Histone deacetylase inhibitor, trichostatin A induces ubiquitin-dependent cyclin D1 degradation in MCF-7 breast cancer cells.
ABSTRACT: Cyclin D1 is an important regulator of G1-S phase cell cycle transition and has been shown to be important for breast cancer development. GSK3beta phosphorylates cyclin D1 on Thr-286, resulting in enhanced ubiquitylation, nuclear export and degradation of the cyclin in the cytoplasm. Recent findings suggest that the development of small-molecule cyclin D1 ablative agents is of clinical relevance. We have previously shown that the histone deacetylase inhibitor trichostatin A (TSA) induces the rapid ubiquitin-dependent degradation of cyclin D1 in MCF-7 breast cancer cells prior to repression of cyclin D1 gene (CCND1) transcription. TSA treatment also resulted in accumulation of polyubiquitylated GFP-cyclin D1 species and reduced levels of the recombinant protein within the nucleus.Here we provide further evidence for TSA-induced ubiquitin-dependent degradation of cyclin D1 and demonstrate that GSK3beta-mediated nuclear export facilitates this activity. Our observations suggest that TSA treatment results in enhanced cyclin D1 degradation via the GSK3beta/CRM1-dependent nuclear export/26S proteasomal degradation pathway in MCF-7 cells.We have demonstrated that rapid TSA-induced cyclin D1 degradation in MCF-7 cells requires GSK3beta-mediated Thr-286 phosphorylation and the ubiquitin-dependent 26S proteasome pathway. Drug induced cyclin D1 repression contributes to the inhibition of breast cancer cell proliferation and can sensitize cells to CDK and Akt inhibitors. In addition, anti-cyclin D1 therapy may be highly specific for treating human breast cancer. The development of potent and effective cyclin D1 ablative agents is therefore of clinical relevance. Our findings suggest that HDAC inhibitors may have therapeutic potential as small-molecule cyclin D1 ablative agents.
Project description:Histone deacetylase inhibitors (HDACIs) have been shown to induce apoptotic and autophagic cell death in vitro and in vivo. The molecular mechanisms that underlie these cytotoxic effects are not yet clearly understood. Recently, HDACIs were shown to induce Akt dephosphorylation by disrupting HDAC-protein phosphatase 1 (PP1) complexes. This disruption results in the increased association of PP1 with Akt, resulting in the dephosphorylation and consequent inactivation of the kinase. Akt enhances cellular survival through the phosphorylation-dependent inhibition of several pro-apoptotic proteins. Akt is an important negative regulator of GSK3beta, a kinase that has been shown to regulate apoptosis in response to various stimuli. In the present study, we investigated the role of GSK3beta in mediating the cytotoxic effects in MCF-7 breast cancer cells treated with trichostatin A (TSA), a prototype HDACI. We show that TSA induces Akt dephosphorylation in a PP1-dependent manner, resulting in activation of GSK3beta in MCF-7 cells. Similarly, knockdown of HDAC1 and-2 by small interfering RNA (siRNA) resulted in the dephosphorylation of Akt and GSK3beta. Selective inhibition of GSK3beta attenuated TSA induced cytotoxicity and resulted in enhanced proliferation following drug removal. Our findings identify GSK3beta as an important mediator of TSA-induced cytotoxicity in MCF-7 breast cancer cells.
Project description:Ubiquitin-dependent proteolysis of cyclin D1 is associated with normal and tumor cell proliferation and survival. The SCFFBXO31 (Skp1-Cul1-Rbx1-FBXO31) ubiquitin ligase complex mediates genotoxic stress-induced cyclin D1 degradation. Previous studies have suggested that cyclin D1 levels are maintained at steady state by phosphorylation-dependent nuclear export and subsequent proteolysis in the cytoplasm. Here we present the crystal structures of the Skp1-FBXO31 complex alone and bound to a phosphorylated cyclin D1 C-terminal peptide. FBXO31 possesses a unique substrate-binding domain consisting of two ?-barrel motifs, whereas cyclin D1 binds to FBXO31 by tucking its free C-terminal carboxylate tail into an open cavity of the C-terminal FBXO31 ?-barrel. Biophysical and functional studies demonstrate that SCFFBXO31 is capable of recruiting and ubiquitinating cyclin D1 in a phosphorylation-independent manner. Our findings provide a conceptual framework for understanding the substrate specificity of the F-box protein FBXO31 and the mechanism of FBXO31-regulated cyclin D1 protein turnover.
