ERalpha Transcriptional Activity Research Articles

Targeting the receptor tyrosine kinase RET sensitizes breast cancer cells to tamoxifen treatment and reveals a role for RET in endocrine resistance

Endocrine therapy is the main therapeutic option for patients with estrogen receptor (ERalpha)-positive breast cancer. Resistance to this treatment is often associated with estrogen-independent activation of ERalpha. In this study, we show that in ERalpha-positive breast cancer cells, activation of the receptor tyrosine kinase RET (REarranged during Transfection) by its ligand GDNF results in increased ERalpha phosphorylation on Ser118 and Ser167 and estrogen-independent activation of ERalpha transcriptional activity. Further, we identify mTOR as a key component in this downstream signaling pathway. In tamoxifen response experiments, RET downregulation resulted in 6.2-fold increase in sensitivity of MCF7 cells to antiproliferative effects of tamoxifen, whereas GDNF stimulation had a protective effect against the drug. In tamoxifen-resistant (TAM(R)-1) MCF7 cells, targeting RET restored tamoxifen sensitivity. Finally, examination of two independent tissue microarrays of primary human breast cancers revealed that expression of RET protein was significantly associated with ERalpha-positive tumors and that in primary tumors from patients who subsequently developed invasive recurrence after adjuvant tamoxifen treatment, there was a twofold increase in the number of RET-positive tumors. Together these findings identify RET as a potentially important therapeutic target in ERalpha-positive breast cancers and in particular in tamoxifen-resistant tumors.

664 KLF4 suppresses estrogen-dependent breast cancer growth by inhibiting the transcriptional activity of ER alpha

The growth factor proepithelin has recently emerged as an important regulator of transformation in several physiological and pathological systems. In this study, we determined the biological roles of proepithelin in prostate cancer cells using purified human recombinant proepithelin as well as proepithelin-depletion strategies. Proepithelin promoted the migration of androgen-dependent and -independent human prostate cancer cells; androgen-independent DU145 cells were the more responsive. In these cells, proepithelin additionally stimulated wound closure, invasion, and promotion of cell growth in vitro. These effects required the activation of both the Akt and mitogen-activated protein kinase pathways. We have analyzed proepithelin expression levels in different available prostate cancer microarray studies using the Oncomine database and found a statistically significant increase in proepithelin mRNA expression levels in prostate cancers compared with nonneoplastic controls. Notably, depletion of endogenous proepithelin by siRNA and antisense strategies impaired the ability of DU145 cells to grow and migrate after serum withdrawal and inhibited anchorage-independent growth. Our results provide the first evidence for a role of proepithelin in stimulating the migration, invasion, proliferation, and anchorage-independent growth of prostate cancer cells. This study supports the hypothesis that proepithelin may play a critical role as an autocrine growth factor in the establishment and initial progression of prostate cancer. Furthermore, proepithelin may prove to be a useful clinical marker for the diagnosis of prostate tumors.

Cooperative Activation of Cyclin D1 and Progesterone Receptor Gene Expression by the SRC-3 Coactivator and SMRT Corepressor

Although the ability of coactivators to enhance the expression of estrogen receptor-alpha (ERalpha) target genes is well established, the role of corepressors in regulating 17beta-estradiol (E2)-induced gene expression is poorly understood. Previous studies revealed that the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor is required for full ERalpha transcriptional activity in MCF-7 breast cancer cells, and we report herein the E2-dependent recruitment of SMRT to the regulatory regions of the progesterone receptor (PR) and cyclin D1 genes. Individual depletion of SMRT or steroid receptor coactivator (SRC)-3 modestly decreased E2-induced PR and cyclin D1 expression; however, simultaneous depletion revealed a cooperative effect of this coactivator and corepressor on the expression of these genes. SMRT and SRC-3 bind directly in an ERalpha-independent manner, and this interaction promotes E2-dependent SRC-3 binding to ERalpha measured by co-IP and SRC-3 recruitment to the cyclin D1 gene as measured by chromatin IP assays. Moreover, SMRT stimulates the intrinsic transcriptional activity of all of the SRC family (p160) coactivators. Our data link the SMRT corepressor directly with SRC family coactivators in positive regulation of ERalpha-dependent gene expression and, taken with the positive correlation found for SMRT and SRC-3 in human breast tumors, suggest that SMRT can promote ERalpha- and SRC-3-dependent gene expression in breast cancer.

