Degradation Of ERalpha Research Articles

995 New Thiazolidinedione Derivatives With PPARgamma-independent Activity – High Inhibition of Breast Cancer Cell Proliferation With Low Toxicity Towards Human Hepatocytes

Background: Breast cancer is the most frequent cancer in women. The development of de novo and acquired resistance to therapies and the absence of targeted therapy for some types of tumours are strong motivations for discovering new therapeutic agents. Thiazolidinediones (TZD) are studied in this context. These PPARg ligands have antiproliferative effects in vitro and in vivo and they were tested in clinical trials. Nevertheless compounds like troglitazone (TGZ) display hepatotoxicity. More recently, it was shown that in fact,TZD exert their anticancer effects mainly in a PPARg-independent manner. Therefore, we synthesized new TZD derivatives (D2 family) devoid of PPARg agonist activity. We studied their mechanism of action and their toxicity towards human hepatocytes before performing in vivo experiments on murine models. Material and Methods: TGZ derivatives were obtained by chemical synthesis. We used hormone-dependent (MCF-7) and hormone-independent (MDA-MB-231)breast cancer cell lines as well as primary cultures of human hepatocytes. Proliferation and toxicity were studied with the Cell titer glo and MTT assays respectively. Gene expressions were studied by microarray, RT-PCR, western blotting and immunocytochemistry. RNA interference was used for gene silencing. Results: The PPARg-independent activity of D2-TZD in breast cancer cells was associated with a very early increase in intracellular calcium (after 2 minutes) followed by ERK1/2 activation (after 5 minutes) and a transient increase in the expression of the transcription factor Early Growth Response gene 1 (maximum after 3 hours). Endoplasmic reticulum stress and proteasomal degradation of cyclin D1 and ERalpha occurred later. D2-TGZ and TGZ had similar antiproliferative efficiencies: their IC50 were 29.7 and 35.4 mM respectively in MCF7 cells and 16.6 and 15.8 mM in MDA-MB-231 cells. Among the new D2-TGZ derivatives that we obtained, some of them were more potent than TGZ and D2-TGZ with IC50 around 5 mM. Furthermore, these derivatives displayed a lower toxicity towards human hepatocytes than the parent compound TGZ (1.6-fold when used at 100 mM). Conclusions: Our work contributed to a better understanding of the PPARg-independent effects of TZD. We were able to obtain derivatives which were more potent to inhibit breast cancer cell proliferation and less toxic towards hepatocytes. This opens the possibility to start in vivo experiments in mouse models of breast cancer.

Selective Estrogen Receptor Down-Regulator and Selective Estrogen Receptor Modulators Differentially Regulate Lactotroph Proliferation

BackgroundWe recently reported that estrogen receptor α (ERα), even in absence of estrogen (E2), plays a critical role in lactotroph homeostasis. The anti-estrogen ICI 182780 (ICI), but not tamoxifen or raloxifene, rapidly promoted the degradation of ERα, and inhibited cell proliferation. However, all three ER antagonists suppressed PRL release, suggesting that receptor occupation is sufficient to inhibit prl gene expression whereas receptor degradation is required to suppress lactotroph proliferation. In this study our objective was to determine whether ERα degradation versus occupation, differentially modulates the biological outcome of anti-estrogens.Principal FindingsUsing the rat lactotroph cell line, GH3 cells, we report that ICI induced proteosome mediated degradation of ERα. In contrast, an ERα specific antagonist, MPP, that does not promote degradation of ERα, did not inhibit cell proliferation. Further, ICI, but not MPP, abolished anchorage independent growth of GH3 cells. Yet, both ICI and MPP were equally effective in suppressing prl expression and release, as well as ERE-mediated transcriptional activity.ConclusionTaken together, our results demonstrate that in lactotrophs, ERα degradation results in decreased cell proliferation, whereas ERα occupation by an antagonist that does not promote degradation of ERα is sufficient to inhibit prl expression.

