Estrogen-independent molecular actions of mutant estrogen receptor 1 in endometrial cancer

Blanchard, Zannel; Vahrenkamp, Jeffery M.; Berrett, Kristofer C.; Arnesen, Spencer; Gertz, Jason

doi:10.1101/gr.244780.118

Cited by 35 publications

(54 citation statements)

References 43 publications

(88 reference statements)

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“…Mutant clones were generated using the CETCH-seq method described by Savic et al (21). We followed procedures outlined by Blanchard et al (22) (Supplemental Fig. S1a) using the same guide RNA/Cas9 vector and WT and D538G pFETCH vectors for mutant and WT ER clone generation.…”

Section: Generation Of Mutant Clonesmentioning

confidence: 99%

“…72 hours post-transfection, cells were treated with G418 (Thermo Fisher Scientific) at 300ug/ml final concentration, which was applied every two days in conjunction with media changes. Single-cell clones were picked and validated using the same procedures as described by Blanchard et al (22) including limiting dilution plating, colony picking, sanger sequencing and FLAG and ER immunoblots. In all, two clones for each genotype (WT, Y537S, and D538G) were validated for both T-47D and MCF-7 cell lines.…”

Section: Generation Of Mutant Clonesmentioning

confidence: 99%

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Estrogen Receptor Alpha Mutations in Breast Cancer Cells Cause Gene Expression Changes through Constant Activity and Secondary Effects

et al. 2021

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While breast cancer patients with tumors that express estrogen receptor α (ER) generally respond well to hormone therapies that block ER activity, a significant number of patients relapse. Approximately 30% of these recurrences harbor activating mutations in the ligand binding domain (LBD) of ER, which have been shown to confer ligand-independent function. However, much is still unclear regarding the effect of mutant ER beyond its estrogen independence. To investigate the molecular effects of mutant ER, we developed multiple isogenic ER mutant cell lines for the most common LBD mutations, Y537S and D538G. These mutations induced differential expression of thousands of genes, the majority of which were mutant allele-specific and were not observed upon estrogen treatment of wildtype cells. These mutant-specific genes showed consistent differential expression across ER mutant lines developed in other laboratories. Wildtype cells with long-term estrogen exposure only exhibited some of these transcriptional changes, suggesting that mutant ER causes novel regulatory effects that are not simply due to constant activity. While ER mutations exhibited minor effects on ER genomic binding, with the exception of ligand independence, ER mutations conferred substantial differences in chromatin accessibility. Mutant ER was bound to approximately a quarter of mutant-enriched accessible regions that were enriched for other DNA binding factors including FOXA1, CTCF, and OCT1. Overall, our findings indicate that mutant ER causes several consistent effects on gene expression, both indirectly and through constant activity..

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Section: Generation Of Mutant Clonesmentioning

confidence: 99%

Section: Generation Of Mutant Clonesmentioning

confidence: 99%

Estrogen Receptor Alpha Mutations in Breast Cancer Cells Cause Gene Expression Changes through Constant Activity and Secondary Effects

et al. 2021

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“…Mutant clones were generated using the CETCH-seq method described by Savic et al (36). We followed procedures outlined by Blanchard et al (22) (Supplemental Fig. S1a) using the same guide RNA/Cas9 vector and WT and D538G pFETCH vectors for mutant and WT ER clone generation.…”

Section: Generation Of Mutant Clonesmentioning

confidence: 99%

Estrogen receptor alpha mutations in breast cancer cells cause gene expression changes through constant activity and secondary effects

Arnesen

Blanchard

Williams

et al. 2020

Preprint

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While breast cancer patients with tumors that express estrogen receptor α (ER) generally respond well to hormone therapies that block ER's actions, a significant number relapse. Approximately 30% of these recurrences harbor activating mutations in ER's ligand binding domain (LBD). ER mutations have been shown to confer ligand-independent function to ER; however, much is still unclear regarding the effect of mutant ER beyond its estrogen independence. To investigate mutant ER's molecular effects, we developed multiple isogenic ER mutant cell lines for the most common ER LBD mutations, Y537S and D538G. We found that these mutations induce differential expression of thousands of genes, the majority of which are mutant allele-specific and are not observed upon estrogen treatment of wildtype cells. The mutant-specific genes show consistent differential expression across ER mutant lines developed in other laboratories. The observed gene expression changes cannot be explained by constitutive ER activity alone, as wildtype cells with long-term estrogen exposure only exhibit some of these transcriptional changes, suggesting that mutant ER causes novel regulatory effects that are not simply due to constant activity. While ER mutations have minor effects on ER genomic binding, with the exception of ligand independence, we observed substantial differences in chromatin accessibility due to ER mutations. Mutant ER is bound to approximately a quarter of mutant-enriched accessible regions that are enriched for other DNA binding factors including FOXA1, CTCF, and OCT1. Our findings indicate that mutant ER causes several consistent effects on gene expression both indirectly and through constant activity.