The protein encoded by the functional steroid receptor RNA activator is a new modulator of ER alpha transcriptional activity

The steroid receptor RNA activator gene (SRA1) encodes for a functional RNA (SRA) as well as a protein (SRAP). While several groups reported on SRA-RNA mechanism of action, SRAP exact function remains to be elucidated, mainly due to a lack of studies investigating the function of the protein independently of its RNA counterpart. Using two independent models to examine its specific functions, SRAP was found to enhance estrogen receptor alpha activity in a ligand and response-element dependent manner. Our data therefore suggest that both transcript and protein products of the SRA1 gene co-modulate the transcriptional activity of steroid receptors.

C-Abl regulates estrogen receptor α transcription activity through its stabilization by phosphorylation

Estrogen receptors are members of the steroid hormone superfamily of nuclear receptors that act as ligand-activated transcription factors. Similar to other steroid hormone receptors, estrogen receptor alpha (ERalpha) is a substrate for protein kinases, and phosphorylation has profound effects on the function of this receptor. In this study, we show that ERalpha associates with c-Abl nonreceptor tyrosine kinase. The direct interaction is mediated by two PXXP motifs of ERalpha and the c-Abl SH3 domain. Mutational analysis and in vitro kinase assays show that ERalpha can be phosphorylated on two sites, tyrosine 52 (Y-52) and tyrosine 219 (Y-219). ERalpha phosphorylation by c-Abl stabilizes ERalpha, resulting in enhanced ERalpha transcriptional activity and increased expression of endogenous ERalpha target genes. Furthermore, ERalpha phosphorylation at the Y-219 site affects DNA binding and dimerization by ERalpha. Both the c-Abl inhibitor and the c-Abl kinase dead mutation abolish the c-Abl-induced accumulation of ERalpha and enhancement of ERalpha transcriptional activity, indicating that c-Abl kinase activity is required for regulation of the ERalpha function. Moreover, the ERalpha (Y52,219F) mutant shows reduced breast cancer cell growth and invasion. Taken together, these results show that c-Abl is a novel kinase that upregulates ERalpha expression and promotes breast cancer cell proliferation, suggesting a great potential for this kinase to function as a therapeutic target for breast cancer.

Adenosine A1 receptor, a target and regulator of estrogen receptorα action, mediates the proliferative effects of estradiol in breast cancer

Estrogen receptor-α (ERα) and its ligand estradiol (E2) play critical roles in breast cancer growth and are key therapeutic targets. Here, we report a novel dual role of the adenosine A1 receptor (Adora1) as an E2/ERα target and a regulator of ERα transcriptional activity. In ERα-positive breast cancer cells, E2 up-regulated Adora1 mRNA and protein levels, an effect that was reversed by the E2 antagonist ICI 182,780. siRNA ablation of Adora1 in ERα-positive cells reduced basal and E2-dependent proliferation, whereas Adora1 over-expression in an ERα-negative cell line induced proliferation. The selective Adora1 antagonist, DPCPX, reduced proliferation, establishing Adora1 as a mediator of E2/ERα-dependent breast cancer growth. Intriguingly, Adora1 ablation decreased both mRNA and protein levels of ERα and, consequently, estrogen responsive element-dependent ERα transcriptional activity. Moreover, Adora1 ablation decreased binding activity of ERα to the promoter of its target gene TFF1 and led to reduced TFF1 promoter activity and mRNA levels, suggesting that Adora1 is required for full transcriptional activity of ERα upon E2 stimulation. Taken together, we demonstrated a short feed-forward loop involving E2, ERα, and Adora1 that favors breast cancer growth. These data suggest that Adora1 may represent an important target for therapeutic intervention in hormone-dependent breast cancer.

PKA-mediated stabilization of FoxH1 negatively regulates ERalpha activity.

