PAX2and AIB1Expression In Breast Cancer And The Roles In The Drug Resistant Mechanism Of Tamoxifen

Objective: Breast cancer is the most common malignant tumor of femalein the world, which seriously threatening those health and life. The incidence ofbreast cancer keeps on rising annually in China and more young females arefaced with it. Breast cancer is typical hormonal dependent tumor, andendocrine therapy, its representative drug tamoxifen（TAM）, has become themost important and successful one in the combined therapies for estrogenreceptor-positive（ER+） breast cancers due to its low toxicity, economy andlong curative effect. However, it was proved that40%ER-positive tumorsbecame resistant induced by long-term tamoxifen therapy, leading to progressionof breast cancer even death. However, the molecular mechanisms of tamoxifenresistance were not completely understood. So it was hot that studies on theresistant mechanism of TAM and its related gene of breast cancer.Some researchs had shown that paired box gene-2（PAX2）and amplifiedin breast cancer-1（AIB1） regulated the transcription of human epidermalgrowth factor receptor-2（HER-2） and the protein expression of HER-2duringthe treatment of tamoxifen for ER+breast cancers,which could account forTAM resistance. To explore the resistance mechanism of TAM and the relativefactors, we first detected the protien expressions of PAX2and AIB1withimmunohistochemistry（IHC） in postoperative ER（+） breast cancer patientstreated with TAM. Combining the follow-up results, we retrospectivelyanalyzed the relationship between PAX2/AIB1and the curative effect ofTAM.Secondly we detected the expressions of PAX2and AIB1in the tumortissues and the nontumorous tissues of breast on the transcriptional and proteinlevel by using reverse transcription-polymerase chain reaction （RT-PCR） andIHC.The relatetionships among the expressions of PAX2/AIB1in ER（+）breast cancers, the clinic pathological characteristics and the expression of ER,PR and HER-2were analyzed, to futher determine if PAX2/AIB1weresensitive indicators for prognosing the effect of antiestrogen therapy. Forfinding out the role of PAX2/AIB1on the antiestrogen treatment in ER+breastcancers,we used RT-PCR and Western blot method to detect the expressions ofPAX2, AIB1and HER-2in ER （+）/ER（-） breast cell lines （MCF-7andMDA-MB-231） stimulated with estrogen and TAM sensitive/TAM resistantbreast cell lines （MCF-7and MCF-7/TAMR） treated with or without TAM.PAX2siRNA and AIB1siRNA were transiently transfected to MCF-7andMCF-7/TAMR respectively, while plasmid of PAX2and AIB1weretransfected to MCF-7/TAMR and MCF-7to explore the regulationmechanisms among PAX2/AIB1, HER-2and TAM resistance.Methods:1The protein expressions of PAX2and AIB1in167postoperative ER（+）breast cancers treated with TAM were detected by IHC, and the relationshipbetween PAX2/AIB1and the recurrence or metastasis after TAM treatmentwas analyzed, to determine if PAX2/AIB1influenced the TAM therapeuticeffect.2The mRNA and protein expression of PAX2and AIB1in70breast tumorand nontumorous tissues were detected by using RT-PCR and IHC. Therelationships between PAX2/AIB1and the clinic pathological characteristicswere analyzed, including age, menses, tumor size, clinical stages,pathological type, histological grades, the metastatic state of the axillarylymph nodes, ER, progestogen receptor（PR） and HER-2in70breast cancers,to futher determine if PAX2/AIB1regulated the expression of HER-2andinfluenced the effect of antiestrogen therapy.3The mRNA expressions of PAX2, AIB1and HER-2were measuredwith RT-PCR in ER （+） MCF-7and ER （-） MDA-MB-231breast cancer celllines stimulated with or without estrogen respectively, so did TAM sensitvecell lines MCF-7and TAM resistant cell lines MCF-7/TAMR stimulated by0.1%anhydrous alcohol and1μM tamoxifen for24h to get the basicexpressions of PAX2, AIB1and HER-2in those cell lines. 4PAX2siRNA and AIB1siRNA were transiently transfected to MCF-7and MCF-7/TAMR respectively, while plasmid of PAX2and AIB1weretransfected to MCF-7/TAMR and MCF-7to observe the expression ofPAX2/AIB1/HER-2and cell proliferation explore the regulation mechanismsamong PAX2/AIB1, HER-2and TAM resistance.Results：The first part: Protein expression of PAX2/AIB1and the relationshipsbetween them and the TAM resistance of ER+breast cancers1Follow-up results: Among167breast cancer patients,110casesappeared tumor-free survival, while57cases appeared local recurrence ormetastasis within5years after therapy, with the mean disease-free survival（DFS） of45months and the mean overall survival （OS） of57months.2Among the different expression level of HER-2（low-expression andover-expression）, the difference of PAX2and AIB1protein expression weresignificant （χ2=8.401, p=0.004; χ2=12.398, p=0.000）．