Estrogen receptor mutations found in breast cancer metastases integrated with the molecular pharmacology of selective ER modulators

Abstract

The consistent reports of mutations at Asp538 and Tyr537 in helix 12 of the ligand-binding domain (LBD) of estrogen receptors (ERs) from antihormone-resistant breast cancer metastases constitute an important advance. The mutant amino acids interact with an anchor amino acid, Asp351, to close the LBD, thereby creating a ligand-free constitutively activated ER. Amino acids Asp 538, Tyr 537, and Asp 351 are known to play a role in either the turnover of ER, the antiestrogenic activity of the ER complex, or the estrogen-like actions of selective ER modulators. A unifying mechanism of action for these amino acids to enhance ER gene activation and growth response is presented. There is a range of mutations described in metastases vs low to zero in primary disease, so the new knowledge is of clinical relevance, thereby confirming an additional mechanism of acquired resistance to antihormone therapy through cell population selection pressure and enrichment during treatment. Circulating tumor cells containing ER mutations can be cultured ex vivo, and tumor tissues can be grown as patient-derived xenografts to add a new dimension for testing drug susceptibility for future drug discovery.

Figure 1.

Mutations and molecular interactions of the estradiol (E₂)–estrogen receptor (ER) complex. A) Schematic representation of the wild-type human ER cDNA. The position initially known for the natural single-point mutations Gly400Val, Asp351Tyr, and Lys303Arg are indicated. The activating function (AF) – two region and various mutant receptors generated by random chemical or site-directed mutagenesis are shown that either cause loss of AF-2 activity (538, 542, and 543) (118) or cause an increase in estrogenic activity if the receptor is liganded with an antiestrogen (540, 543, 544, 547, and 548) (81–85). The red arrow connecting 538 to the anchor Asp351 illustrates the current finding of Toy et al. (3) that D538G interacts and closes the empty ER pocket. B) The interaction of E₂ (blue) in the ligand-binding domain (LBD) with relevant amino acids and the associated amino acids in the vicinity from helix 12 (19). C) A space filled model from the top of the E₂ LBD showing the closed helix 12 (yellow) securing E₂ within. Three amino acids of relevance are indicated Asp(D)351 on the surface of the LBD complex and Tyr(Y)537 and Asp(D)538.D. The selective ER modulators 4-hydroxytamoxifen (4-OHT) and raloxifene (Ral) secured within the LBD of the ER by the same two amino acids, Glu353 and Arg394, via a phenolic hydroxyl on both 4-OHT and Ral, as noted with the 3 phenolic hydroxyl on ring A of E₂ (B). E) A space filler model from the top of the Ral LBD showing helix 12 pushed back (yellow) and the piperidine ring of Ral-neutralizing Asp(D)351. The amino acids Asp(D)538 and Tyr(Y)537 are far away from potential interactions to influence the position of helix 12 closure.

Figure 2.

A summary of interactions of selective estrogen receptor (ER) modulator (SERM) antiestrogen side chains. Interaction of key amino acids at 351 or 537 and 538 amino acids in helix 12 that may hypothetically secure the closing of the ligand-binding domain (LBD) for coactivator binding and subsequent modulation of estrogen action by SERMs. A) Substitution of different amino acids at 351 to modulate a raloxifene ER complex to be estrogenic (68). This research was originally published in (70) the American Society for Biochemistry and Molecular Biology. B) The role of the antiestrogenic side chain of raloxifene to modulate the estrogenic SERM ER complex by neutralizing and shielding the carboxylic group of the surface amino acid Asp351 to produce no estrogen action. C) Long polar amino acids glutamic acid and D. tyrosine at 351 form estrogen-like complexes with raloxifene.

Figure 3.

Representations of the experimental (gray) and molecular dynamics-modeled structures (blue) of ERα-4OHT complex, helix 12 colored in magenta. The experimental structure of the wild-type ERα-4OHT complex, PDB code 3ERT (A), mutant forms D351Y (B) and D351G (C) and the modeled structure of the ERα in close conformation accommodating 4OHT (C atoms colored in purple). In each image, the amino acids important for binding or to stabilize helix 12 are shown as sticks with the C atoms colored in blue, and the H-bonds are depicted as black dashed lines. D) This network is initiated in the ligand-binding site by the side chain of His524, and it connects Lys531 of helix 11 with Glu339 of helix 3 via Glu449. E) Two proposed routes for the development of tamoxifen-stimulated tumor growth. Cells (shown as ovals) with excess production of transcriptional coactivators are stimulated to divide by the action of 4-hydroxytamoxifen (4OH tamoxifen)–estrogen receptor (ER complexes, or cells with both an excess production of coactivators) and a mutant receptor can superamplify the 4OH tamoxifen ER signal [from (92)].