Experimental Study On The Roles Of PAX2and AIB1in The Mechanisms Of Tamoxifen Resistance In Breast Cancer

Objective: Breast cancer is the most common malignant tumor occurringin women and has become the primary death cause of female cancerworldwide. At present, the trend lines of breast cancer are continuing to risegradually, especially among younger women. However, clinical studies haves h o w n t h a t e n d o c r i n e t h e r a p y h a s b e c o m e o n e o f t h e m o s teffective treatments based on its long-term therapeutic benefit and lowi n c i d e n c e o f s i d e e ff e c t s. Ta m o x i f e n, w h i c h i s a m e m b e r o fselective estrogen receptor modulators (SERMs), is considered to be the mostcommonly used endocrine therapy for the premenopausal breast cancerpatients. According to clinical researches,40%of ER-positive tumorsbecome resistant induced by long-term tamoxifen therapy, leading tobreast cancer progression and death. However, the molecular mechanisms oftamoxifen resistance are not completely understood.PAX2gene is localized to the conserved region of human chromosome10q24. PAX2encodes paired box gene2, one of many human homologues ofthe Drosophila melanogaster gene. PAX2is believed to be a target oftranscriptional suppression of the tumor suppressor gene WT1. According toseveral previous retrospective studies, both quantitative relationship betweenPAX2and AIB1indicated the expression level of HER2protein.Over-expression of HER2protein related to the tamoxifen resistance in breasttumor. However, it is not yet to be determined how PAX2and AIB1work inthe tamoxifen resistance mechanism of breast cancer. Therefore, based on thesystematic experimental research, the following article has shown that theexpression of PAX2and AIB1and their function in the regulation mechanismon HER2in estrogen-receptor-positive breast cancer cells. In addition, theresearch also provides a mechanistic insight into the molecular basis of tamoxifen resistance in breast cancer.Methods:1Cell culture and treatmentIn this research, we chose wild-type human breast cancer MCF-7cell lineand tamoxifen-resistant human breast cancer MCF7/TAMR cell line(whichwere derived by long-term exposure to tamoxifen). The MCF-7andMCF7/TAMR cells then treated with solvent (anhydrous ethanol) alone orwith1μM TAM for24h respectively. The inhibitor of HER2Lapatinib wasadded to the medium at a final concentration of9nM.3Transfection with small interfering RNA or plasmidMCF-7cells were reversely transfected with NC siRNA or siRNAtargeting PAX2at a concentration of100pmol respectively usingLipofectamine RNA iMAX (Invitrogen) according to the manufacturer’sinstructions. And at24hours after treatment, medium was discarded, thencells were treated with solvent (anhydrous ethanol) alone or with1μM TAMfor24h respectively. The AIB1siRNA was then transiently transfected intoMCF7/TAMR cells with the same method processing.Exponentially growing MCF-7cells (1×105cells/well) were plated for24h. The AIB1expression vectors and the pCMV6-ENTRY vector were thentransiently transfected into MCF-7cells using Lipofectamine2000(Invitrogen)for24h respectively, and lastly, then cells were treated with solvent(anhydrous ethanol) alone or with1μM TAM for24h respectively. The PAX2expression vectors were then transiently transfected into MCF7/TAMR cellswith the same method processing.4Western blot analysisIn the following step, after all proteins isolated from the cells, theexpression of various protein levels in the cells was determined by Westernblot，and analyzed with computer Odyssey imaging system.5Quantitative real-time polymerase chain reaction (PCR)After the treatment of TAM on the MCF-7and MCF7/TAMR cells, totalRNA was extracted from cells and reverse transcribed into cDNA, the expression of PAX2, AIB1and HER2mRNA level in the cells in differentgroups were determined using Quantitative Real-time PCR method andanalyzed with Gene Amp PCR system.6MTT Cell Proliferation AssayThe cells were collected and used the MTT cell proliferation assaymeasures the cell proliferation rate.7Statistical analysisAnalysis was performed in SPSS V13.0(SPSS Inc.). Results wereexpressed as thex±SD. Statistical calculations were performed with ANOVA.Differences in measured variables between experimental and control groupswere assessed by paired-samples t-test. For each protocol, at least threeindependent experiments were performed. P<0.05was considered statisticallysignificant.Results:1The expression of PAX2, AIB1and HER2in human estrogen-receptor positive breast cancer cellsWestern bolt results showed that，PAX2、AIB1、HER2protein expressionwere examined in both MCF-7and MCF7/TAMR cell lines. The expressionsof AIB1protein are not unaltered, while PAX2protein levels were lower inMCF7/TAMR cells than it of MCF-7cells. Furthermore, tamoxifen-resistantbreast cancer cell line MCF7/TAMR was characterized by elevated HER2levels in comparison with MCF-7.2Cell proliferation and the AIB1, PAX2and HER2expression after TAMtreatmentMTT assay result: Exposure of MCF-7cells to TAM at increasingconcentrations caused distinct decrease of cell proliferation. Compared toparent MCF-7cells, MCF7/TAMR cells had greater growth responses to thelow concentration of TAM, but present inhibition effect to the higher ones. Theproliferation of two groups of cells has a statistically significant difference bythe treatment of each concentration of TAM.Cells were starved in0.5%serum medium for24h followed by1μM TAM stimulation