A novel mechanism whereby BRCA1/1a/1b fine tunes the dynamic complex interplay between SUMO-dependent/independent activities of Ubc9 on E2-induced ERα activation/repression and degradation in breast cancer cells

BRCA1 dysfunction is associated with hormone-responsive cancers. We have identified a consensus SUMO modification site in the amino-terminal region of BRCA1/1a/1b proteins and the mutation in this potential SUMO acceptor site (K 109 to R) impaired their ability to bind and repress ligand-dependent ERalpha transcriptional activity in breast cancer cells. Furthermore, we have found SUMO E2-conjugating enzyme Ubc9 to bind BRCA1 proteins. We have mapped BRCA1 [within amino acids (aa) 1-182] as the minimum domain that is sufficient for in vitro binding to Ubc9 as well as for regulating ERalpha activity. BRCA1 Mutant #1 (K109 to R) was impaired in its ability to both bind, as well as modulate Ubc9 mediated SUMO-dependent/independent E2-induced ERalpha transcriptional activity in breast cancer cells. Similarly, BRCA1 cancer-predisposing mutation (61Cys-Gly) abrogated the ability to both bind Ubc9 as well as inhibit ERalpha activity suggesting physiological significance. Addition of BRCA1 but not Mutant #1 to E2-induced ERalpha in the presence of SUMO-1 and Ubc9 resulted in the degradation of ERalpha suggesting BRCA1 to be a putative SUMO-1 and Ubc9-dependent E3 ubiquitin ligase for ERalpha. This is the first report demonstrating the participation of Ubc9 in BRCA1 E3 ubiquitin ligase mediated degradation of ERalpha. These results suggest a novel function for BRCA1 in regulating the dynamic cycles of SUMO and ubiquitin modifications required for ERalpha turn over and deregulation of this molecular switch due to lack of BRCA1 results in ERalpha-negative/positive breast cancers. This study will help in designing novel BRCA1 function-based targeted treatment for breast cancers.

ER Re-expression and Re-sensitization to Endocrine Therapies in ER-negative Breast Cancers

Breast cancer is the leading cause of cancer amongst women in the westernized world. The presence or absence of ERalpha in breast cancers is an important prognostic indicator. About 30-40% of breast cancers lack detectable ERalpha protein. ERalpha- breast cancers are resistant to endocrine therapies and have a worse prognosis than ERalpha+ breast cancers. Since expression of ERalpha is necessary for response to endocrine therapies, investigational studies are ongoing in order to understand the generation of the ERalpha- phenotype and develop interventions to restore ERalpha expression in ERalpha- breast cancers. DNA methylation and chromatin remodeling are two epigenetic mechanisms that have been linked with the lack of ERalpha expression and in these cases; demethylation of the ERalpha promoter or treatment with HDAC inhibitors shows promise in restoring ERalpha expression in ERalpha- breast cancers. Two additional potential mechanisms underlying generation of the ERalpha- phenotype involve E6-AP and Src, both of which have been shown to be elevated in ERalpha- breast cancer and can drive the proteasomal degradation of ERalpha. Recently, studies have demonstrated that upregulated growth factor signaling due to hyperactive MAPK activity significantly contributes to generation of the ERalpha- phenotype and that inhibition of MAPK activity can cause re-expression of the ERalpha and restore sensitivity to endocrine therapies. Given the challenges in treating ERalpha- breast cancer, understanding and manipulating the cellular mechanisms that effect expression of ERalpha are imperative in order to restore sensitivity to endocrine therapies and to design novel therapeutics for the treatment of ERalpha- breast cancers.

