The Empirical Study Of AIB1 For Tamoxifen Resistance In Estrogen Receptor-Positive Breast Cancer

BackgroundBreast cancer is one of the most common malignant tumors in women,and its incidence is the highest for all the female tumors in European and American developed countries.According to national statistics,in recent years the incidence of breast cancer in China continued to rise significantly,which located in the second place of female malignant tumors in China.With the in-depth study for the biological characteristics of breast cancer,we also have a new concept for breast cancer.Since the current study suggest that breast cancer is a systemic disease from it begin,and the treatment regimen is transited from initial simple surgical treatment to adjuvant radiotherapy and chemotherapy for surgical treatment,and then to current comprehensive treatment theory.In the comprehensive treatment,the position of dominant surgery was shaken,while endocrine therapy has suppressed chemotherapy and became one of the most important treatment ways due to the long lasting effect of the endocrine therapy.A number of studies have proved that endocrine therapy greatly improved the curative effect of early breast cancers.And it also significantly improved the life quality and extended the survival time of the patients because of the rational application of the endocrine therapy in advanced breast cancers.Oophorectomy was first used for treatment of pre-menopausal breast cancer patients by Beatson in 1896,which created a precedent for endocrine treatment of breast cancer.In 1960s,Toft isolated mouse ER protein.Tamoxifen was found to have therapeutic effects on breast cancer in 1970s.And it has been approved by FDA in 1977 for the treatment of woman with advanced breast cancer.Then it was applied for adjuvant treatment of primary breast cancer in 1986.For ER positive breast cancer patients,the recurrence rate could be decreased to 50%and the motality could be reduced by 28%by use of tamoxifen.Tamoxifen has been used to treat breast cancer for more than 30 years,which is the preferred endocrine therapy for all the ER-positive and post-menopausal breast cancers,and also has been the gold standard for endocrine therapy of breast cancer. Tamoxifen and estrogen compete to bind ER hormone binding domain,then the tamoxifen-ER complex was formed which then induced the changes of ER conformation,and closed the DNA binding domain,so that the gene transcription could not be activated,thus inhibited the estrogen's activities.Tamoxifen can be used as adjuvant endocrine therapy for ER positive breast cancer patients with node negative and/or positive.A wide range of endocrine therapy drugs has been found currently,whereas the standard treatment status of tamoxifen still can not be completely replaced.Nowadays,the characteristics of tamoxifen and ER are still the research hotspot.A large-scale randomized clinical trial confirmed that hundreds of thousands of breast cancer patients have remained disease free survival(DFS) because of using tamoxifen.Some other studies reported that postoperative adjuvant endocrine therapy is better than or equal to chemotherapy for ER positive breast cancers.However,ER positive breast cancer patients who is primary sensitive to tamoxifen may still become tamoxifen resistance.Lots of studies found that 5 years of tamoxifen treatment for ER positive breast cancer could acquire long term survival rate and its effect can be extended to 5 years after stopping the drug.However, tamoxifen could acquire resistance to breast cancer for more than 5 years of application.Only 50%of ER positive breast cancer was initially sensitive to tamoxifen therapy in the clinic,and all of them would become tamoxifen resistance, and finally resulted in tumor progress and death.Therefore,it has great clinical significance to study the resistance mechanisms of endocrine therapy and to find ways to overcome the resistance.AIB1 gene is p160 steroid receptor transcription coactivator(SRC) family mumber which is a new definite proto-oncogene discovered in recent years.Because it was first found in amplified chromosome area of human breast cancer,so it was first named to be amplified in breast cancer 1(AIB1).AIB 1 is the only amplified member of SRC family in the progression of human epithelial tumors,which can enhance the estrogen agonist activity of tamoxifen.The transfection experiments in vitro showed that P160 SRCs could enchance the activity of the nuclear recptor.Lots of studies suggested that AIB1 played an important role on many biological processes,such as cell proliferation,migration,differentiation,sexual maturation,as well as the occurrence and development of some cancers.However,at present,we have known little about the