Project description:Bazedoxifene (BZA) is a third-generation selective estrogen receptor modulator (SERM) that has been approved for the prevention and treatment of postmenopausal osteoporosis. It has antitumor activity; however, its mechanism of action remains unclear. In the present study, we characterized the effects of BZA and several other SERMs on the proliferation of hormone-dependent MCF-7 and T47D breast cancer cells and hormone-independent MCF-7:5C and MCF-7:2A cells and examined its mechanism of action in these cells. We found that all of the SERMs inhibited the growth of MCF-7, T47D, and MCF-7:2A cells; however, only BZA and fulvestrant (FUL) inhibited the growth of hormone-independent MCF-7:5C cells. Cell cycle analysis revealed that BZA and FUL induced G(1) blockade in MCF-7:5C cells; however, BZA down-regulated cyclin D1, which was constitutively overexpressed in these cells, whereas FUL suppressed cyclin A. Further analysis revealed that small interfering RNA knockdown of cyclin D1 reduced the basal growth of MCF-7:5C cells, and it blocked the ability of BZA to induce G(1) arrest in these cells. BZA also down-regulated estrogen receptor-? (ER?) protein by increasing its degradation and suppressing cyclin D1 promoter activity in MCF-7:5C cells. Finally, molecular modeling studies demonstrated that BZA bound to ER? in an orientation similar to raloxifene; however, a number of residues adopted different conformations in the induced-fit docking poses compared with the experimental structure of ER?-raloxifene. Together, these findings indicate that BZA is distinct from other SERMs in its ability to inhibit hormone-independent breast cancer cell growth and to regulate ER? and cyclin D1 expression in resistant cells.
Project description:The transcription factor CCAAT/enhancer binding protein delta (C/EBPdelta, CEBPD, NFIL-6beta) has tumor suppressor function; however, the molecular mechanism(s) by which C/EBPdelta exerts its effect are largely unknown. Here, we report that C/EBPdelta induces expression of the Cdc27 (APC3) subunit of the anaphase promoting complex/cyclosome (APC/C), which results in the polyubiquitination and degradation of the prooncogenic cell cycle regulator cyclin D1, and also down-regulates cyclin B1, Skp2, and Plk-1. In C/EBPdelta knockout mouse embryo fibroblasts (MEF) Cdc27 levels were reduced, whereas cyclin D1 levels were increased even in the presence of activated GSK-3beta. Silencing of C/EBPdelta, Cdc27, or the APC/C coactivator Cdh1 (FZR1) in MCF-10A breast epithelial cells increased cyclin D1 protein expression. Like C/EBPdelta, and in contrast to cyclin D1, Cdc27 was down-regulated in several breast cancer cell lines, suggesting that Cdc27 itself may be a tumor suppressor. Cyclin D1 is a known substrate of polyubiquitination complex SKP1/CUL1/F-box (SCF), and our studies show that Cdc27 directs cyclin D1 to alternative degradation by APC/C. These findings shed light on the role and regulation of APC/C, which is critical for most cellular processes.