Estrogen receptor alpha (ERalpha) mediates the mitogenic effects of estrogen. ERalpha signaling regulates the normal growth and differentiation of mammary tissue, but uncontrolled ERalpha activation increases the risk to breast cancer. Estrogen binding induces ligand-dependent ERalpha activation, thereby facilitating ERalpha dimerization, promoter binding and coactivator recruitment. ERalpha can also be activated in a ligand-independent manner by many signaling pathways, including protein kinase A (PKA) signaling. However, in several ERalpha-positive breast cancer cells, PKA inhibits estrogen-dependent cell growth. FoxH1 represses the transcriptional activities of estrogen receptors and androgen receptors (AR). Interestingly, FoxH1 has been found to inhibit the PKA-induced and ligand-induced activation of AR. In the present study, we examined the effects of PKA activation on the ability of FoxH1 to represses ERalpha transcriptional activity. We found that PKA increases the protein stability of FoxH1, and that FoxH1 inhibits PKA-induced and estradiol-induced activation of an estrogen response element (ERE). Furthermore, in MCF7 cells, FoxH1 knockdown increased the PKA-induced and estradiol-induced activation of the ERE. These results suggest that PKA can negatively regulate ERalpha, at least in part, through FoxH1.

Retracted: CK1δ modulates the transcriptional activity of ERα via AIB1 in an estrogen-dependent manner and regulates ERα–AIB1 interactions

Oncogenesis in breast cancer often requires the overexpression of the nuclear receptor coactivator AIB1/SRC-3 acting in conjunction with estrogen receptor-α (ERα). Phosphorylation of both ERα and AIB1 has been shown to have profound effects on their functions. In addition, proteasome-mediated degradation plays a major role by regulating their stability and activity. CK1δ, a member of the ubiquitous casein kinase-1 family, is implicated in the progression of breast cancer. In this study, we show that both ERα and AIB1 are substrates for CK1δ in vitro, and identify a novel AIB1 phosphorylation site (S601) targeted by CK1δ, significant for the co-activator function of AIB1. CK1δ is able to interact with ERα and AIB1 in vivo, while overexpression of CK1δ in breast cancer cells results in an increased association of ERα with AIB1 as confirmed by co-immunoprecipitation assays from cell lysates. Using an siRNA-based approach, luciferase reporter assays and qRT-PCR, we observe that silencing of CK1δ leads to reduced ERα transcriptional activity, despite increased ERα levels, similarly to proteasome inhibition. We provide evidence that AIB1 protein levels are reduced by CK1δ silencing, in an estradiol-dependent manner; such destabilization can be inhibited by pre-treatment with the proteasome inhibitor MG132. We propose that differing activities adopted by ERα and AIB1 as a consequence of their interactions with and phosphorylation by CK1δ, particularly AIB1 stabilization, influence the transcriptional activity of ERα, and therefore have a role in breast cancer development.

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.

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.

The Gαi and Gαq proteins mediate the effects of melatonin on steroid/thyroid hormone receptor transcriptional activity and breast cancer cell proliferation

Melatonin, via its MT1 receptor, but not the MT2 receptor, can modulate the transcriptional activity of various nuclear receptors - estrogen receptor alpha (ERalpha) and retinoic acid receptor alpha (RARalpha), but not ERbeta- in MCF-7, T47D, and ZR-75-1 human breast cancer cell lines. The anti-proliferative and nuclear receptor modulatory actions of melatonin are mediated via the MT1 G protein-coupled receptor expressed in human breast cancer cells. However, the specific G proteins and associated pathways involved in the nuclear receptor transcriptional regulation by melatonin are not yet clear. Upon activation, the MT1 receptor specifically couples to the G(alphai2), G(alphai3), G(alphaq), and G(alphall) proteins, and via activation of G(alphai2) proteins, melatonin suppresses forskolin-induced 3',5'-cyclic adenosine monophosphate production, while melatonin activation of G(alphaq), is able to inhibit phospholipid hydrolysis and ATP's induction of inositol triphosphate production in MCF-7 breast cancer cells. Employing dominant-negative and dominant-positive) forms of these G proteins, we demonstrate that G(alphai2) proteins mediate the suppression of estrogen-induced ERalpha transcriptional activity by melatonin, while the G(q) protein mediates the enhancement of retinoid-induced RARalpha transcriptional activity by melatonin. However, the growth-inhibitory actions of melatonin are mediated via both G(alphai2) and G(alphaq) proteins.

