Natalia M. Solodin

Differential Regulation of Estrogen-Inducible Proteolysis and Transcription by the Estrogen Receptor α N Terminus

ABSTRACT The ubiquitin-proteasome pathway has emerged as an important regulatory mechanism governing the activity of several transcription factors. While estrogen receptor α (ERα) 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 ERα proteolysis that is in turn necessary for its transcriptional activity. Here, we investigated the role of other regions of ERα and identified S118 within the N-terminal AF-1 transactivation domain as an additional element for regulating estrogen-induced ubiquitination and degradation of ERα. Significantly, different S118 mutants revealed that degradation and transcriptional activity of ERα are mechanistically separable functions of ERα. We find that proteolysis of ERα correlates with the ability of ERα 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 ERα proteolysis and transcription.

Increases in estrogen receptor-α concentration in breast cancer cells promote serine 118/104/106-independent AF-1 transactivation and growth in the absence of estrogen

A common phenotype in breast cancer is the expansion of the estrogen receptor‐α (ER+) cell population and an inappropriate elevation of ERα protein, the latter predisposing patients for a poorer prognosis than those with lower levels of the receptor. A tetracycline‐inducible ERα overexpression model was developed in the MCF‐7 cell line to assess induction of endogenous gene activation and growth in response to elevations in ERα protein. Heightened levels of ERα resulted in aberrant promoter occupancy and gene activation in the absence of hormone, which was independent of ligand and AF‐2 function. This increased receptor activity required the amino‐terminal A/B domain and was not inhibited by tamoxifen, which supports an enhancement of AF‐1 function, yet was independent of serine‐104, 106, and 118 phosphorylation. Ligand‐independent transcription was accompanied by an increase in growth in the absence of hormonal stimulation. The results suggest that elevated levels of ERα in breast cancer cells can result in activation of receptor transcriptional function in a manner distinct from classical mechanisms that involve ligand binding or growth factor‐induced phosphorylation. Further, they describe a potential mechanism whereby increases in ERα concentration may provide a proliferative advantage by augmenting ERα function regardless of ligand status.—Fowler, A. M., Solodin, N., Preisler‐Mashek, M. T., Zhang, P., Lee, A. V., Alarid, E. T. Increases in estrogen receptor‐α concentration in breast cancer cells promote serine 118/104/106‐independent AF‐1 transactivation and growth in the absence of estrogen. FASEB J. 18, 81–93 (2004)

Repression of ESR1 through actions of estrogen receptor alpha and Sin3A at the proximal promoter.

ABSTRACT Gene expression results from the coordinated actions of transcription factor proteins and coregulators. Estrogen receptor alpha (ERα) is a ligand-activated transcription factor that can both activate and repress the expression of genes. Activation of transcription by estrogen-bound ERα has been studied in detail, as has antagonist-induced repression, such as that which occurs by tamoxifen. How estrogen-bound ERα represses gene transcription remains unclear. In this report, we identify a new mechanism of estrogen-induced transcriptional repression by using the ERα gene, ESR1. Upon estrogen treatment, ERα is recruited to two sites on ESR1, one distal (ENH1) and the other at the proximal (A) promoter. Coactivator proteins, namely, p300 and AIB1, are found at both ERα-binding sites. However, recruitment of the Sin3A repressor, loss of RNA polymerase II, and changes in histone modifications occur only at the A promoter. Reduction of Sin3A expression by RNA interference specifically inhibits estrogen-induced repression of ESR1. Furthermore, an estrogen-responsive interaction between Sin3A and ERα is identified. These data support a model of repression wherein actions of ERα and Sin3A at the proximal promoter can overcome activating signals at distal or proximal sites and ultimately decrease gene expression.

Temporal variation in estrogen receptor-α protein turnover in the presence of estrogen

