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Pair of Studies Point to ESR1 Mutation in Hormone-Resistant Breast Cancer

NEW YORK (GenomeWeb News) – A pair of studies that appeared in Nature Genetics yesterday implicated mutations in the ESR1 gene in the development of hormone-resistant metastatic breast cancer.

Both teams, one from the University of Michigan and the other from Memorial Sloan-Kettering Cancer Center, reported that mutations affecting the ligand-binding domain of ESR1 seem to be linked to acquired resistance to hormone therapies.

"This is the tumor's way of evading hormonal therapy. These mutations activate the estrogen receptor when there is no estrogen — as is the case when a patient takes an aromatase inhibitor," said Dan Robinson, a research assistant professor of pathology who was the first author on the Michigan effort. "It's essentially an on-switch for the estrogen receptor."

About 70 percent of all breast cancers express estrogen receptors, and such tumors are commonly treated with ER antagonists like tamoxifen and fulvestrant as well as aromatase inhibitors that suppress estrogen production such as letrozole and exemestane. After a period of time though, many patients' tumors develop resistance to those therapies.

As part of the Michigan team's MI-ONCOSEQ program, which enrolls patients with advanced cancers for integrative sequencing, the researchers homed in on a subset of patients with metastatic ER-positive breast cancer. They sequenced tumor and normal samples from those 11 patients and uncovered an array of mutations, including ones in PIK3CA, BRCA1, and FGFR2.

Of those patients, though, six had nonsynonymous mutations in ESR1 that affected the ligand-binding domain. The Michigan investigators uncovered the mutations through exome sequencing on either the Illumina HiSeq 2000 or HiSeq 2500 and confirmed them through transcriptome sequencing — ESR1 was expressed at moderate to high levels — also on the Illumina platform.

Similarly, the Sloan-Kettering investigators linked ESR1 to resistant ER-positive breast cancer. They gathered a cohort of 38 tumor samples from individuals with metastatic ER-positive breast cancer who had received hormonal therapy for at least three months. Drawing on a sequencing panel run on the Illumina HiSeq 2000 that searched for mutations and copy number changes in some 230 commonly affected genes, the researchers found a mean 4.3 mutations per tumor, as compared to the matched normal controls.

They compared the mutations found in their cohort to their prevalence in the Cancer Genome Atlas. Three genes — ESR1, RPTOR, and ERBB3 — were more commonly mutated in the Sloan-Kettering cohort than the TCGA one.

"The fact that these mutations are enriched among tumors from patients who had relapsed while on hormonal therapy suggests that they may have a role in the development of acquired resistance," said the MSKCC team, which was led by Sarat Chandarlapaty.

Chandarlapaty and his colleagues noted that the ESR1 mutations they saw also clustered in the ligand-binding domain, and most of them affected amino acid 537 or 538 in helix 12 of the protein.

They confirmed their findings in a cohort of patients enrolled in the BOLERO-2 clinical trial that examined patients with ER-positive metastatic breast cancer. In that group, 11 percent of the cases had ESR1 mutations and, again, most of those mutations affected Tyr537 or Asp538 in patients who had undergone a long course of hormone therapy.

The Michigan team also noted that the mutations in their cohort typically affected Tyr537 and Asp538, as well as one case with an affected Leu536. For three of their patients, they had samples that were taken at the time of their primary diagnosis, which showed that their tumors did not originally harbor those mutations. Additionally, the patients were all treated with anti-estrogens and aromatase inhibitors.

They also turned to other cohorts, including the TCGA and others, to find that ESR1 mutations did not appear to be present in primary resections of ER-positive breast cancer or triple-negative breast cancers.

Both sets of researchers examined the functional role of those mutated regions of the ESR1 ligand-binding domain.

By cloning the five mutations it identified into expression vectors, the Michigan team found that all five led to strong constitutive activation of an estrogen-response element-luciferase reporter system, suggesting to the group that the mutations developed during a time of estrogen deprivation.

In addition, in a dose-response study, the team found that wild-type ESR1 was inhibited in a dose-response fashion by the anti-estrogens fulvestrant and endoxifen. The mutated forms of ESR1 also responded in a dose-depended fashion to the drugs. "One could speculate that the corresponding mutations did not arise under selective pressure of anti-estrogen treatment but rather in the context of an estrogen deprivation setting, such as treatment with aromatase inhibitors and/or oophorectomy," the team noted.

The mutations, as the research teams noted, clustered in helix 12 of the ESR1 protein. That region is involved in the response of the estrogen receptor to agonists and antagonists.

Using site-directed mutagenesis in breast cancer cell lines, the Sloan-Kettering group found that mutations at residues 537 and 538 can lead to the activation of ER signaling in the absence of the hormone. Mutations at those locations also led to increased Ser118 phosphorylation, greater association with AIB1, and reduced response to HSP90 inhibitors.

Through computer modeling, the MSKCC team found that the mutations also affected the conformation of the ESR1 protein. Both p.Tyr537Ser and p.Asp538Gly mutations appeared to favor the agonist conformation of ERα. Interestingly, the investigators noted that hydrogen bonds appeared to form between Ser537 or Gly538 and Asp351 on H3.

"The newly discovered hydrogen bond involving Gly538 is likely caused by altered backbone conformations inaccessible to all natural amino acids except glycine," Chandarlapaty and his colleagues said. "Furthermore, these stimulations agree with the activity levels of the mutants, suggesting that these hydrogen bonds stabilize the agonist conformation, especially for the coactivator-recruiting H12 helix and its surroundings, and may contribute to the elevated activity of the mutants."

They noted, though, that the conformational changes don't point out an obvious way in which it prevents ER antagonists from binding.

Still, since higher doses of receptor agonists like tamoxifen and fulvestrant appeared to antagonize the mutant isoforms as well as ER signaling, the Sloan-Kettering team suggested that more potent or more specific mutant antagonists might benefit patients.

The Michigan team suggested that patients could be monitored via circulating tumor DNA for these mutations. "In this manner, treatment could be shifted to head off evolving tumor resistance," it said.