Repression of Estrogen Receptor alpha by CDK11p58 Through Promoting its Ubiquitin-Proteasome Degradation

Estrogen receptor alpha (ERalpha) is a ligand-dependent transcription factor that mediates physiological responses to 17beta-estradiol (E(2)). These responses of cells to estrogen are regulated in part by degradation of ERalpha. In this report, we found that CDK11(p58) repressed ERalpha transcriptional activity. And we further demonstrated that ERalpha protein level was down-regulated by CDK11(p58) in mammalian cells in a ligand independent manner. This effect could be abrogated by treatment with proteasome inhibitor MG132. Our results indicated that the ubiquitin/proteasome-mediated degradation of ERalpha was promoted by CDK11(p58). Furthermore, the interaction between ERalpha and CDK11(p58) was detected. This interaction was necessary for the polyubiquitination and degradation of ERalpha. On the contrary, the other isoform of CDK11, CDK11(p110) and the kinase dead mutant of CDK11(p58), D224N, did not associate with ERalpha and failed to reduce the ERalpha protein level. These data identified a new negative regulatory protein of ERalpha and provided a new pathway by which CDK11(p58) negatively regulated cells.

Disruption of ERα signalling pathway by PPARγ agonists: evidences of PPARγ-independent events in two hormone-dependent breast cancer cell lines

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that can be activated by natural ligands such as 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ(2)) as well as synthetic drugs such as thiazolidinediones. The treatment of human breast cancer cell lines with PPARgamma agonists is known to have antiproliferative effects but the role of PPARgamma activation in the process remains unclear. In the present study, we investigated the effects of four PPARgamma agonists, Rosiglitazone (RGZ), Ciglitazone (CGZ), Troglitazone (TGZ) and the natural agonist 15d-PGJ(2), on estrogen receptor alpha (ERalpha) signalling pathway in two hormone-dependent breast cancer cell lines, MCF-7 and ZR-75-1. In both of them, TGZ, CGZ and 15d-PGJ(2) induced an inhibition of ERalpha signalling associated with the proteasomal degradation of ERalpha. ZR-75-1 cells were more sensitive than MCF-7 cells to these compounds. Treatments that induced ERalpha degradation inhibited cell proliferation after 24 h. In contrast, 24 h exposure to RGZ, the most potent activator of PPARgamma disrupted neither ERalpha signalling nor cell proliferation. 9-cis retinoic acid never potentiated the proteasomal degradation of ERalpha. PPARgamma antagonists (T0070907, BADGE and GW 9662) did not block the proteolysis of ERalpha in MCF-7 and ZR-75-1 cells treated with TGZ. ERalpha proteolysis still occurred in case of PPARgamma silencing as well as in case of treatment with the PPARgamma-inactive compound Delta2-TGZ, demonstrating a PPARgamma-independent mechanism. The use of thiazolidinedione derivatives able to trigger ERalpha degradation by a PPARgamma-independent pathway could be an interesting tool for breast cancer therapy.

Glycogen Synthase Kinase-3 Protects Estrogen Receptor α from Proteasomal Degradation and Is Required for Full Transcriptional Activity of the Receptor

Glycogen synthase kinase-3 (GSK-3) plays a key role in the regulation of transcription factors including steroid receptors. Having identified estrogen receptor-alpha (ERalpha) as substrate for GSK-3, the impact of GSK-3 on ERalpha function and activity upon 17beta-estradiol (E2)-dependent activation remains to be clarified. Here we show by using small interfering technology in combination with immunoblot, gene expression analysis, and luciferase reporter assays that silencing of GSK-3alpha or GSK-3beta results in the reduction of ERalpha levels and transcriptional activity in ERalpha-positive breast cancer cells. Using MCF-7 cells we demonstrate that reduction of ERalpha levels upon GSK-3 silencing was due to increased proteasomal degradation of ERalpha rather than inhibition of ERalpha protein synthesis. Indeed, under this condition, ERalpha protein was rescued using the proteasome inhibitor MG132 in presence of the protein synthesis inhibitor cycloheximide. In addition, strong accumulation of ubiquitinated ERalpha was obtained after GSK-3 silencing in the presence of MG132. We conclude that GSK-3 protects ERalpha from proteasomal degradation and plays a crucial role in ERalpha protein stabilization and turnover. Furthermore, in vitro kinase assay depicted that GSK-3beta phosphorylates ERalpha at Ser-118. GSK-3 silencing resulted in decrease of E2-induced nuclear ERalpha phosphorylation at Ser-118 and E2-induced estrogen response element-dependent luciferase reporter gene expression. Neither Ser-118 phosphorylation nor luciferase activity was restored by use of MG132. Moreover, the expression of estrogen-responsive genes (pS2 and progesterone receptor) was decreased upon GSK-3 silencing. These findings demonstrated that GSK-3 is required for E2-induced ERalpha phosphorylation at Ser-118 and full transcriptional activity of the receptor upon E2 stimulation.

