The Role Of Androgen Receptor In Fulvestrant Resistance In Estrogen Receptor-positive Breast Cancer Cells

1.Background and ObjectiveBreast cancer is currently the top malignant tumor among women worldwide.Estrogen receptor（ER）-positive breast cancer accounts for nearly 75%of all types of breast cancer.In past decades,the development of endocrine therapy,which specifically targets at ER,has reshaped the treatment of ER-positive breast cancer.Fulvestrant,the first selective ER down-regulator （SERD）,has been demonstrated potent in treating ER-positive breast cancer.By down-regulating and degrading ER,it can effectively block ER pathway and inhibit activation of ER pathway. However,despite the potency of endocrine therapy,the treatment for ER-positive advanced and/or metastatic breast cancer still remains as a challenge.Secondary resistance to Fulvestrant is one of the difficulties in treating ER-positive advanced and/or metastatic breast cancer. Therefore,in order to develop corresponding targeted therapy to better treat these patients,it is urgent to understand the underlying mechanisms that may be involved in secondary resistance to Fulvestrant. In this study,ER-positive Fulvestrant-resistant breast cancer cells were established by being cultured in long-term estrogen-deprived condition with Fulvestrant.We found that the androgen receptor（AR）pathway in Fulvestrant-resistant cells was activated,while ER pathway was inhibited.A blockade of AR in Fulvestrant-resistant cells could increase the sensitivity to Fulvestrant,and dual inhibition of AR and ER was more effective than blocking either pathway alone.Furthermore,we observed that Fulvestrant-resistant cells exhibited a mesenchymal-like appearance,indicating a potential epithelial-mesenchymal transition（EMT）.Verified by m RNA expression profiles,significant changes in cell adhesion-related biological process were also observed.SOX9,a transcription factor associated with cell adhesion change,was predicted to be involved with AR.Further experiments verified AR,by upregulating SOX9,could promote cell migration and proliferation.This study aims to reveal the role of AR in Fulvestrant resistance and provides an insight into potential clinical treatment for ER-positive breast cancer resistant to Fulvestrant.2.Materials and Methods1.Establish Fulvestrant-resistant breast cancer cells and detect its IC₅₀The ER-positive Fulvestrant-resistant breast cancer cells was established in long-term estrogen-deprived culture medium containing Fulvestrant.By using CCK-8,we detected the cell viability of both Fulvestrant-resistant and sensitive breast cancer cells in the condition with Fulvestrant,and calculated the IC₅₀ to see if there was any significant difference.2.Explore changes of ER and AR pathways in Fulvestrant-resistant cellsqRT-PCR and Western blot were used to detect the expressions of ER and AR,as well as their downstream proteins in Fulvestrant-resistant and sensitive cells.Both Fulvestrant-resistant and sensitive cells were treated with Dihydrotestosterone（DHT）and Bicalutamide to see the effects of these two drugs on cells.3.Explore Bicalutamide’s effect on Fulvestrant-resistant cellsFulvestrant-resistant cells were treated with different concentrations of Bicalutamide,and the sensitivity to Fulvestrant after being treated by Bicalutamide was measured by CCK-8 method.4.Detect the combined effects of Bicalutamide and FulvestrantFulvestrant-resistant cells were treated with different concentrations of both Bicalutamide and Fulvestrant.Cell viability after treatment was detected by CCK-8 method.The combination index（CI）of Bicalutamide and Fulvestrant was calculated using Compu Syn software,and a value below 1 is considered synergistic.Flow cytometry was used to detect the effects of Bicalutamide and Fulvestrant on cell apoptosis.5.Predict the molecules that might be involved in AR-related EMT in Fulvestrant-resistant cells Morphological changes of Fulvestrant-resistant cells were observed under microscope,and expressions of EMT-related proteins in Fulvestrant-resistant and sensitive cells were detected by Western blot.Top 10 up-and down-regulated proteins associated with cell adhesion regulation were selected based on m RNA expression profile.String prediction algorithm（http://string- db.org）was used to predict if any molecules among the 20 proteins might interact with AR.6.Explore the regulation between AR and SOX9 in ER-positive breast cancer Western blot was used to verify SOX9 expression in Fulvestrant-resistant cells.Fulvestrant- resistant cells were treated with DHT and Bicalutamide respectively,and Western blot was used to detect the expressions of AR and