HSP90 Inhibitor AUY922 Can Reverse Fulvestrant Induced Feedback Reaction In Human Breast Cancer Cells

Background and Objective: Hormone therapy has been one of the most important treatment strategy in breast cancer,and it can improve the survival significantly in ER+ breast cancer patients[1].fulvestrant is one of those drugs[2].Proteomics assay has showed that,PI3K/AKT signal pathway is overexpressed in long term estrogen deprived cells(LTED cells)due to the activation of Erb B family members.And others has also found that PI3K/AKT signal pathway has been activated in hormone therapy resistance breast cells[3-7],thus promotes cell growth.However,only EGFR inhibitor gefitinib can't reverse the feedback activation of AKT in hormone resistance cells[8].The cell survival and drug resistance in cancer cells seems more complexed when considering the network of signal pathways.The effect seems limited or none when trying to kill cancer cells with only one single agent or targeting only one protein or gene.Therefore,targeting multi critical proteins simultaneously which related in cell growth seems more promising in the future treatment.HSP90 is a universally expressed chaperone protein in cells helping protein folding.Evidence shows that the expression of HSP90 in tumor tissue is 2-3 times higher than that in normal tissue,and the high expression is related with adverse survival[9,10].It is the same condition in breast cancer patients,and high expressed HSP90 is also related with HER2+,ER+,large tumor size and lymph nodes positive[11].Moreover,HSP90 has been found invovled in hormone therapy resistance[12].AUY922 is a second generation of HSP90 inhibitor,which can bind to ATP-binding domain competitively,as a consequence,many key proteins in the cells can't be folded properly and thus lost their functions.Although fulvestrant's feedback activation is the main cause of hormone resistance,and many proteins may relate in this process,there is no research about if AUY922 can reverse these feedback effects.We propose that AUY922 can reverse this feedback activation after Fulvestrant treatment and hope this will become the molecular mechanism for a new therapeutic strategy.Methods: 1.Western blotting was used to test the ER changes,the feedback activation of Erb B family and downstream PI3K/AKT and ERk pathway in MCF-7 and T47 D cells after fulvestrant treatment.RT-PCR was also used to test the m RNA changes in Erb B family after treatment of fulvestrant.2.Western blotting was used to test AUY922 could inhibit PI3K/AKT and ERk pathway.3.Western blotting was used to test if AUY922 could reverse the feedback reaction of Erb B family and downstream PI3K/AKT and ERK pathway after treatment of fulvestrant.4.MTT assay and clone forming assay were used to test combination effects of these two drugs in cell growth inhibition.Combination effect in cell cycle arrest was tested via Flow cytometry.5.Western blotting was used in human tumor assay was used to consolidate the results in breast cancer cell lines that combination of AUY922 could reverse feedback activation of fulvestrant.Results: 1.ER was degraded in MCF-7 and T47 D cells after 48 h treatment of fulvestrant.2.Erb B family members were reactivated and phosphorylated Erb B were increased in MCF-7 and T47 D cells after 48 h treatment of fulvestrant.3.AUY922 could inhibit the activity of HSP90 in MCF-7 and T47 D cells,while fulvestrant had no influence on HSP90.Moreover,AUY922 could inhibit PI3K/AKT and ERK pathway due to the inhibition of HSP90.4.Based on the 24 h treatment of fulvestrant,combination of AUY922 successfully inhibited and reversed the feedback activation of Erb B family and downstream PI3K/AKT and ERK signal pathway.5.In the cell growth assay and clone forming assay,combination of AUY922 and fulvestrant were more effective than each alone.Moreover,the effect in cell cycle arrest was much better after combination of AUY922 and fulvestrant.6.We conducted human tumor assay to consolidate the results in breast cancer cell lines,and fulvestrant did reactivate the Erb B family and downstream PI3K/AKT pathway in human tumor tissue,and AUY922 could reverse this feedback effect.Conclusion: Generally speaking,we demonstrate Erb B receptors and downstream PI3K/AKT and ERK pathway are reactivated after treatment of Fulvestrant,and AUY922 can reverse these feedback effects in both human breast cell lines and human fresh tumor samples,which provides a new mechanism of combination use of Fulvestrant and AUY922.