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“…Beyond breast cancer, three potentially-pathogenic ESR1 mutations were very recently identified in cervical squamous cell carcinoma samples [ 48 ]; thus, it would be interesting to use this highly sensitive and specific methodology in cervical cancer samples and potentially pre-malignant cases of endometriosis as well. Very recently, a de Novo ESR1 Hotspot Mutation was detected in a patient with endometrial cancer treated with an aromatase inhibitor [ 49 ], while it has been reported that—in endometrial cancer— ESR1 mutations are associated with worse outcomes and less obesity [ 50 ]. However, the experimental investigation of ESR1 mutations in endometrial cancer has not been performed.…”

Section: Discussionmentioning

confidence: 99%

ESR1 NAPA Assay: Development and Analytical Validation of a Highly Sensitive and Specific Blood-Based Assay for the Detection of ESR1 Mutations in Liquid Biopsies

Stergiopoulou

Markou

Tzanikou

et al. 2021

Cancers

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A considerable number of estrogen receptor-positive breast cancer (ER+ BrCa) patients develop resistance to endocrine treatment. One of the most important resistance mechanisms is the presence of ESR1 mutations. We developed and analytically validated a highly sensitive and specific NaME-PrO-assisted ARMS (NAPA) assay for the detection of four ESR1 mutations (Y537S, Y537C, Y537N and D538G) in circulating tumour cells (CTCs) and paired plasma circulating tumour DNA (ctDNA) in patients with ER+ BrCa. The analytical specificity, analytical sensitivity and reproducibility of the assay were validated using synthetic oligos standards. We further applied the developed ESR1 NAPA assay in 13 ER+ BrCa primary tumour tissues, 13 non-cancerous breast tissues (mammoplasties) and 64 liquid biopsy samples: 32 EpCAM-positive cell fractions and 32 paired plasma ctDNA samples obtained at different time points from 8 ER+ metastatic breast cancer patients, during a 5-year follow-up period. Peripheral blood from 11 healthy donors (HD) was used as a control. The developed assay is highly sensitive (a detection of mutation-allelic-frequency (MAF) of 0.5% for D538G and 0.1% for Y537S, Y537C, Y537N), and highly specific (0/13 mammoplasties and 0/11 HD for all mutations). In the plasma ctDNA, ESR1 mutations were not identified at the baseline, whereas the D538G mutation was detected in five sequential ctDNA samples during the follow-up period in the same patient. In the EpCAM-isolated cell fractions, only the Y537C mutation was detected in one patient sample at the baseline. A direct comparison of the ESR1 NAPA assay with the drop-off ddPCR using 32 identical plasma ctDNA samples gave a concordance of 90.6%. We present a low cost, highly specific, sensitive and robust assay for blood-based ESR1 profiling. The clinical performance of the ESR1 NAPA assay will be prospectively evaluated in a large number of well-characterized patient cohorts.

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Estrogen-independent molecular actions of mutant estrogen receptor 1 in endometrial cancer

Cited by 35 publications

References 43 publications

Estrogen Receptor Alpha Mutations in Breast Cancer Cells Cause Gene Expression Changes through Constant Activity and Secondary Effects

Estrogen Receptor Alpha Mutations in Breast Cancer Cells Cause Gene Expression Changes through Constant Activity and Secondary Effects

Estrogen receptor alpha mutations in breast cancer cells cause gene expression changes through constant activity and secondary effects

ESR1 NAPA Assay: Development and Analytical Validation of a Highly Sensitive and Specific Blood-Based Assay for the Detection of ESR1 Mutations in Liquid Biopsies

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