| Breast cancer is the most common form of malignant disease in women in the Western World, and the numbers of breast cancer patients are growing year by year in China. Endocrine therapy is the most effective systemic treatment for patients with ERα-positive breast cancer. Tamocifen is now a standard component of front-line thereay for ER+ breast cancers, inducing remissions of over half of patients treated at first presentation, however, most tumors eventually become resistant to it with the progression of the disease. The major clinical problem in endocrine therapy is tumor resistance. Recently, the cross-talk between ERαsignaling and signal transduction pathways has been shown to be associated with endocrine resistance. Therefore, depletion of ERαin order to block cross talk between ERαand signal transduction, is maybe a new target for effective therapeutic strategies for tumor resistance.Molecular chaperone Hsp90 is responsible for the stability of special substrate proteins which are targets of anti-cancer treatments because these proteins are associated with malignant behavior. It has been shown that inhibition of Hsp90 function results in degradation of Hsp90 client proteins. Therefore, Hsp90 has been an ideal target for anti-cancer therapy. Her-2 as a member of tyrosine kinase overexpresses in many types of human cancer. It has been demonstrated that overexpression of Her-2 increases metastatic potential and resistance to anti-cancer agents, and is associated with a poor prognosis. Previous reports showed that Hsp90 inhibitors induces Her-2 dissociation with Hsp90 by blocking Hsp90 function and CHIP, cochaperone of Hsp90, a E3 ligase, mediates Hsp90 inhibitor-induced Her-2 degradation by proteasome pathway. HDAC inhibitors are regarded as one of the most promising classes of new anticancer agents with minimal toxicity in normal cells. HDAC inhibitors, including FK228, LAQ824, and suberoylanilide hydroxamic acid (SAHA), have been in I and II clinical trials and exert significant anti-cancer effects. Treatment with HDACi such as TSA has been shown to down-regulate ERαin MCF-7 cells. However, the mechanisms by which HDAC inhibitors mediate depletion of ERαin MCF-7 cells have not been fully elucidated. In our study, the data showed that SAHA exerts anti-breast cancer effect through depletion of ERαby ubiquitin-proteasome pathway.Histone deacetylase inhibitor, a new class of anti-cancer agents, has been in phase I/II of clinical trials including FK228, LAQ824 and SAHA. HDAC inhibitors have been recognized for their antiproliferative and apoptotic effects on cancer cells, which are mediated in part by selective alternation in genes expression. Recently, treatment with HDAC inhibitors was also shown to induce acetylation of Hsp90, which inactivates its ATP binding and chaperone association with its client proteins, including Her2, AKT, c-Raf.ERa is one of Hsp90 substrate proteins, as well as Her-2. It has been reported that HDACi TSA depletes ERαprotein in MCF-7 cells, however, the mechanism of TSA inducing ERαdegradation is not clear. In our studies, we also confirmed that HDACi SAHA depletes ERαeffectively, and inducing ERαdegradation via ubiqutin-proteasome. Our data showed that treatment with SAHA effectively inhibited cell proliferation and induced apoptosis of MCF-7 cells.The aim of our studies is to reveal the molecular mechanism of SAHA inducing ERαdegradation and killing breast cancer MCF-7 cells and to investigate whether E3 ligase CHIP participates in SAHA-induced ERαdegradation.Results:1. FK228 inhibits MCF-7 cells growth in a time-dependent manner by MTT assay as well as increases the percentage of cells in G2/M phase of the cell cycle by flow cytometry assay and the morphological change of MCF-7 cells is observed by Wright-Giemsa staining.2. The molecular mechanism of SAHA-inducing ERαdegradation. 1) SAHA depletes the levels of ERα;Treatment with SAHA depletes the mRNA and protein levels of ERα, and SAHA induces ERαdegradation via ubiquitin-proteasome by coimmunoprecipitation. Accompanied with down-regulation of ERα, transcription activity of ERαalso is decreased.2) SAHA induces Hsp90 acetylation and inactivates Hsp90 chaperone in MCF-7 cells which leads to dissociation of ERα-Hsp90 complex. SAHA also decreases the levels of client proteins of Hsp90 including Raf-1,Cdk4 and Survivin.3) FK228 inhibits MCF-7 cells growth and induces apoptosis by inactivation of Raf-Mek-Erk and PI3K/Akt survival signal pathways; SAHA decreases the levels of client proteins of Hsp90 and inhibition of p-AKT and p-ERK, leads to block PI3K-Akt and Raf-Mek-Erk signaling, inducing apoptosis of MCF-7 cells.3. E3 ligase CHIP participates in SAHA-induced ERαdegradation.Transient transfection and stable transfection shows that CHIP could induces ERαdegradation, however, overexpression of CHIP doesn't alter the transcription of ERα.4. E3 ligase CHIP enhances SAHA-induced ERαdegradation.To further investigate the role of CHIP in SAHA-induced ERαdegradation, we established MCF-7 breast cancer cell lines stably expressing CHIP and two CHIP mutants. We chose stable cell line MCF-7WT as model which expresses high levels of CHIP. Coimmunoprecipitation analysis showed that treatment of SAHA enhanced the association of ERα-CHIP complex and CHIP promtes ERαubiqutination. The levels of ERαis lower in MCF-7-CHIP cells than that in other two mutants transfectants, which indicates that CHIP exerts its E3 ligase activity depending on its TPR and U-box domain. Taken together, the results indicated that CHIP mediates SAHA-induced ERαdegradation.5. The effects of ligands on SAHA-induced CHIP- ERαinteraction.We investigated whether ligand binding could interfere with SAHA-induced CHIP-ERαinteraction. Coimmunoprecipitation analysis showed that Estrodial(E2) and tamocifen(4-OHT) reduce association of CHIP- ERα, but ICI182,780(ICI) enhances this association. These results indicated that distinct downstream pathways exist for ERαdegradation.Conclusions:1. Treatment with SAHA depletes the mRNA and protein levels of ERα, and induces ERαdegradation via ubiquitin-proteasome. Accompanied with down-regulaton of ERα, transcription activity of ERαalso is decreased;2. SAHA inactivates Hsp90 chaprerone by inducing acetylation of Hsp90, leading to degradation of its client protein and inhibition of p-AKT and p-ERK. Disruption of PI3K-Akt and Raf-Mek-Erk results in cytostasia and apoptosis;3. The mechanism of SAHA killing MCF-7 cells is maybe through two pathways, including inducing ERαdegradation as well as repressing the transcriptional activity and blocking the survival signaling pathway;4. E3 ligase CHIP enhances SAHA-induced ERαdegradation in MCF-7 cells.
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