SOX9.Fulvestrant-resistant cells were transfected with siRNA（Small interfering RNA）targeting SOX9.QRT-PCR and Western blot were used to detect the expressions of AR and/or SOX9.7.Verify SOX9’s effects on cell migrationFulvestrant-sensitive cells were transfected with overexpressed SOX9.Wound healing assay and transwell assay validated the effect of SOX9 overexpression on cell migration.8.Explore SOX9’s effects on cell proliferation and cell cycleAfter SOX9 overexpression or SOX9 interference,cell proliferation of Fulvestrant-sensitive and resistant cells on days 1,3 and 5 was detected by CCK-8 assay,and cell cycle was detected using cell cycle assay.3.Results1.The ER-positive Fulvestrant-resistant breast cancer cells was established in long-term estrogen-deprived culture medium containing Fulvestrant.IC₅₀ of the Fulvestrant-resistant and sensitive breast cancer cells was detected by CCK-8.IC₅₀ of Fulvestrant-resistant cells was found significantly higher than that of Fulvestrant-sensitive breast cancer cells（P<0.05）.2.qRT-PCR and Western blot were used to detect expressions of ER and its downstream proteins in both the resistant and sensitive cells.It was found that,in resistant cells,m RNA and protein expressions of ER both decreased,as well as the expressions of ER downstream proteins;however,the expressions of AR and its downstream protein both increased,with an increase of nuclear translocation of AR.Resistant and sensitive cells were then both treated with DHT or Bicalutamide.After DHT treatment,the expression of AR in both cells incresead;however, while DHT increased cell proliferation in the resistant cells,it inhibited cell proliferation in the sensitive cells.After the treatment with Bicalutamide,the expression of AR in both cells decreased,with an inhibition of cell proliferation in the resistant cells and an increase of cell proliferation in the sensitive cells.3.Different concentrations of Bicalutamide were used to treat Fulvestrant-resistant cells.CCK-8assay showed that Bicalutamide increased the responsiveness to Fulvestrant in resistant cells.As the concentrations of Bicalutamide increased,the inhibitory effects of Fulvestrant on cell proliferation was also enhanced.4.The resistant cells were treated with different concentrations of Bicalutamide and Fulvestrant.The CCK-8 method showed that the combination was more effective than either drug alone in terms of inhibiting cell proliferation.The CI values of Bicalutamide and Fulvestrant calculated by Compu Syn software were mostly found less than 1,which indicated that there was a synergistic effect between Bicalutamide and Fulvestrant.Flow cytometry also showed that the combination significantly increased cell apoptosis compared with Fulvestrant monotherapy.5.By observing the cell morphology of the resistant cells,we found these cells exhibited a mesenchymal-like appearance.Western blot revealed that in resistant cells,E-cadherin was down-regulated,while N-cadherin,Snail and Vimentin were up-regulated.The m RNA expression profile showed significant changes in the cell adhesion-related biological process in resistant cells.Top ten up-and down-regulated cell adhesion-associated proteins were selected for further study.String prediction algorithm was used to predict if there was any association between AR and these proteins,and it was found there might be a correlation between SOX9 and AR.6.Western blot confirmed that the expression of SOX9 in resistant cells was significantly increased.After the treatment with DHT or Bicalutamide,AR and SOX9 expressions changed in tandem in the resistant cells,that is,either simultaneously increased or decreased.Interfering the expression of SOX9 using si SOX9 did not significantly change AR expression,showed by Western blot.7.The wound healing assay and transwell assay showed that cell migration was significantly enhanced in cells overexpressed with SOX9.8.Cell proliferation in SOX9-overexpressed cells and SOX9-interferred cells on days 1,3 and 5was examined by CCK-8.It was found that overexpressing SOX9 significantly increased cell proliferation and interfering SOX9 significantly inhibited cell proliferation.No significant changes were observed in cell cycle after either SOX9 overexpression or interference.4.Conclusion4.1.Activation of AR pathyway,rather than ER pathway,contributed to the proliferation of Fulvestrant-resistant ER-positive breast cancer cells;4.2.Inhibiting AR could restore the sensitivity to Fulvestrant in ER-positive breast cancer cells;dual inhibition of AR and ER was more effective than inhibiting either pathway alone;4.3.AR could further promote cell migration and proliferation by upregulating SOX9.