COUP-TFI modulates estrogen signaling and influences proliferation, survival and migration of breast cancer cells

We previously showed that COUP-TFI interacts with the Estrogen Receptor alpha (ER alpha) to recruit Extracellular signal Regulated Kinases (ERKs) in an Estradiol (E2)-independent manner, resulting in an enhancement of ER alpha transcriptional activity. However, the involvement of COUP-TFI in physiologically relevant functions of ER alpha, such as the mitogenic activity that E2 has on breast cancer cells, remains poorly understood. Here, we first showed that the amounts of COUP-TFI protein are higher in dedifferentiated mammary cell lines (MDA-MB-231) and tumor breast cells as compared to the differentiated MCF-7 cell line and normal breast cells. To evaluate the functional relevance of the COUP-TFI/ER alpha interplay in mammary cells, we generated MCF-7 cells that stably over-express COUP-TFI. We found that the over-expression of COUP-TFI enhances motility and invasiveness of MCF-7 cells. COUP-TFI also promotes the proliferation of MCF-7 cells through ER alpha-dependent mechanisms that target cell cycle progression and cell survival. To further investigate the mechanisms underlying these effects of COUP-TFI, we evaluated the expression of known E2-target genes in breast cancer, and found that COUP-TFI differentially regulated genes involved in cell proliferation, apoptosis, and migration/invasion. Notably, Cathepsin D (CTSD) transcript and protein levels were significantly higher in presence and absence of E2 in MCF-7 over-expressing COUP-TFI. Chromatin Immunoprecipitation assays showed that ER alpha, phospho-RNA Polymerase II, as well as p68 RNA Helicase, a phospho-Serine 118 dependent co-activator of ER alpha, were preferentially recruited onto the CTSD gene proximal promoter in COUP-TFI over-expressing cells. These results suggest that COUP-TFI selectively regulates the expression of endogenous E2-target genes and consequently modifies ER alpha positive mammary cells response to E2.

Role for HER2/neu and HER3 in fulvestrant-resistant breast cancer.

Tamoxifen resistance is common for estrogen receptor alpha (ERalpha) positive breast cancer. Second-line therapies include aromatase inhibitors or fulvestrant. We have shown previously that fulvestrant reversed 17beta-estradiol-induced tumor regression of tamoxifen-stimulated MCF-7 xenografts (MCF-7TAMLT) treated for >5 years with tamoxifen in athymic mice and paradoxically stimulated growth. We investigated mechanisms responsible for growth by fulvestrant in the presence of physiologic estradiol and therapeutic strategies in vivo. The results demonstrated that only estradiol increased expression of the estrogen-responsive genes, c-myc, igf-1, cathepsin D, and pS2 mRNAs, in MCF-7E2 and MCF-7TAMLT tumors. Tamoxifen or fulvestrant decreased the estradiol-induced increase of these mRNAs in both tumor models. However, tyrosine-phosphorylated HER2/ neu, HER3, phospho-extracellular-regulated kinase-1/2 (ERK-1/2), and phospho-glycogen synthetase kinase 3alpha (GSK3alpha) and beta proteins were increased in MCF-7TAMLT tumors treated with fulvestrant compared to estradiol, control, or tamoxifen. Phospho-HER2/neu interacted with HER3 protein in MCF-7TAMLT tumors. In order to determine whether the functional interaction of HER2/neu with HER3 is critical for growth of fulvestrant-stimulated MCF-7TAMLT tumors, pertuzumab (an antibody that blocks HER2/neu-HER3 interaction) was used in an in vivo xenograft growth assay. Only growth of fulvestrant-treated MCF-7TAMLT xenografts was decreased significantly by 37.2% in response to pertuzumab (P=0.004). Pertuzumab specifically decreased the interaction of HER2/neu protein with HER3 in fulvestrant-stimulated MCF-7TAMLT tumors. These results suggested growth of MCF-7TAMLT tumors by tamoxifen or fulvestrant is potentially independent of ERalpha transcriptional activity as evidenced by lack of induction of four estrogen-responsive genes. The results suggested that growth of MCF-7TAMLT tumors treated with fulvestrant in the presence of physiologic estradiol is in part mediated through enhanced signaling from the HER2/neu-HER3 pathway as pertuzumab partially inhibited growth and the interaction of HER2/neu with HER3 in vivo.

Chaperoning of estrogen receptor and induction of mammary gland proliferation by neuronal protein synuclein gamma

Synucleins are emerging as central players in the formation of pathologically insoluble deposits characteristic of neurodegenerative diseases. However, synuclein gamma (SNCG), previously identified as a breast cancer specific gene (BCSG1), is also highly associated with breast cancer progression. Using transgenic mouse model, we demonstrated a role of SNCG in induction of highly proliferative pregnancy-like phenotype of mammary epithelial cells and branching morphology. SNCG participated in the heat shock protein-based multiprotein chaperone complex for steroid receptor signaling. Expression of SNCG in mammary epithelium resulted in a significant stimulation of ERalpha transcriptional activity. SNCG-induced mammary gland proliferation can be effectively blocked by antiestrogen and ovariectomy, indicating that the induced proliferation is mediated by ERalpha signaling and requires estrogen stimulation. These data indicate the chaperone activity of SNCG on stimulation of steroid receptor signaling in mammary gland and, thus induces extensive mammary gland proliferation and contributes to the hormonal impact on mammary tumorigenesis.

