The estrogen receptor alpha nuclear localization sequence is critical for fulvestrant-induced degradation of the receptor

Highlights

• Deletion of ERα NLS domain (ERΔNLS) causes cytoplasmic localization of the receptor.

• NLS deletion does not impair ligand binding.

• ERΔNLS is degraded by E2 but is resistant to fulvestrant-induced degradation.

• NLS, but not merely ERα localization is critical for fulvestrant-induced degradation.

Abstract

Fulvestrant, a selective estrogen receptor down-regulator (SERD) is a pure competitive antagonist of estrogen receptor alpha (ERα). Fulvestrant binds ERα and reduces the receptor's half-life by increasing protein turnover, however, its mechanism of action is not fully understood. In this study, we show that removal of the ERα nuclear localization sequence (ERΔNLS) resulted in a predominantly cytoplasmic ERα that was degraded in response to 17-β-estradiol (E2) but was resistant to degradation by fulvestrant. ERΔNLS bound the ligands and exhibited receptor interaction similar to ERα, indicating that the lack of degradation was not due to disruption of these processes. Forcing ERΔNLS into the nucleus with a heterologous SV40-NLS did not restore degradation, suggesting that the NLS domain itself, and not merely receptor localization, is critical for fulvestrant-induced ERα degradation. Indeed, cloning of the endogenous ERα NLS onto the N-terminus of ERΔNLS significantly restored both its nuclear localization and turnover in response to fulvestrant. Moreover, mutation of the sumoylation targets K266 and K268 within the NLS impaired fulvestrant-induced ERα degradation. In conclusion, our study provides evidence for the unique role of the ERα NLS in fulvestrant-induced degradation of the receptor.

Introduction

The female sex hormone estradiol is implicated in breast cancer pathogenesis. The effects of 17-β-estradiol (E2), the most potent estrogen, are mediated via estrogen receptors (ERs) ERα and ERβ. Endocrine therapy targets ERα and approximately 70% of breast cancers are ERα-positive (McGuire, 1975). Moreover, the clinical value of ERα status in determining response to endocrine therapy has been established (Maynard et al., 1978).

ERα has a modular structure with several distinct domains, including an amino-terminally located ligand-independent transcriptional activation function (AF-1) domain (amino acids 1–184), a DNA binding domain (DBD; amino acids 185–250), a hinge region (amino acids 251–354), and a ligand-dependent AF-2 domain (amino acids 355–549). The hinge region of several nuclear receptors was originally thought of as a flexible linker between the DBD and the AF-2 domain (Khorasanizadeh and Rastinejad, 2001). However, for many nuclear receptors, including ERα, this region also serves important regulatory functions, serving as a site for a number of post-translational modifications, including acetylation (Wang et al., 2001), phosphorylation (Cui et al., 2004), sumoylation (Sentis et al., 2005), methylation (Zhou et al., 2009), and ubiquitination (Berry et al., 2008). Additionally, it is important for both ERα DNA binding (Schultz et al., 2002, Melvin et al., 2002, Melvin et al., 2004) and receptor subcellular localization (Ylikomi et al., 1992). Its importance for localization stems from the fact that the ERα nuclear localization sequence (NLS) is located within the hinge region.

Deletion and fusion experiments have identified the amino acids within ERα that are critical for its nuclear accumulation. ERα amino acids 256–303 are sufficient to target a heterologous protein, β-galactosidase, to the nucleus (Picard et al., 1990). However, deletions within the context of the endogenous ERα protein showed that amino acids 274–298 do not possess any NLS function (Ylikomi et al., 1992), but that an ERα mutant with a deletion of amino acids 250–274 (identical to the deletion in our ERΔNLS) was completely cytoplasmic in the absence of ligand. Thus, the functional ERα NLS lies within these amino acids. An ERα mutant protein with every lysine and arginine between amino acid positions 253–271 (9 residues in total) mutated to alanine was completely cytoplasmic in the absence of hormone (Burns et al., 2011). Interestingly, a hormone-inducible NLS has also been identified within the ligand binding domain (LBD) of ERα (Ylikomi et al., 1992). While this NLS can cooperate with the hinge region NLS, on its own, it is insufficient to promote nuclear localization of the receptor.

Regulation of ERα target genes is critical for breast cancer progression. Fulvestrant, belongs to the class of anti-estrogens known as selective estrogen receptor down-regulators (SERDs). Fulvestrant (ICI 182,780) is currently approved by the United States Food and Drug Administration for the treatment of ERα-positive metastatic breast cancer in postmenopausal women with disease progression following prior anti-estrogen therapy (Bross et al., 2003, Bross et al., 2002). Fulvestrant is a competitive antagonist with a very similar structure to the endogenous ligand E2 and thus competes with E2 for binding to the LBD of ERα. However, a long hydrophobic side chain gives the drug its unique anti-estrogenic properties. The binding of both E2 and fulvestrant results in ERα degradation via the ubiquitin/proteasome pathway (Nawaz et al., 1999, Wijayaratne and McDonnell, 2001). However, while the turnover induced by E2 is associated with an actively functioning state of ERα (Nawaz and O'Malley, 2004), the degradation elicited by fulvestrant is associated with receptor inhibition (Osborne et al., 2004), suggesting different mechanisms of action. Despite this, the exact mechanism of fulvestrant action, including whether or not receptor degradation is actually required for its anti-estrogenic function, is currently unknown. In fact, several reports have attributed some of fulvestrant's properties to its ability to influence ERα subcellular localization. One report suggested that fulvestrant disrupts ERα nucleo-cytoplasmic shuttling, resulting in cytoplasmic accumulation of the receptor (Dauvois et al., 1993). On the other hand, other reports argue that fulvestrant treatment results in receptor immobilization and strong interaction of ERα with the nuclear matrix (Stenoien et al., 2000, Stenoien et al., 2001, Long and Nephew, 2006, Long et al., 2010, Kocanova et al., 2010).

Given the conflicting reports regarding the effect of fulvestrant on ERα localization, we decided to examine this relationship in more detail. We generated an ERα mutant with a deletion of the NLS domain (amino acids 250–274). Not only did the deletion promote receptor cytoplasmic localization, but it also influenced the degradation response elicited by fulvestrant. Mutation of two ERα sumoylation targets within the NLS does not affect nuclear localization, but significantly impaired fulvestrant induced degradation of the receptor suggesting that sumoylation of the NLS domain is required for fulvestrant-induced degradation of ERα.