The clock gene Per2 links the circadian system to the estrogen receptor

Circadian rhythms regulate diverse physiological processes including homeostatic functions of steroid hormones and their receptors. Estrogen receptor-alpha (ERalpha) is essential for normal mammary gland physiology and is a prognostic marker for the treatment of breast cancer. We report that Per2, a core clock gene, links the circadian cycle to the ERalpha signaling network. Binding of enhances ERalpha degradation, while suppression of Per2 levels leads to ERalpha stabilization. In turn, Per2 itself is estrogen inducible in these cells, suggesting a feedback mechanism to attenuate stimulation by estrogen. In addition, overexpression of Per2 in breast cancer cells leads to significant growth inhibition, loss of clonogenic ability and apoptosis. Taken together, these results further support a critical role for peripheral circadian regulation in tissue homeostasis and suggest a novel role for clock genes in estrogen receptor-positive breast cancer.

Estrogen treatment of spinal cord injury attenuates calpain activation and apoptosis

Spinal cord injury (SCI) is a devastating neurologic injury, and currently, the only recommended pharmacotherapy is high-dose methylprednisolone, which has limited efficacy. Estrogen is a multi-active steroid with anti-oxidant and anti-apoptotic effects. Estrogen may modulate intracellular Ca2+ and prevent inflammation. For this study, male rats were divided into three groups. Sham-group animals received a laminectomy at T12. Injured rats received both laminectomy and 40 gram centimeter force SCI. Estrogen-group rats received 4 mg/kg 17beta-estradiol (estrogen) at 15 min and 24 hr post-injury, and vehicle-group rats received equal volumes of dimethyl sulfoxide. Animals were sacrificed at 48 hr post-injury, and 1-cm segments of the lesion, rostral penumbra, and caudal penumbra were excised. The degradation of 68 kD neurofilament protein (NFP) and estrogen receptors (ER) was examined by Western blot analysis. Protein levels of calpain and the activities of calpain and caspase-3 were also examined. Levels of cytochrome c were determined in both cytosolic and mitochondrial fractions. Cell death with DNA fragmentation was examined using the TUNEL assay. At the lesion, samples from both vehicle and estrogen treated animals showed increased levels of 68 kD NFP degradation, calpain content, calpain activity, cytochrome c release, and degradation of ERalpha and ERbeta, as compared to sham. In the caudal penumbra, estrogen treatment significantly attenuated 68 kD NFP degradation, calpain content, calpain activity, levels of cytosolic cytochrome c, and ERbeta degradation. At the lesion, vehicle-treated animals displayed more TUNEL+ cells, and estrogen treatment significantly attenuated this cell death marker. We conclude that estrogen may inhibit cell death in SCI through calpain inhibition.

Antagonist-Induced, Activation Function-2-Independent Estrogen Receptor α Phosphorylation

