Calcineurin regulates the stability and activity of estrogen receptor α

Estrogen receptor α (ER-α) mediates estrogen-dependent cancer progression and is expressed in most breast cancer cells. However, the molecular mechanisms underlying the regulation of the cellular abundance and activity of ER-α remain unclear. We here show that the protein phosphatase calcineurin regulates both ER-α stability and activity in human breast cancer cells. Calcineurin depletion or inhibition down-regulated the abundance of ER-α by promoting its polyubiquitination and degradation. Calcineurin inhibition also promoted the binding of ER-α to the E3 ubiquitin ligase E6AP, and calcineurin mediated the dephosphorylation of ER-α at Ser²⁹⁴ in vitro. Moreover, the ER-α (S294A) mutant was more stable and activated the expression of ER-α target genes to a greater extent compared with the wild-type protein, whereas the extents of its interaction with E6AP and polyubiquitination were attenuated. These results suggest that the phosphorylation of ER-α at Ser²⁹⁴ promotes its binding to E6AP and consequent degradation. Calcineurin was also found to be required for the phosphorylation of ER-α at Ser¹¹⁸ by mechanistic target of rapamycin complex 1 and the consequent activation of ER-α in response to β-estradiol treatment. Our study thus indicates that calcineurin controls both the stability and activity of ER-α by regulating its phosphorylation at Ser²⁹⁴ and Ser¹¹⁸. Finally, the expression of the calcineurin A–α gene (PPP3CA) was associated with poor prognosis in ER-α–positive breast cancer patients treated with tamoxifen or other endocrine therapeutic agents. Calcineurin is thus a promising target for the development of therapies for ER-α–positive breast cancer.

Estrogen receptor α (ER-α) plays a central role in the proliferation of breast cancer cells by increasing the expression of oncogenes, such as those encoding cyclin D1 and c-Myc (1). The expression and activity of ER-α are increased in >70% of breast cancer cases, and the receptor is targeted by drugs such as tamoxifen (2, 3). A substantial proportion of ER-α–positive breast cancer cells become resistant to anti‐estrogens, however, resulting in the progression of the disease. The mechanisms by which the cancer cells acquire resistance to these agents include the generation of splice variants of ER-α, the mutation of the ER-α gene (ESR1), and changes in stability of the ER-α protein (4).Increased protein stability appears to be a key contributor to the up-regulation of ER-α in breast cancer. The ubiquitination of ER-α is one mechanism responsible for ER-α degradation. Several E3 ligases that mediate the degradation of ER-α have been identified and include E6-associated protein (E6AP) (5), carboxyl terminus of Hsp70-interacting protein (CHIP) (6), breast cancer type 1 (BRCA1) (7), BRCA1-associated RING domain 1 (8), S phase kinase–associated protein 2 (SKP2) (9), and mouse double minute 2 homolog (10). On the other hand, other E3 ligases—such as RING finger protein (RNF) 31, shank-associated RH domain–interacting protein, and RNF8 (11–13)—have been shown to promote ER-α signaling by stabilizing ER-α protein.The residues Lys³⁰² and Lys³⁰³ of ER-α are targeted for ubiquitination (14). The ubiquitination of ER-α is associated with its phosphorylation, with several kinases such as cyclin-dependent kinase (CDK) 11 (15), Src (5), protein kinase C (16), p38 mitogen-activated protein kinase (9), and extracellular signal–regulated kinase 7 (17) having been shown to phosphorylate the protein. The phosphorylation of ER-α at Ser²⁹⁴ has thus been related to its ubiquitination by SKP2 (9), with the Ser²⁹⁴-phosphorylated form of ER-α being a preferred substrate for ubiquitination by SKP2 in vitro. However, the expression level of ER-α was found to be unaltered in cells depleted of SKP2, suggesting that other E3 ligases may contribute to the degradation of ER-α subsequent to its phosphorylation at Ser²⁹⁴.Calcium is an important regulator of signaling pathways that control oncogenesis and cancer progression, and Ca²⁺ signaling has been linked to signaling by ER-α. β-estradiol (E2) has been shown to induce rapid Ca²⁺ influx in cells, and the Ca²⁺-binding protein calmodulin interacts with ER-α, increases its stability, and modulates E2-regulated gene expression (18). Calcineurin is a Ca²⁺/calmodulin-activated serine–threonine phosphatase that plays a major role in the regulation of immediate cellular responses and gene expression by Ca²⁺ signaling (19). It is also a target of immunosuppressive drugs administered in clinical practice, such as cyclosporine A and FK506. Calcineurin is composed of two subunits: a catalytic subunit, designated calcineurin A, that is encoded by three genes (PPP3CA, PPP3CB, and PPP3CC), and a regulatory subunit, designated calcineurin B, that is encoded by two genes (PPP3R1 and PPP3R2).In the present study, we found that calcineurin plays a previously unrecognized role as a positive regulator of the stability and activity of ER-α in breast cancer cells by mediating its dephosphorylation at Ser²⁹⁴, as well as the activation of mechanistic target of rapamycin complex 1 (mTORC1) and the consequent phosphorylation of ER-α at Ser¹¹⁸, respectively. Furthermore, a high-expression level of PPP3CA was associated with poor prognosis in a subset of breast cancer patients, suggesting that the selective inhibition of calcineurin might be an effective approach to the treatment of ER-α–positive breast cancer.