Phosphatidylinositol 3-Kinase and Glycogen Synthase Kinase 3 Regulate Estrogen Receptor-Mediated Transcription in Neuronal Cells

In addition to 17beta-estradiol binding, estrogen receptor (ER) transcriptional activity could be controlled by intracellular kinase signaling pathways activated by growth factors. In this report we present evidence suggesting that glycogen synthase kinase 3 (GSK3), an effector kinase of the phosphatidylinositol 3-kinase (PI3K) pathway, may affect ERalpha activity in N2a neuroblastoma cells. LiCl, sodium valproate, and SB415286, three inhibitors of GSK3, dose-dependently blocked ERalpha-mediated transcription. In contrast, overexpression of wild-type GSK3, but not of a mutant inactive form, increased ER-dependent gene expression. Pharmacological or genetic inhibition of the PI3K/Akt pathway, whose activity is inversely correlated with that of GSK3, increased ERalpha-mediated transcription, and this effect was blocked by GSK3 inhibitors. As in other cell types, IGF-I increased ERalpha activity in absence of estradiol by a mechanism independent of PI3K. In contrast, IGF-I decreased ERalpha activity in the presence of estradiol, and this effect was mediated by PI3K. We also observed a regulated interaction between beta-catenin, one of the main GSK3 nuclear targets, and ERalpha. Transfection with a nondegradable mutant of beta-catenin blocked the increase in ERalpha transcriptional activity induced by the PI3K inhibitor wortmannin, suggesting a role for beta-catenin in estrogen signaling. In addition, we investigated the regulation of ER protein levels as a potential mechanism for its regulation by the PI3K/GSK3 pathway; GSK3 blockade increased ERalpha protein stability, whereas PI3K inhibition decreased it. In summary, our findings suggest that ER-dependent gene expression in N2a cells is controlled by the PI3K/Akt/GSK3 signaling pathway.

Prognostic Significance of Estrogen Receptor Beta in Epithelial Hyperplasia of Usual Type With Known Outcome

The prognostic significance of ER-alpha expression in benign proliferative breast disease has been confirmed in epithelial hyperplasia of usual type (HUT). However, little is known about the role of ER-beta in these lesions. Therefore, this study was performed to test the hypothesis that, in HUT lesions, the ratio of ER-alpha:ER-beta is an accurate determinant of breast cancer risk and of predicting subsequent progression to invasive breast cancer. This case-control study analyzed a cohort of benign proliferative breast lesions and foci of ductal HUT in 117 patients with long follow-up (20 years). These foci were analyzed by morphometric image analysis together with immunohistochemistry using monoclonal antibodies to ER-beta1 and to ER-alpha. The data were compared with ER-beta expression in all breast carcinomas that subsequently developed in the same patients as well as to ER-alpha expression in the corresponding tissues. In cases that progressed to carcinoma, the ratio of ER-alpha to ER-beta in HUT was significantly higher (P < 0.001) than in those that did not progress. None of the HUT foci from patients who progressed to breast cancer were simultaneously ER-alpha negative and ER-beta positive. Using both ER-beta and ER-alpha in a logistic model demonstrated a 75% correct classification rate for the cohort studied. These findings confirm the diagnostic and prognostic value of defining the ER-alpha and ER-beta status of HUT lesions identified morphologically. The data support the hypothesis that high ER-alpha:ER-beta levels characterize those cases within HUT likely to progress to breast cancer. The data also reveal that a reduced level of ER-beta relative to ER-alpha is an accurate predictor of individual cases of HUT likely to progress to invasive breast carcinoma, thus supporting the concept that ER-alpha transcriptional activity is directly modulated by ER-beta.