Estrogen receptor alpha (ERalpha) serine 118 (Ser118) phosphorylation modulates activation function-1 (AF1) function. Correct positioning of helix 12 promotes agonist-dependent recruitment of cyclin-dependent kinase-7 to catalyze this event. In this study we show robust cyclin-dependent kinase-7-independent, AF2 antagonist-induced Ser118 phosphorylation. Estradiol (E2) and ICI-182,780 (ICI-780) induce Ser118 phosphorylation of wild-type ERalpha and either of two helix 12 mutants, suggesting AF2-independent action, probably via shedding of 90-kDa heat shock protein. With E2 treatment, the predominantly nuclear, phosphorylated ERalpha in COS-1 cells is detergent soluble. Although levels of ICI-780-induced phosphorylation are profound, Ser118-phosphorylated ERalpha is aggregated over the nucleus or in the cytoplasm, fractionating with the cell debris and making detection in cleared lysates improbable. Selective ER modulators (SERMs) elicit a mixed response with phosphorylated ERalpha in both detergent-soluble and -insoluble compartments. Apparent ligand-induced loss of ERalpha protein from cleared lysates is thus due to ligand-induced redistribution into the pellet, not degradation. The COS-1 response to ICI-780 can be mimicked in MCF-7 cells treated with a proteasome inhibitor to block authentic ligand-induced degradation. With SERMs and antagonists, the magnitude of Ser118-phosphorylated receptor redistribution into the insoluble fraction of COS-1 cells correlates with the magnitude of authentic ERalpha degradation in MCF-7 cells. A strong inverse correlation with ligand-induced uterotropism in vivo (P < 0.0001) and direct correlation with AF2-independent transrepression of the matrix metalloprotease-1 promoter in endometrial cells in vitro are seen. These data suggest that ligand-induced Ser118 phosphorylation of ERalpha can be AF2 independent. Furthermore, they identify translocation of Ser118-phosphorylated ERalpha out of the nucleus, leading to cytoplasmic aggregation, as an antagonist pathway that may precede receptor degradation.

CHIP (Carboxyl Terminus of Hsc70-Interacting Protein) Promotes Basal and Geldanamycin-Induced Degradation of Estrogen Receptor-α

In estrogen target cells, estrogen receptor-alpha (ERalpha) protein levels are strictly regulated. Although receptor turnover is a continuous process, dynamic fluctuations in receptor levels, mediated primarily by the ubiquitin-proteasome pathway, occur in response to changing cellular conditions. In the absence of ligand, ERalpha is sequestered within a stable chaperone protein complex consisting of heat shock protein 90 (Hsp90) and cochaperones. However, the molecular mechanism(s) regulating ERalpha stability and turnover remain undefined. One potential mechanism involves CHIP, the carboxyl terminus of Hsc70-interacting protein, previously shown to target Hsp90-interacting proteins for ubiquitination and proteasomal degradation. In the present study, a role for CHIP in ERalpha protein degradation was investigated. In ER-negative HeLa cells transfected with ERalpha and CHIP, ERalpha proteasomal degradation increased, whereas ERalpha-mediated gene transcription decreased. In contrast, CHIP depletion by small interference RNA resulted in increased ERalpha accumulation and reporter gene transactivation. Transfection of mutant CHIP constructs demonstrated that both the U-box (containing ubiquitin ligase activity) and the tetratricopeptide repeat (TPR, essential for chaperone binding) domains within CHIP are required for CHIP-mediated ERalpha down-regulation. In addition, coimmunoprecipitation assays demonstrated that ERalpha and CHIP associate through the CHIP TPR domain. In ERalpha-positive breast cancer MCF7 cells, CHIP overexpression resulted in decreased levels of endogenous ERalpha protein and attenuation of ERalpha-mediated gene expression. Furthermore, the ERalpha-CHIP interaction was stimulated by the Hsp90 inhibitor geldanamycin (GA), resulting in enhanced ERalpha degradation; this GA effect was further augmented by CHIP overexpression but was abolished by CHIP depletion. Finally, ERalpha dissociation from CHIP by various ERalpha ligands, including 17beta-estradiol, 4-hydroxytamoxifen, and ICI 182,780, interrupted CHIP-mediated ERalpha degradation. These results demonstrate a role for CHIP in both basal and GA-induced ERalpha degradation. Furthermore, based on our observations that CHIP promotes ERalpha degradation and attenuates receptor-mediated gene transcription, we suggest that CHIP, by modulating ERalpha stability, contributes to the regulation of functional receptor levels, and thus hormone responsiveness, in estrogen target cells.

Differential regulation of estrogen-inducible proteolysis and transcription by the estrogen receptor alpha N terminus.