Estrogen receptor-a (ERa) is essential in the maintenance of cellular responsiveness to estrogen in the reproductive system. It is established that ligand binding induces downregulation of ERa protein by targeting receptor for destruction by the 26S proteasome. However, ERa is preserved in cells chronically exposed to estrogen and it is unknown how receptor levels are maintained in the continued presence of the signal that induces degradation. A modified pulsechase analysis was developed using a tet-inducible ERa expression system to determine the rate of ERa protein decay following both acute and chronic estrogen treatments. Upon initial hormone treatment, ERa half-life is shortened from 3 to 1 h. However, ERa half-life increases over time, achieving a half-life of w6 h in 72 h of estrogen treatment. Analysis of ERa half-life in the presence and absence of proteasome inhibitor, MG132, revealed that the increased stability is due in part to a decreased rate of proteolysis. In addition, we observed a time-dependent increase in phospho-S118 ERa and showed that the half-life of the phosphomimetic ERa mutant, S118E-ER, is identical to that of wild-type receptor under conditions of chronic estrogen treatment. These data provide evidence that as cells adapt to chronic stimulation, ERa protein is stabilized due first to a decreased rate of proteolysis, and secondarily, to the accumulation of proteasome-resistant, phosphorylated form of receptor. This temporal control of proteolysis allows for the establishment of steady-state levels of receptor and provides a protective mechanism against loss of hormone responsiveness.

Regulation of Estrogen Receptor α N-Terminus Conformation and Function by Peptidyl Prolyl Isomerase Pin1

ABSTRACT Estrogen receptor alpha (ERα), a key driver of growth in the majority of breast cancers, contains an unstructured transactivation domain (AF1) in its N terminus that is a convergence point for growth factor and hormonal activation. This domain is controlled by phosphorylation, but how phosphorylation impacts AF1 structure and function is unclear. We found that serine 118 (S118) phosphorylation of the ERα AF1 region in response to estrogen (agonist), tamoxifen (antagonist), and growth factors results in recruitment of the peptidyl prolyl cis/trans isomerase Pin1. Phosphorylation of S118 is critical for Pin1 binding, and mutation of S118 to alanine prevents this association. Importantly, Pin1 isomerizes the serine118-proline119 bond from a cis to trans isomer, with a concomitant increase in AF1 transcriptional activity. Pin1 overexpression promotes ligand-independent and tamoxifen-inducible activity of ERα and growth of tamoxifen-resistant breast cancer cells. Pin1 expression correlates with proliferation in ERα-positive rat mammary tumors. These results establish phosphorylation-coupled proline isomerization as a mechanism modulating AF1 functional activity and provide insight into the role of a conformational switch in the functional regulation of the intrinsically disordered transactivation domain of ERα.

Pin1 modulates ERα levels in breast cancer through inhibition of phosphorylation-dependent ubiquitination and degradation.

Estrogen receptor-alpha (ERα) is an important biomarker used to classify and direct therapy decisions in breast cancer (BC). Both ERα protein and its transcript, ESR1, are used to predict response to tamoxifen therapy, yet certain tumors have discordant levels of ERα protein and ESR1, which is currently unexplained. Cellular ERα protein levels can be controlled post-translationally by the ubiquitin-proteasome pathway through a mechanism that depends on phosphorylation at residue S118. Phospho-S118 (pS118-ERα) is a substrate for the peptidyl prolyl isomerase, Pin1, which mediates cis-trans isomerization of the pS118-P119 bond to enhance ERα transcriptional function. Here, we demonstrate that Pin1 can increase ERα protein without affecting ESR1 transcript levels by inhibiting proteasome-dependent receptor degradation. Pin1 disrupts ERα ubiquitination by interfering with receptor interactions with the E3 ligase, E6AP, which also is shown to bind pS118-ERα. Quantitative in situ assessments of ERα protein, ESR1, and Pin1 in human tumors from a retrospective cohort show that Pin1 levels correlate with ERα protein but not to ESR1 levels. These data show that ERα protein is post-translationally regulated by Pin1 in a proportion of breast carcinomas. As Pin1 impacts both ERα protein levels and transactivation function, these data implicate Pin1 as a potential surrogate marker for predicting outcome of ERα-positive BC.

Peptidylprolyl Isomerase Pin1 Directly Enhances the DNA Binding Functions of Estrogen Receptor α