In the group of protienover-expression of HER-2, the protein over-expression rate of PAX2waslower, while that of AIB1was higher.3Relationships between the protein expression of PAX2, AIB1andrecurrence or metastasis of breast cancers:Among167cases,57cases appeared recurrence or metastasis, while110cases were disease-free survival within5years. After statistical analysis,recurrence or metastasis rates had significant difference between differentgroups of PAX2（χ2=4.198, p=0.040） and AIB1（χ2=5.336, p=0.021）. Breastcancers with over-expression of PAX2and low-expression of AIB1had lowerrecurrence or metastasis rate, so PAX2and AIB1might be sensitive prognosticindex for prognosing TAM treatment effect.The second part: Expression of PAX2/AIB1in breast cancers and therelationships between them and the clinic characteristics1Expression of PAX2/AIB1on the mRNA level in breast cancer andnontumorous tissuesThe value of AIB1mRNA in breast cancer and nontumorous tissues were （1.7390±0.7687） and （1.2742±0.6836）, which the difference wassignifant（Z=-2.031,p=0.030）. While there had no significant difference ofPAX2mRNA between those tissues （Z=-0.184, p=0.830）.2The relationship between the mRNA and protein expressions ofPAX2/AIB1and clinic pathological characteristics of70breast cancersAccording to the age（<50years and≥50years）, the menstrual status（premenopause and postmenopause）, tumor size （≤2cm,>2cm）, the clinicalstage （stage I,II and III）, the pathological types （infiltrating ductal carcinoma,infiltrating lobular carcinoma and others）, the histological grade （grade I^IIand III） and the metastatic state of the axillary lymph nodes （negative,1³，and＞3）, the mRNA and protein expression of PAX2had no significantdifference among groups of those factors（p>0.05）．The mRNA expression ofAIB1had positive correlation with the histological grades and the metastaticstate of the axillary lymph nodes, while the protein expression of AIB1onlyhad positive correlation with the latter.3The relationships between the expression of PAX2/AIB1and HER-2,ER, PR on mRNA and protein levelThere were19,32and19cases of HER-2（-）, HER-2（+⁺+） and HER-2（+++） respectively. The mRNA and protein expression of PAX2had negativecorrelation with the expression of HER-2and the difference of each group wassignificant（p=0.037; χ2=7.614, p=0.022）. The mRNA expression of AIB1hadno significant difference between different expression of HER-2, while theprotein expression of AIB1increased with the rising expression of HER-2（χ2=11.564，p=0.003）.There were32,38,46and24cases of ER （+⁺+）, ER （+++）, PR （+⁺+）and PR （+++） respectively. It was showed that the mRNA and proteinexpression of PAX2and AIB1had no relationships with the expressions of ERand PR after statistical analysis.4The relationships between protein expression of PAX2and AIB1The over-expression rate of PAX2was35.85%（19/53） in the low-expression of AIB1（-）⁺⁺, and that was5.88%（1/17） in the over-expression of AIB1（+++）, which had the significant difference. The protein expression ofPAX2had negative correlation with that of AIB1（χ2=5.664；p=0.017；r=-0.306）.5Follow-up results: In all the70patients there were12cases out ofcontact and6cases with recurrence or metastasis including2cases dead. TheDFS was44months and OS was46months. So there were26cases treatedwith TAM after operation including2cases with metastasis, whose DFS was46.7months and OS was46.5months.Among the26cases,9cases with over-expression of AIB1had2casesoccuring metastasis and17cases with low-expression of AIB1had nometastasis, which suggested that patients with over-expression of AIB1hadhigh risk of recurrence and metastasis（χ2=4.093；p=0.043）.The third part:The empirical study on the roles of PAX2/AIB1on theresistance mechanism of TAM1The mRNA expressions of AIB1/HER-2increased and that of PAX2decreased in the ER （+） breast cell line MCF-7after estrogen stimulation,while there had no significant changes in ER （-） breast cell line MDA-MB-231after estrogen stimulation. It suggested that PAX2, AIB1and HER-2took partin the estrogen dependent signal conduction path and the cell proliferationswere companied by the decreased expression of PAX2and the increased thatof AIB1and HER-2.2PAX2, AIB1and HER-2could