The ubiquitin-proteasome pathway has emerged as an important regulatory mechanism governing the activity of several transcription factors. While estrogen receptor alpha (ERalpha) is also subjected to rapid ubiquitin-proteasome degradation, the relationship between proteolysis and transcriptional regulation is incompletely understood. Based on studies primarily focusing on the C-terminal ligand-binding and AF-2 transactivation domains, an assembly of an active transcriptional complex has been proposed to signal ERalpha proteolysis that is in turn necessary for its transcriptional activity. Here, we investigated the role of other regions of ERalpha and identified S118 within the N-terminal AF-1 transactivation domain as an additional element for regulating estrogen-induced ubiquitination and degradation of ERalpha. Significantly, different S118 mutants revealed that degradation and transcriptional activity of ERalpha are mechanistically separable functions of ERalpha. We find that proteolysis of ERalpha correlates with the ability of ERalpha mutants to recruit specific ubiquitin ligases regardless of the recruitment of other transcription-related factors to endogenous model target genes. Thus, our findings indicate that the AF-1 domain performs a previously unrecognized and important role in controlling ligand-induced receptor degradation which permits the uncoupling of estrogen-regulated ERalpha proteolysis and transcription.

Ligand-dependent switching of ubiquitin–proteasome pathways for estrogen receptor

Recent evidence indicates that the transactivation of estrogen receptor alpha (ERalpha) requires estrogen-dependent receptor ubiquitination and degradation. Here we show that estrogen-unbound (unliganded) ERalpha is also ubiquitinated and degraded through a ubiquitin-proteasome pathway. To investigate this ubiquitin-proteasome pathway, we purified the ubiquitin ligase complex for unliganded ERalpha and identified a protein complex containing the carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP preferentially bound to misfolded ERalpha and ubiquitinated it to induce degradation. Ligand binding to the receptor induced the dissociation of CHIP from ERalpha. In CHIP-/- cells, the degradation of unliganded ERalpha was abrogated; however, estrogen-induced degradation was observed to the same extent as in CHIP+/+ cells. Our findings suggest that ERalpha is regulated by two independent ubiquitin-proteasome pathways, which are switched by ligand binding to ERalpha. One pathway is necessary for the transactivation of the receptor and the other is involved in the quality control of the receptor.

Inhibiting proteasomal proteolysis sustains estrogen receptor-alpha activation.

Estrogen receptor-alpha (ER alpha) is a ligand-dependent transcription factor that mediates physiological responses to 17 beta-estradiol (E2). Ligand binding rapidly down-regulates ER alpha levels through proteasomal proteolysis, but the functional impact of receptor degradation on cellular responses to E2 has not been fully established. In this study, we investigated the effect of blocking the ubiquitin-proteasome pathway on ER alpha-mediated transcriptional responses. In HeLa cells transfected with ER alpha, blocking either ubiquitination or proteasomal degradation markedly increased E2-induced expression of an ER-responsive reporter. Time course studies further demonstrated that blocking ligand-induced degradation of ER alpha resulted in prolonged stimulation of ER-responsive gene transcription. In breast cancer MCF7 cells containing endogenous ER alpha, proteasome inhibition enhanced E2-induced expression of endogenous pS2 and cathepsin D. However, inhibiting the proteasome decreased expression of progesterone receptor (PR), presumably due to the heterogeneity of the PR promoter, which contains multiple regulatory elements. In addition, in endometrial cancer Ishikawa cells overexpressing steroid receptor coactivator 1, 4-hydroxytamoxifen displayed full agonist activity and stimulated ER alpha-mediated transcription without inducing receptor degradation. Collectively, these results demonstrate that proteasomal degradation is not essential for ER alpha transcriptional activity and functions to limit E2-induced transcriptional output. The results further indicate that promoter context must be considered when evaluating the relationship between ER alpha transcription and proteasome inhibition. We suggest that the transcription of a gene driven predominantly by an estrogen-responsive element, such as pS2, is a more reliable indicator of ER alpha transcription activity than a gene like PR, which contains a complex promoter requiring cooperation between ER alpha and other transcription factors.

