The Human Estrogen Receptor-α Is a Ubiquitinated Protein Whose Stability Is Affected Differentially by Agonists, Antagonists, and Selective Estrogen Receptor Modulators

Abstract

The human estrogen receptor ␣-isoform (ER␣) is a nuclear transcription factor that displays a complex pharmacology. In addition to classical agonists and antagonists, the transcriptional activity of ER␣ can be regulated by selective estrogen receptor modulators, a new class of drugs whose relative agonist/antagonist activity is determined by cell context. It has been demonstrated that the binding of different ligands to ER␣ results in the formation of unique ER␣-ligand conformations. These conformations have been shown to influence ER␣-cofactor binding and, therefore, have a profound impact on ER␣ pharmacology. In this study, we demonstrate that the nature of the bound ligand also influences the stability of ER␣, revealing an additional mechanism by which the pharmacological activity of a compound is determined. Of note we found that although all ER␣-ligand complexes can be ubiquitinated and degraded by the 26 S proteasome in vivo, the mechanisms by which they are targeted for proteolysis appear to be different. Specifically, for agonist-activated ER␣, an inverse relationship between transcriptional activity and receptor stability was observed. This relationship does not extend to selective estrogen receptor modulators and pure antagonists. Instead, it appears that with these compounds, the determinant of receptor stability is the ligand-induced conformation of ER␣. We conclude that the different conformational states adopted by ER␣ in the presence of different ligands influence transcriptional activity directly by regulating cofactor binding and indirectly by modulating receptor stability. ER␣1 resides within the nuclei of target cells in an inactive form in the absence of hormone. Upon binding its cognate ligand estradiol, the receptor undergoes an activating conformational change permitting it to interact with specific cofactors and bind DNA response elements within target gene promoters (1, 2). The DNA-bound receptor-ligand complex is then capable of either activating or repressing target gene transcription, depending on both the cell and the promoter context. The classical model of ER␣ action suggests that the role of agonists, such as estradiol, is that of a switch converting the receptor from an inactive to an active form. It now appears that ER␣ pharmacology is more complex, since it has been observed that different ER␣-ligands induce different changes in receptor conformation and that target cells can distinguish between these complexes (3-5). For instance, the anti-estrogen tamoxifen opposes estrogen action in the breast, whereas it manifests estrogenic activities in bone, the cardiovascular system, and the uterus. Reflecting its complex pharmacology, tamoxifen has recently been reclassified as a selective estrogen receptor modulator (SERM). Additional SERMs have been identified, such as raloxifene, GW5638, TSE424, lasofoxifene, and arzoxifene, each of which has distinct agonist/antagonist profiles (6). The challenge, therefore, has been to understand the mechanism(s) underlying SERM-mediated action an...

Eliminating epigenetic barriers induces transient hormone-regulated gene expression in estrogen receptor negative breast cancer cells

“…Simultaneous treatment of the cells that had been exposed to TSA, AZA or a combination treatment, with estradiol and the pure anti-estrogen ICI 182 780 abolished the hormoneinduced increase of pS2/TFF1 or PR transcription ( Figure 2b). As ICI binds to ERa and induces its degradation (Wijayaratne and McDonnell, 2001), this inhibition demonstrates that hormone-regulated activation of ERa target gene expression is dependent on ERa. We noted, however, that in the absence of estradiol, expression levels of both PR and pS2/TFF1 were significantly higher in AZA-treated cells compared to untreated cells and identical to the levels observed for cells treated with E2/ICI (a 5-or 15-fold induction; Figure 2b).…”

Antiestrogens: structure-activity relationships and use in breast cancer treatment

“…1991, Dauvois et al . 1992, El Khissiin & Leclercq 1999, Wijayaratne & McDonnell 2001). However, despite the pure antiestrogenic character of fulvestrant, it did not compare advantageously with tamoxifen when used as a first-line therapy for advanced or metastatic breast cancer (Howell et al .…”

“…Degradation takes place with faster kinetics than that in the presence of agonists in MCF-7 cells. Although 4-hydroxytamoxifen stabilizes ERα protein levels (Wijayaratne & McDonnell 2001, Lupien et al . 2007, Kocanova et al .…”

“…2013). Affinity purification of ERα modified by tagged ubiquitin showed that ICI 182,780 triggers a 2-fold enhancement of ERα ubiquitination compared with basal levels (Wijayaratne & McDonnell 2001). Agonists induce or are permissive for recruitment of several E3 ubiquitin ligases, such as E6-AP, CHIP, MDM2, BRCA1/BARD1, EFP/TRIM25, SPOP, RBCK1, CUEDC2, SKP2, VHL and RNF31 by ERα (Helzer et al .…”

Development and evolution of therapies targeted to the estrogen receptor for the treatment and prevention of breast cancer

“…Fulvestrant, a pure antiestrogen is a derivative of the natural hormone estradiol with a long hydrophobic side chain substituted at the 7α position on the B ring of the steroid nucleus [72]. The steroid binds to the ligand binding domain of the ER in an inverted configuration with the hydrophobic side chain disrupting the ER shape by binding with the grove that can be occupied by Helix 12 of the ER [73] As a result of the unusual shape of the ER complex, it is rapidly ubiquitinilated and destroyed by the proteosome [74]. Pure antiestrogens cause a down regulation of ER so no genes can be activated.…”