mechanism of AIB1 function.In human epithelial tumors,AIB1 is the unique overexpressed members in SRC family,which could enchance the estrogen-agonist activity of tamoxifen.AIB1 is also an important coactivator for ER, which could stimulate tumorigensis in transgenic animal models when AIB1 is overexpressed.The loss of AIB1 will influence ER signals via inhibiting its receptor degradation and deactivating transcription.As the coactivator of the ER,AIB1 was only overexpressed in ER positive breast cancer cell lines,and was not expressed in ER negative breast cancer cell lines.Clinical studies also proved that ABI1 was widely existed in ER positive breast tumors.Moreover,the expression of AIB1 was positively correlated with ERαand negatively correlated with ERβ.The binding of estrogen and ER was the main pathways to promote the proliferation of breast cancer in estrogen dependent breast cancers.However,besides the E-ER signal pathways,the EGFR family signal pathways were also another important mechanism.As one of the important EGFR family numbers,HER2 was a widely used tumor marker in clinical to predict endocrine therapy and recurrence of breast cancer. Kent Osborne has proved that AIB1 and HER2 is worked together to decrease the effect of tamoxifen therapy for breast cancer patients.AIB1 and HER2 protein levels in tumors from 316 breast cancer patients were determined using western blot analysis; they found that high AIB1 expression in patients who had not received adjuvant tarnoxifen therapy was associated with better prognosis and longer DFS.In contrast, for patients who had received tamoxifen therapy,high AIB1 expression was associated with worse DFS,which is indicative of tamoxifen resistance.In multivariable analyses of these primarily ER positive patients,AIB1 expression was an even more important predictor of tamoxifen responsiveness than expression of PR or HER2.Another study suggests that the antagonist activity of tamoxifen is reduced and its agonist activity is enhanced by the phosphorylation of AIB1 and ER via activating MAPK by HER2 in the patients with high level of AIB1 and HER2,which resulted in tamoxifen resistance.In this situation,tamoxifen not only fails to provide the protective antitumor signals but may instead even stimulate the tumor because of its agonist activity.A recent report showed that the expression of AIB1 in HER1-3 positive breast cancers would predict early relapse and death of the breast cancers, which acquires important guiding significance for the endocrine therapy and prognosis of breast cancers.These studies showed that AIB1 played an important role in antiestrogen-resistant breast tumors.However,a unifying mechanism of how AIB1 alters tamoxifen activity from an antagonist to an agonist has not been elucidated.The purpose of the present study was to identify the mechanism for the tamoxifen resistance displayed by ER positive tumors that express high levels of both AIB1 and HER2.We examined the effect of AIB1 on estrogen agonist activity of Tam-bound ER in tumor cells induced by tamoxifen,in order to take the experimental basis for clinical application.Partâ… Construction and identification of RNAi eukaryotic expression vectors AIB1ObjectiveAIB1 specific shRNA expressing vector is constructed by the technique of gene engineer,so as to study its inhibition role on AIB1 gene in breast cancer BT474 cell line.Methods(1) Four chains of oligonucleotides were synthesized correspondingly according to the AIB1-specific siRNA sequence and the negative control sequence;(2) The complementary 4 oligonucleotides chains were phosphorylated,and then the complementary double strands were formed after the annealing;(3) The linerarization of plasmid vector pGenesil-1-U6 was formed after the digestion by both BamHI and Hindâ…¢enzyme;(4) The complementary template strand of siRNA and the linear plasmid vector was ligated by T4 DNA ligating enzyme;(5) The competence cell was prepared by the bacillus coli DH5α,and the recombinant plasmids were transformed into the competence;(6) The bacillus coli were amplified,and the depurated plasmid was extracted from the bacterium by the plasmid extraction kit;(7) The extracted plasmids DNA were performed for electrophoresis under agarose gel,the concentration and purity of the plasmids were analyzed by ultraviolet photometer;(8) The recombinant plasmids were identified by SalI enzyme digestion,and the digested products were performed for electrophoresis under agarose gel;(9) The identified positive bacterium was sent to Wuhan Crystal Biotech Company for DNA sequencing.Results(1) The constructed pGenesil-shAIB1 and pGenesil-shControl plasmid expression vector was digested by SalI enzyme and performed for agarose gel electrophoresis,a 400bp