for24h. Whole cell extracts were then prepared by lysisagent and analyzed by Western blot. The results showed that PAX2and HER2protein level was significantly decreased in MCF-7cells with TAMstimulation than the control group, but the level of AIB1protein was increased.It’s indicating that TAM can suppress HER2and PAX2expression, inducedthe expression of AIB1. In contrast, changes of PAX2and AIB1proteinlevel in MCF7/TAMR cells had no statistic significance before andafter treatment, tamoxifen no longer repress the HER2protein level butinduce.3The biological function of PAX2in breast cancer3.1The effects of PAX2in breast cancer with technique of RNA interferencein vitro3.1.1The expect of depletion of PAX2expression on HER2, AIB1expressionunder the influence of TAM in MCF-7cells:In comparison with NC siRNA group, depletion of PAX2by specificsiRNA transfection effectively suppressed HER2mRNA down-regulationinduced by TAM treatment in MCF-7cells. Western blot shown showed thesame result on HER2protein. After transfection with PAX2siRNA in MCF-7cells, the down-regulation of endogenous PAX2expression by PAX2siRNAled to decreased the expression of AIB1in PAX2siRNA+TAM group relativeto that in NC siRNA+TAM group.3.1.2The effect on TAM treatment sensitivity of MCF-7cells in thepresence of PAX2siRNAMTT assay results showed that treatment of PAX2siRNA-transfectedcells with tamoxifen has no effect on cell number, relative to the PAX2siRNA+TAM group. Pretreatment of cells with an HER2inhibitor(Lapatinib)induced the down-regulatory effect of cell growth in PAX2siRNA+TAMgroup.3.2The effects of PAX2in breast cancer with technique of plasmidtransfection in vitro3.2.1The expect of over-express the PAX2expression on HER2, AIB1 expression under the influence of TAM in MCF7/TAMR cellsHER2mRNA expression in PAX2plasmid+TAM group was dramaticallydecreased to the PAX2plasmid+Vehicle group. Western blot also proved thatthe HER2protein expression decrease in PAX2plasmid+TAM group, relativeto the Vector+TAM group. After reintroducing PAX2into MCF7/TAMR cells,the AIB-1protein level was decreased after1μM TAM treatment for24h.3.2.2The effect on TAM treatment sensitivity of MCF7/TAMR cellstransfected by PAX2plasmidMTT assay results showed that over-express of PAX2with technique ofplasmid transfection can regain the ability of tamoxifen to inhibit cell growthin these previously resistant cells. The results showed treatment of PAX2+TAM cells number dramatically decreased, relative to the PAX2+Vehiclegroup.4The biological function of AIB1in breast cancer4.1The effects of AIB1in breast cancer with technique of plasmidtransfection in vitro4.1.1The expect of over-express the AIB1expression on HER2, AIB1expression under the influence of TAM in MCF-7cellsWhile TAM could down-regulate the HER2mRNA expression in MCF-7cells, we can see AIB1plasmid abrogated this inhibition and consequentlyelevated HER2transcription with treatment of TAM. Western blot also provethat the HER2protein expression in AIB1+TAM group hadno statistic significance change relative to the AIB1+Vehicle group.Western blot revealed that the PAX2protein expression was decreased inAIB1plasmid+TAM group, relative to the AIB1plasmid+Vehicle group, butincreased compared with Vector+TAM group.4.1.2The effect on TAM treatment sensitivity of MCF-7cells transfected byAIB1plasmidMTT assay results showed that treatment of AIB1plasmid-transfectedcells with tamoxifen had no more slow down the cell proliferation, unlike theAIB1+Vehicle group. Pretreatment of cells with the HER2inhibitor (Lapatinib), the cells growth rate of AIB1+TAM prominently slower thanAIB1+Vehicle group. It suggested that elevated AIB-1levels could reversethe anti-proliferation of tamoxifen, while it can be reversed by the HER2inhibitor Lapatinib.4.2.1The expect of AIB1siRNA on HER2, PAX2expression under theinfluence of TAM in MCF7/TAMR cellsWhile TAM showed no effects on the HER2mRNA expression inMCF7/TAMR cells, we can see AIB1siRNA recovered this inhibition abilityof TAM to suppress HER2transcription. Western blot also prove that AIB1siRNA+TAM group had no statistic significance effect on the HER2proteinexpression, relative to the AIB1siRNA+Vehicle group.Western blot revealed that the PAX2protein expression was decreased inAIB1siRNA+TAM group, relative to the AIB1siRNA+Vehicle group.4.2.2The effect on TAM treatment sensitivity of MCF7/TAMR cellstransfected by AIB1siRNAMTT assay results showed that the cells growth rate of AIB1siRNA+TAM prominently slower than the AIB1+Vehicle group.Conclusion:1The expression of PAX2、AIB1protein were examined in both MCF-7and MCF7/TAMR cell lines. The expressions of AIB1protein are notunaltered, while the expressions of PAX2protein levels were lower inMCF7/TAMR cells than that in MCF-7cells. Furthermore, MCF7/TAMR wascharacterized by elevated HER2levels.2The over-expression of PAX2enhanced the tamoxifen efficacy toinhibit the growth of breast cancer cells, but AIB1do the opposite. It suggeststhat AIB1and PAX2may be as the crucial predictors to measure theeffectiveness of breast cancer endocrine therapy.3PAX2might be functioning as a general transcriptional repressor ofHER2by the treatment of tamoxifen. PAX2expected to be a new target tosolve the problem of tamoxifen resistance in breast cancer.4PAX2and AIB1may interact with each other for binding the cis-regulatory element of HER2, the outcome of which regulates thetranscription of HER2, therefore, determines tamoxifen response in breastcancer cells.