Background: Estrogen receptor α (ERα) activity in breast cancer cells is increased by Pin1, an isomerase that alters protein conformation. Results: Pin1-mediated isomerization increases ERα DNA binding affinity. Conclusion: Pin1 allosterically regulates ERα DNA binding directly via isomerization of phosphorylated receptor. Significance: Pin1 regulation of ERα provides a framework for understanding regulation by intrinsically disordered domains on transcription factor function. The transcriptional activity of estrogen receptor α (ERα), the key driver of breast cancer proliferation, is enhanced by multiple cellular interactions, including phosphorylation-dependent interaction with Pin1, a proline isomerase, which mediates cis-trans isomerization of the N-terminal Ser(P)118-Pro119 in the intrinsically disordered AF1 (activation function 1) domain of ERα. Because both ERα and Pin1 have multiple cellular partners, it is unclear how Pin1 assists in the regulation of ERα transactivation mechanisms and whether the functional effects of Pin1 on ERα signaling are direct or indirect. Here, we tested the specific action of Pin1 on an essential step in ERα transactivation, binding to specific DNA sites. DNA binding analysis demonstrates that stable overexpression of Pin1 increases endogenous ERα DNA binding activity when activated by estrogen but not by tamoxifen or EGF. Increased DNA binding affinity is a direct effect of Pin1 on ERα because it is observed in solution-based assays with purified components. Further, our data indicate that isomerization is required for Pin1-modulation of ERα-DNA interactions. In an unbiased in vitro DNA binding microarray with hundreds of thousands of permutations of ERα-binding elements, Pin1 selectively enhances the binding affinity of ERα to consensus DNA elements. These studies reveal that Pin1 isomerization of phosphorylated ERα can directly regulate the function of the adjacent DNA binding domain, and this interaction is further modulated by ligand binding in the ligand-binding domain, providing evidence for Pin1-dependent allosteric regulation of ERα function.

A kinetic model identifies phosphorylated estrogen receptor-α (ERα) as a critical regulator of ERα dynamics in breast cancer

Receptor levels are a key mechanism by which cells regulate their response to stimuli. The levels of estrogen receptor‐α (ERα) impact breast cancer cell proliferation and are used to predict prognosis and sensitivity to endocrine therapy. Despite the clinical application of this information, it remains unclear how different cellular processes interact as a system to control ERα levels. To address this question, experimental results from the ERα‐positive human breast cancer cell line (MCF‐7) treated with 17‐β‐estradiol or vehicle control were used to develop a mass‐action kinetic model of ERα regulation. Model analysis determined that RNA dynamics could be captured through phosphorylated ERα (pERα)‐dependent feedback on transcription. Experimental analysis confirmed that pERα‐S118 binds to the estrogen receptor‐1 (ESR1) promoter, suggesting that pERα can feedback on ESR1 transcription. Protein dynamics required a separate mechanism in which the degradation rate for pERα was 8.3‐fold higher than nonphosphorylated ERα. Using a model with both mechanisms, the root mean square error was 0.078. Sensitivity analysis of this combined model determined that while multiple mechanisms regulate ERα levels, pERα‐dependent feedback elicited the strongest effect. Combined, our computational and experimental results identify phosphorylation of ERα as a critical decision point that coordinates the cellular circuitry to regulate ERα levels.—Tian, D., Solodin, N. M., Rajbhandari, P., Bjorklund, K., Alarid, E. T., Kreeger, P. K. A kinetic model identifies phosphorylated estrogen receptor‐α (ERα) as a critical regulator of ERα dynamics in breast cancer. FASEB J. 29, 2022‐2031 (2015). www.fasebj.org

The proteasome inhibitor bortezomib induces an inhibitory chromatin environment at a distal enhancer of the estrogen receptor-α gene.

Expression of the estrogen receptor-α (ERα) gene, ESR1, is a clinical biomarker used to predict therapeutic outcome of breast cancer. Hence, there is significant interest in understanding the mechanisms regulating ESR1 gene expression. Proteasome activity is increased in cancer and we previously showed that proteasome inhibition leads to loss of ESR1 gene expression in breast cancer cells. Expression of ESR1 mRNA in breast cancer cells is controlled predominantly through a proximal promoter within ∼400 base pair (bp) of the transcription start site (TSS). Here, we show that loss of ESR1 gene expression induced by the proteasome inhibitor bortezomib is associated with inactivation of a distal enhancer located 150 kilobases (kb) from the TSS. Chromatin immunoprecipitation assays reveal several bortezomib-induced changes at the distal site including decreased occupancy of three critical transcription factors, GATA3, FOXA1, and AP2γ. Bortezomib treatment also resulted in decreased histone H3 and H4 acetylation and decreased occupancy of histone acetyltransferase, p300. These data suggest a mechanism to explain proteasome inhibitor-induced loss of ESR1 mRNA expression that highlights the importance of the chromatin environment at the −150 kb distal enhancer in regulation of basal expression of ESR1 in breast cancer cells.