of DNA band can be seen under the ultraviolet,which showed that the constructed plasmid vector was identical to the design requirement;(2) The enzyme-identified positive bacterium was sent to Wuhan Crystal Biotech Company for DNA sequencing,the results showed that the inserted segments in the recombinant plasmids were in accordance with the designed sequences.ConclusionsWe have successfully constructed the RNAi expression vector of target gene pGenesil- shAIB1 and negative control pGenesil-shControl.Partâ…¡Role of AIB1 for tamoxifen resistance in ER-positive breast cancer cellsObjective(1) To explore the role of AIB1-specific RNAi expression vector on inhibition of AIBI gene in human breast cancer BT474 cells;(2) To observe the changes of biological characteristics of BT474 cells after the AIB1 is silenced by RNAi,and to explore the mechanisms of AIB1 on tamoxifen resistance in ER positive breast cancer cells by evaluating the expression of AIB1,ERα,HER2 and pS2 proteins in BT474 cells.Methods(1) BT474 cell line is cultured routinely,and the pshAIB1 and pshControl vector was transfected into the BT474 cells by Lipofectamine 2000.The cells with stable expression of AIB1 shRNA was screened by G418.(2) The expression level of AIB1 mRNA and protein was analyzed in BT474, BT474/shAIB1 and BT474/shControl cells by RT-PCR and Western blot analysis.(3) The cells were counted using a Vi-cell XR automated cell viability analyzer according to the trypan blue dye exclusion method,and then drew the cell growth curves.(4) The cell proliferation of MCF-7,BT474,BT474/shAIB1 and BT474/shControl cells were determined in the presence of different concentrations of tamoxifen,so as to identify the tamoxifen responsiveness of these cells.And the changes of estradiol sensitivity on the cells were determined between pre- and post-RNAi.(5) The cell cycle distribution of the BT474/shAIB1 and BT474/shControl cells was assessed in absence or presence of estradiol by flow cytometry.(6) The expression of AIB1,ERa,HER2 and pS2 protein in BT474/shAIB1 and BT474/shControl cells was evaluated in the presence of Tam and E₂ by Western blot analysis.Results(1) The vector was successfully transfected into the BT474 cells by Lipofectamine 2000 and the positive clone cell lines was screened by G418.(2) Compared to BT474 and BT474/shControl cells,the expression of AIB1 mRNA and protein was significantly decreased in BT474/shAIB1 cells,while their expression level has no difference between BT474 and BT474/shControl cells.(3) The cell growth curve showed that there is no significant difference between on the cell growth of BT474 and BT474/shControl cells(p＞0.05);while the growth of BT474/shAIB1 cells was significantly reduced compared to the other two groups (p＜0.05).(4) ER-positive breast cancer BT474 cells are primary resistance to tamoxifen contrasted by the tamoxifen-sensitive MCF-7 cells.BT474/shControl cells become sensitive to tamoxifen with the inhibition of AIB1;whereas the BT474/shControl cells which transfected negative control vector is still resistance to tamoxifen.(5) The cell cycle analysis showed that the cell entry into the S phase was strongly inhibited in BT474/shAIB1 cells compared with the BT474/shControl cells both in the absence or presence of E₂.(6) Western bolt analysis showed that the expression level of ERαwas comparable in BT474/shControl and BT474/shAIB1 cells;E₂ treatment resulted in degradation of ERα,whereas tamoxifen stabilized the receptor.When the AIB1 levels were reduced by RNAi,ERαwas stabilized in the presence of E₂.The antiestrogen tamoxifen upregulated the expression of AIB1 in both of the BT474/shControl and BT474/shAIB1 cells.Compared to BT474/shControl cells,the BT474/shAIB1 cells expressed a considerable level of HER2.There are also no significant changes in HER2 expression in E₂ or tamoxifen treated cells.Knockdown of AIB1 resulted in a concomitant loss of expression of pS2 in BT474 cells.E₂ treatment increased pS2 expression in both of the BT474/shControl and BT474/shAIB1 cell lines,and tamoxifen alone has no significant effect on pS2 protein expression.Conclusions(1) The constructed RNAi expression vector to AIB1 was demonstrated to have inhibiting effect on the expression of AIB1 mRNA and protein in breast cancer BT474 cells;(2) AIB1 gene is intimate associated with the cell growth of BT474 cells,and the effect is hormone independent;the role of AIB1 on stimulating the cell growth is partly caused by accelerating the cell cycle progression.(3) Tamoxifen behaves like an estrogen agonist in ER-positive breast cancer cells that express high levels of both AIB1 and HER2,resulting in de novo resistance. Knockdown of AIB1 can eliminate this cross talk and restore the antitumor effects of tamoxifen.