Protein Kinase A Activation of Estrogen Receptor α Transcription Does Not Require Proteasome Activity and Protects the Receptor from Ligand-Mediated Degradation

17beta-Estradiol (E2)-stimulated estrogen receptor (ERalpha) transcription is accompanied by protein degradation via the 26S-proteasome pathway. Inhibition of proteasome activity stabilizes ERalpha protein and abolishes E2-activated transcription, suggesting functional linkages between transcription and degradation. It is not known whether ligand-independent ERalpha activation is coupled to proteolysis. In pituitary cells, forskolin (FSK) stimulates ERalpha transcription through the protein kinase A (PKA) pathway. This study examined interactions between E2-dependent and PKA-stimulated pathways in GH(3) cells by measuring transcription of a transfected reporter gene and endogenous ERalpha levels. E2 stimulated estrogen response element-mediated transcription 2- to 3-fold and decreased ERalpha protein levels to 40%. In contrast, FSK stimulated ERalpha transcription without decreasing ERalpha protein. Treatment with FSK plus E2 resulted in synergistic ERalpha transactivation, and FSK specifically prevented E2-induced ERalpha degradation. PKA is required for protection and was prevented by H89 (a PKA inhibitor), but not PD98059 (a MAPK kinase inhibitor). Propyl-pyrazole-triol and R,R-diethyl-tetrahydrochrysene, selective ERalpha agonists, reduced ERalpha protein by 50% while stimulating ERalpha transcriptional activity 4- to 8-fold. The antagonist ICI 182,780 similarly decreased ERalpha levels, but prevented ER activation. FSK prevented all ligand-induced ERalpha degradation. Lactacystin, a proteasome inhibitor, abolished E2-stimulated, but not FSK-stimulated, ERalpha transcription. Thus, stimulation of ERalpha transcription by the PKA-dependent pathway is dissociated from receptor degradation and proteasome activity. These data suggest a mechanism of ERalpha transcriptional activation by PKA that is distinct from E2 activation and that may contribute to the synergistic transcriptional activation of ERalpha by ligand-dependent and PKA-dependent pathways.

The NEDD8 pathway is required for proteasome-mediated degradation of human estrogen receptor (ER)-alpha and essential for the antiproliferative activity of ICI 182,780 in ERalpha-positive breast cancer cells.

Steroid hormone receptors, including estrogen receptor-alpha (ERalpha), are ligand-activated transcription factors, and hormone binding leads to depletion of receptor levels via preteasome-mediated degradation. NEDD8 (neural precursor cell-expressed developmentally down-regulated) is an ubiquitin-like protein essential for protein processing and cell cycle progression. We recently demonstrated that ubiquitin-activating enzyme (Uba)3, the catalytic subunit of the NEDD8-activating enzyme, inhibits ERalpha transcriptional activity. Here we report that Uba3-mediated inhibition of ERalpha transactivation function is due to increased receptor protein turnover. Coexpression of Uba3 with ERalpha increased receptor degradation by the 26S proteasome. Inhibition of NEDD8 activation and conjugation diminished polyubiquitination of ERalpha and blocked proteasome-mediated degradation of receptor protein. The antiestrogen ICI 182,780 is known to induce ER degradation. In human MCF7 breast cancer cells modified to contain a disrupted NEDD8 pathway, ICI 182,780 degradation of ERalpha was impaired, and the antiestrogen was ineffective at inhibiting cell proliferation. This study provides the first evidence linking nuclear receptor degradation with the NEDD8 pathway and the ubiquitin-proteasome system, suggesting that the two pathways can act together to modulate ERalpha turnover and cellular responses to estrogens. Based on our observation that an intact NEDD8 pathway is essential for the antiproliferation activity of the ICI 182,780 in ERalpha positive breast cancer cells, we propose that disruptions in the NEDD8 pathway provide a mechanism by which breast cancer cells acquire antiestrogen resistance while retaining expression of ERalpha.