Project description:Objective: The tyrosine phosphatase SHP2 has a dual role in cancer initiation and progression in a tissue type-dependent manner. Several studies have linked SHP2 to the aggressive behavior of breast cancer cells and poorer outcomes in people with cancer. Nevertheless, the mechanistic details of how SHP2 promotes breast cancer progression remain largely undefined. Methods: The relationship between SHP2 expression and the prognosis of patients with breast cancer was investigated by using the TCGA and GEO databases. The expression of SHP2 in breast cancer tissues was analyzed by immunohistochemistry. CRISPR/Cas9 technology was used to generate SHP2-knockout breast cancer cells. Cell-counting kit-8, colony formation, cell cycle, and EdU incorporation assays, as well as a tumor xenograft model were used to examine the function of SHP2 in breast cancer proliferation. Quantitative RT-PCR, western blotting, immunofluorescence staining, and ubiquitination assays were used to explore the molecular mechanism through which SHP2 regulates breast cancer proliferation. Results: High SHP2 expression is correlated with poor prognosis in patients with breast cancer. SHP2 is required for the proliferation of breast cancer cells in vitro and tumor growth in vivo through regulation of Cyclin D1 abundance, thereby accelerating cell cycle progression. Notably, SHP2 modulates the ubiquitin-proteasome-dependent degradation of Cyclin D1 via the PI3K/AKT/GSK3? signaling pathway. SHP2 knockout attenuates the activation of PI3K/AKT signaling and causes the dephosphorylation and resultant activation of GSK3?. GSK3? then mediates phosphorylation of Cyclin D1 at threonine 286, thereby promoting the translocation of Cyclin D1 from the nucleus to the cytoplasm and facilitating Cyclin D1 degradation through the ubiquitin-proteasome system. Conclusions: Our study uncovered the mechanism through which SHP2 regulates breast cancer proliferation. SHP2 may therefore potentially serve as a therapeutic target for breast cancer.
Project description:Growth factor-dependent accumulation of the cyclin D1 proto-oncogene is balanced by its rapid phosphorylation-dependent proteolysis. Degradation is triggered by threonine 286 phosphorylation, which promotes its ubiquitination by an unknown E3 ligase. We demonstrate that Thr286-phosphorylated cyclin D1 is recognized by a Skp1-Cul1-F box (SCF) ubiquitin ligase where FBX4 and alphaB crystallin govern substrate specificity. Overexpression of FBX4 and alphaB crystallin triggered cyclin D1 ubiquitination and increased cyclin D1 turnover. Impairment of SCF(FBX4-alphaB crystallin) function attenuated cyclin D1 ubiquitination, promoting cyclin D1 overexpression and accelerated cell-cycle progression. Purified SCF(FBX4-alphaB crystallin) catalyzed polyubiquitination of cyclin D1 in vitro. Consistent with a putative role for a cyclin D1 E3 ligase in tumorigenesis, FBX4 and alphaB crystallin expression was reduced in tumor-derived cell lines and a subset of primary human cancers that overexpress cyclin D1. We conclude that SCF(FBX4-alphaB crystallin) is an E3 ubiquitin ligase that promotes ubiquitin-dependent degradation of Thr286-phosphorylated cyclin D1.
Project description:This study identifies a novel mechanism by which thiazolidinediones mediate cyclin D1 repression in prostate cancer cells. Based on the finding that the thiazolidinedione family of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists mediated PPARgamma-independent cyclin D1 degradation, we developed a novel PPARgamma-inactive troglitazone derivative, STG28, with high potency in cyclin D1 ablation. STG28-mediated cyclin D1 degradation was preceded by Thr-286 phosphorylation and nuclear export, which however, were independent of glycogen synthase kinase 3beta. Mutational analysis further confirmed the pivotal role of Thr-286 phosphorylation in STG28-induced nuclear export and proteolysis. Of several kinases examined, inhibition of IkappaB kinase alpha blocked STG28-mediated cytoplasmic sequestration and degradation of cyclin D1. Pulldown of ectopically expressed Cul1, the scaffold protein of the Skp-Cullin-F-box E3 ligase, in STG28-treated cells revealed an increased association of cyclin D1 with beta-TrCP, whereas no specific binding was noted with other F-box proteins examined, including Skp2, Fbw7, Fbx4, and Fbxw8. This finding represents the first evidence that cyclin D1 is targeted by beta-TrCP. Moreover, beta-TrCP expression was up-regulated in response to STG28, and ectopic expression and small interfering RNA-mediated knock-down of beta-TrCP enhanced and protected against STG28-facilitated cyclin D1 degradation, respectively. Because cyclin D1 lacks the DSG destruction motif, mutational and modeling analyses indicate that cyclin D1 was targeted by beta-TrCP through an unconventional recognition site, (279)EEVDLACpT(286), reminiscent to that of Wee1. Moreover, we obtained evidence that this beta-TrCP-dependent degradation takes part in controlling cyclin D1 turnover when cancer cells undergo glucose starvation, which endows physiological relevance to this novel mechanism.