be detected in both MCF-7（TAMsensitive cell lines） and MCF-7/TAMR （TAM resistant cell lines） and theexpression of AIB1between these two cell lines had no significant difference（p>0.05）. But the expression of PAX2in MCF-7was higher than that inMCF-7/TAMR,while the expression of HER-2was opposite. It suggested thatthe resistant mechanism of TAM might be related with the expression of PAX2and HER-2.The expression of AIB1visibly increased and that of PAX2/HER-2decreased in MCF-7treated with TAM, and the difference was significant （p＜0.05）. While only HER-2expression increased in MCF-7/TAMR treated with TAM, and the changes of PAX2/AIB1had no significant difference.3MCF-7transiently transfected by PAX2siRNA and MCF-7/TAMRtransiently transfected by AIB1siRNA （down-regulation of PAX2/AIB1）After MCF-7was transiently transfected by PAX2siRNA, the mRNA andprotein expression of HER-2in PAX2siRNA+TAM group increased morethan those of HER-2in PAX2siRNA+Vehicle one, while the proteinexpression of AIB1decreased （p<0.05）, and the cell proliferation was notobviously depressed. It suggested that down-regulation of PAX2in MCF-7cell line inhibited the role of TAM on depressing the HER-2expression andcell proliferation, while it reversed up-regulation of AIB1induced by TAM.After MCF-7/TAMR was transiently transfected by AIB1siRNA, themRNA and protein expression of HER-2, the protein expression of PAX2andthe cell proliferation in AIB1siRNA+TAM group decreased more than thosein AIB1siRNA+Vehicle one（p<0.05）. It suggested that down-regulation ofAIB1in MCF-7/TAMR cell line refreshed the role of TAM on depressing theHER-2expression and the over-expression of AIB1would inhibit theantiestrogen of TAM in breast cancer cell.4MCF-7/TAMR transfected by plasmids of PAX2and MCF-7transfected by those of AIB1（up-regulation of PAX2/AIB1）After MCF-7/TAMR was transfected by plasmids of PAX2, the mRNAand protein expression of HER-2, the expression of AIB1and the cellproliferation in PAX2plasmid+TAM group decreased more than those inPAX2plasmid+Vehicle one （p<0.05）. It suggested that up-regulation of PAX2reversed the role of TAM on promoting the expression of HER-2inMCF-7/TAMR cell and refreshed the sensitivity of TAM. Augument of PAX2would reverse the TAM resistance.After MCF-7/TAMR was transfected by plasmids of AIB1, the mRNAand protein expression of HER-2and the cell proliferation in AIB1plasmid+TAM group increased more than those in AIB1plasmid+Vehicle one, whilethe expression of PAX2decreased（p<0.05）. It suggested that up-regulation ofAIB1didn’t change the role of TAM on depressing the expression of PAX2 but reversed the role of TAM on depressing the expression of HER-2inMCF-7cell. Augument the expression of AIB1would induce TAM resistance.Conclusions:1Among the ER（+） breast cancers treated with TAM, the patients withover-expression of PAX2and low-expression of AIB1had lower recurrenceor metastasis rate within5years. PAX-2and AIB1were good index forprognosis. PAX2and AIB1were related with HER-2expression and TAMresistance.2The mRNA expression of PAX2had no significant difference betweentumor and nontumorous breast tissues. Expression of PAX2had no significantrelationships with age, menses, tumor size, clinical stage, histological grade,metastatic state of the axillary lymph nodes, pathology type and ER/PR onmRNA and protein level.3The mRNA expression of AIB1in breast cancers were higher than thatin nontumorous tissues. The mRNA expression of AIB1had positivecorrelation with the histological grade and the metastatic state of the axillarylymph nodes and the protein expression of AIB1only had positive relationshipwith the latter, but had no significant relationships with other factors.4PAX2and AIB1were all detected in both MCF-7and MCF-7/TAMRcells. The expression of PAX2in MCF-7was higher than that in MCF-7/TAMR, while the expression of AIB1had no difference in these two cell lines.Over-expression of PAX2would increase the TAM sensitivity of cell, whilethe over-expression of AIB1would decrease it.5PAX2was the inhibitor of HER-2transcription on the therapy of TAM.The specific activation of PAX2would be a new target for curing TAMresistance. AIB1had cooperation with PAX2, which regulated the HER-2transcription by competively binding cis-regulatory element of HER-2andthus influeced the sensitivity of TAM.6Raising the expression of PAX2and depressing the expression of AIB1might improve the resistant condition of TAM, so PAX2and AIB1might benovel therapeutic targets.