BCRP promoter was sub-cloned into pEGFP/C-BCRP with SnaB I/NheI digestion to destroy the CMV promoter. All constructs were sequenced to confirm the right promoter and cDNA sequences.3. The expression of PR protein in breast cancer tissue:We checked the expression of PR in MCF-7,T47D,MDA-MB-231cells by Western Blotting.4. Cell lines and culture and transfection:The constructing plasmids of pEGFP-C1/P-BCRP,pEGFP-C1/C-BCRP were transient-transfected to PR (+)-MCF-7and PR (-)-MDA-MB-231cells, respectively and the blank control group was set up. At the same time, the two types of plasmids were stable-transfected to PR (+)-T47D and the G418was added to select stable expression cell lines. In transient-transfected group, the treated cells were collected and in the stable-transfected group, after about4weeks cells of T47D/P-BCRP, T47D/C-BCRP which stably expressing BCRP were established, the cells were collected, too. All types of the cells were checked the expression of BCRP protein by western blotting to evaluate the establishment of the multidrug resistance cell lines.5. After treated by PROG, the BCRP mRNA and protein levels and the efflux function of BCRP were observed.After the treatment of PROG or RU-486, the total RNA and total protein were extracted. The expression of BCRP mRNA and proteins were examed by PCR and western blotting, respectively. Cytometric analysis was used to evaluate the BCRP-mediated drug efflux and MTT method was performed to observe the50%inhibition of concentration of mitoxantrone.6. Electrophoretic mobility shift assay (EMSA):It was confirmed that the specific binding complex was verified in PR(+)-MCF-7, but not in PR(-)-MDA-MB-231after incubated with the consensus oligonucleotide containing PRE sequence and anti-PR antibody.[Results]1.Immunohistochemistry results showed that29of95patients were BCRP positive (30.53%),45of95were PR positive (47.37%),65of95were ERa positive (68.42%) and15of95were Her-2positive (15.79%). Moreover, expression of BCRP was negatively associated with that of PR (P=0.045) and ERa (P=0.017) and positively related with lymph node metastasis in breast invasive ductal carcinoma (P=0.025). While the expression of BCRP was not associated with that of Her-2, also not related with age and tumor size.2.The breast cancer cells of MCF-7and T47D were PR(+)and MDA-MB-231was PR (-)3. After transfected with pEGFP-C1/P-BCRP and pEGFP-C1/C-BCRP, the cells of T47D/P-BCRP,T47D/C-BCRP,MCF-7/P-BCRP,MCF-7/C-BCRP,MDA-MB-231/P-BCRP and MDA-MB-231/C-BCRP over-expressed BCRP proteins by western blotting in them.4. treatment of PROG(1)In transient-transfected group:24hours after treatment, progesterone could inhibit the BCRP mRNA levels by12.5%,19.8%,32.43%,42.6%and50.1%respectively compared with the control cells (P<0.01, respectively). However, the progesterone did not have significant effects on BCRP mRNA level in MCF-7/C-BCRP cells (P>0.05). Similar results were found in MDA-MB-231/P-BCRP and MDA-MB-231/C-BCRP cells (P>0.05). After the addition of progesterone for48hrs, the expression of BCRP protein was decreased by33.3%,50.0%,59.5%,66.7%and80.9%respectively in MCF-7/P-BCRP (P<0.01). While in MCF-7/C-BCRP cells, progesterone showed little impact on BCRP protein levels (P>0.05). Moreover, progesterone had little influence on BCRP protein in both MDA-MB-231/P-BCRP and MDA-MB-231/C-BCRP cells (P>0.05).(2)In stable-transfected group:The cells of T47D/P-BCRP and T47D/C-BCRP were treated with different concentration of PROG from0to10-5M. In T47D/P-BCRP cells, expression of BCRP was inhibited in a dose-dependent manner (P<0.01) with the addition of PROG; while the inhibition of PROG at10-5M was almost completely abolished by the ahead addition of RU-486at10-4M (P<0.01). In T47D/C-BCRP stable cells, though PROG treatment at10-11M and10-9M induced the BCRP expression level, treatment with other concentrations showed little impact on BCRP protein level. Furthermore, both10-5M PROG and10-4M RU-486induced expression of BCRP to levels significantly higher than DMSO treated group (P<0.05).(3)After treatment of PROG, the fluorescence peak was shifted rightward in T47D/P-BCRP cells compared to negative control of T47D cells, which may be caused by the low expression of BCRP. However, when the cells were pretreated with10-4M RU-486, the PROG-mediated efflux of mitoxantrone was greatly abrogated, suggesting PROG inhibits the BCRP efflux activity while RU-486reverses this effect. Interestingly, in T47D/C-BCRP cells, after treating with10-5M PROG, the fluorescence peak shifted leftward and this effect was not abolished by RU-486, which may be incited by the CMV promoter.(4)The results of MTT showed that IC50was0.06±0.02μmol/L,0.26±0.03μmol/L,1.36±0.36μmol/L,1.25±0.26μmol/L and1.40±0.17μmol/L in T47D, T47D/P-BCRP+PROG, T47D/P-BCRP, T47D/C-BCRP+PROG and T47D/C-BCRP cells, respectively. Compared with T47D cells, the sensitivity of T47D/C-BCRP cells to mitoxantrone increased24.40fold (P<0.01); after treating with PROG, the resistance rate increased22.61fold (P<0.01). While in T47D/P-BCRP cell, the sensitivity to mitoxantrone increased25.08fold (P<0.01); after treating with PROG, the sensitivity was decreased4.63fold, which was not significantly different compard to T47D cells.5. EMS A demonstrated that nuclear protein of PR can specifically bind to the identified PRE in BCRP promoter, resulting in the negative regulation of BCRP in PR positive MCF-7. But this phenomenon was not shown in PR negative MDA-MB-231cells.[Conclusion]1. Considered with the status of BCRP protein, only membrane staining of the tumor cell was judged as BCRP positive in breast cancer tissue. The expression of BCRP was negatively related with those of PR and ERa and positively associated with lymph node metastasis in invasive ductal carcinoma. While the expression of BCRP was not related with that of Her-2, also not associated with age and tumor size.2. PROG can negatively regulate BCRP mRNA and protein levels in a dose-dependent manner in PR positive breast cancer cells. It suggested that PROG, similar to the function of estrogen receptor antagonist, can reverse the BCRP-mediated MDR.3.PROG can negatively regulate the expression of BCRP, which mediated by progesterone-PR complexes binding to the PRE in BCRP promoter through the typical pathway to inactivate transcription of the human BCRP gene, leading to reversing BCRP-mediated drug resistance in PR positive breast cancer cells.