Project description:Liriodenine has wide pharmacological functions in phytochemistry, pharmacology and antitumor activities. The anticancer effects of liriodenine on the cell growth and apoptosis of human breast cancer MCF-7 cells, and the underlying mechanisms, are yet to be elucidated. Therefore, the present study investigated the anticancer effects of liriodenine on the cell growth and apoptosis of human breast cancer MCF-7 cells. We used MTT assay to measure cell growth, and flow cytometer and DAPI staining was used to analyze cell apoptosis. Then, Western blot analysis was executed to measure B-cell lymphoma-2 protein (Bcl-2), cyclin D1, vascular endothelial growth factor (VEGF), and p53 protein expression. The effect of liriodenine induced significant apoptosis and suppression of cell growth of the MCF-7 cells. Furthermore, the potential mechanism underlying its antitumor effect on MCF-7 cells may result from activation of caspase-3 activity, Bcl-2, cyclin D1 and VEGF, and promotion of p53 protein expression in MCF-7 cells. Therefore, the present results indicated that the anticancer effects of liriodenine suppress cell growth and induce the apoptosis of human breast cancer MCF-7 cells through inhibition of Bcl-2, cyclin D1 and VEGF expression, and upregulation of p53 expression. Therefore, liriodenine may be a potential therapy for the treatment of human breast cancer.
Project description:SCF(Fbx4) was recently identified as the E3 ligase for cyclin D1. We now describe cell-cycle-dependent phosphorylation and dimerization of Fbx4 that is regulated by GSK3beta and is defective in human cancer. We present data demonstrating that a pathway involving Ras-Akt-GSK3beta controls the temporal phosphorylation and dimerization of the SCF(Fbx4) E3 ligase. Inhibition of Fbx4 activity results in accumulation of nuclear cyclin D1 and oncogenic transformation. The importance of this regulatory pathway for normal cell growth is emphasized by the prevalence of mutations in Fbx4 in human cancer that impair dimerization. Collectively, these data reveal that inactivation of the cyclin D1 E3 ligase likely contributes to cyclin D1 overexpression in a significant fraction of human cancer.
Project description:The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. Here we used gene targeting to "knock in" PIK3CA mutations into human breast epithelial cells to identify new therapeutic targets associated with oncogenic PIK3CA. Mutant PIK3CA knockin cells were capable of epidermal growth factor and mTOR-independent cell proliferation that was associated with AKT, ERK, and GSK3beta phosphorylation. Paradoxically, the GSK3beta inhibitors lithium chloride and SB216763 selectively decreased the proliferation of human breast and colorectal cancer cell lines with oncogenic PIK3CA mutations and led to a decrease in the GSK3beta target gene CYCLIN D1. Oral treatment with lithium preferentially inhibited the growth of nude mouse xenografts of HCT-116 colon cancer cells with mutant PIK3CA compared with isogenic HCT-116 knockout cells containing only wild-type PIK3CA. Our findings suggest GSK3beta is an important effector of mutant PIK3CA, and that lithium, an FDA-approved therapy for bipolar disorders, has selective antineoplastic properties against cancers that harbor these mutations.