Altered Target Gene Regulation Controlled by Estrogen Receptor-α Concentration

Estrogen receptor-alpha (ERalpha) is a transcriptional activator whose concentration is tightly regulated by the cellular environment. In breast tumors of postmenopausal women, elevated receptor concentrations can be associated with negative clinical outcomes, yet it remains poorly understood how such high levels impact ERalpha function. We previously demonstrated that high nuclear concentrations of ERalpha in breast cancer cells bypass the requirement for ligand and are sufficient to activate transcription and accelerate proliferation. Here, we extended those studies and asked whether the transcriptional targets and activation mechanism are similar or different from that of estrogen-stimulated ERalpha. We found that at elevated levels, ERalpha activated, but could not repress, known estrogen-responsive genes. Moreover, the set of activated genes was expanded to include the uterine-restricted target gene, complement component 3. The activation mechanism of ERalpha under these conditions depends both on activation function-1 and residues in the proximal region of the ligand-binding domain. Mutations of aspartate 351 and leucine 372 can inhibit ERalpha transcriptional activity gained at high concentrations and discriminate concentration-inducible ERalpha function from that induced by estrogen. Moreover, we demonstrate that at high levels, ERalpha stimulates transcription without recruiting steroid receptor coactivator-3 and without interference by a Gal4-receptor interaction domain box fusion protein containing LxxLL motifs, further distinguishing this mode of regulation from known activation mechanisms. Together these results demonstrate that the concentration of receptor in breast cancer cells can influence the pattern of target gene expression through a noncanonical activation mechanism.

In vitro and in vivo Effects of Combination of Trastuzumab (Herceptin) and Tamoxifen in Breast Cancer

Extensive interactions between estrogen receptor alpha (ERalpha) and HER2 signaling pathways have been described. Using BT-474 human breast cancer cells, we have previously shown that the combination of tamoxifen (TAM) and Herceptin results in strong synergistic growth inhibition, enhancement of G(0)-G(1) cell cycle accumulation, inhibition of HER2 activity and a cytostatic effect without cell death. To further examine the underlying mechanism of synergy, we investigated the effect of this drug combination on ERalpha function and growth factor downstream signaling. TAM caused a small increase in ERalpha levels while Herceptin had no effect, and both drugs caused an increase in the level of Ser118-phosphorylated ERalpha. However, both TAM and Herceptin individually inhibited ERalpha transcriptional activity, although the combination did not have a greater effect than either single agent. Herceptin inhibited MAPK and Akt activity, while TAM had no effect on these either as a single agent or when added to Herceptin. Using a BALB/c athymic BT-474 in vivo xenograft model, the drug combination (Herceptin 0.3 mg/kg i.p. twice weekly, TAM 1.0 mg/mouse i.p. three times per week) showed a greater inhibition of tumor growth compared to either single agent. Tumor extracts and fixed sections were examined at the end of the treatment period for treatment-specific alterations: we noted a paradoxical proliferation-inducing effect of TAM that was reversed by the addition of Herceptin. Our results indicate that combined targeting of both peptide growth factor receptors and ERalpha represents a promising breast cancer treatment strategy.

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.

The breast cell growth inhibitor, estrogen down regulated gene 1, modulates a novel functional interaction between estrogen receptor alpha and transcriptional elongation factor cyclin T1

Estrogen receptor alpha (ERalpha) regulates transcription of specific genes and is believed to play a major role in breast tumorigenesis. We previously identified estrogen down regulated gene 1 (EDG1 (also known as HEXIM1)) using the C-terminus of ERalpha (E/F domain) as bait in yeast two-hybrid screenings. Here we report on the role of EDG1 as a coregulator of ERalpha transcriptional activity. We observe an interaction between EDG1 and ERalpha. EDG1 inhibits the transcriptional activity of ERalpha and this is dependent upon the C-terminus of EDG1. The C-terminus of EDG1/HEXIM1 was recently shown to inhibit the positive transcription elongation factor b (P-TEFb) by interacting with the cyclin T1 subunit. Here we show that ERalpha interacts with cyclin T1, cyclin T1 and ER co-occupancy on the promoter region of an ER target gene, and that this interaction plays an important role in ERalpha-induced gene expression. The interaction of ERalpha with cyclin T1 also allows ERalpha to compete with EDG1 for cyclin T1, and may release cyclin T1 from EDG1 repression. Conversely, increased EDG1 expression results in inhibition of cyclin T1 recruitment and ERalpha DNA binding. Our results support a novel functional interaction between ERalpha and cyclin T1 that is modulated by EDG1.