Abstract S4-02: The Y537S ESR1 mutation is a dominant driver of distant ER-positive breast cancer metastasis
Authors: Fuqua SAW , Gu G , Rechoum Y , Gelsomino L , Dustin DJ , Corona-Rodriguez A , Beyer AR , Pejerrey SM , Gao M , Tsimelzon A , Tian L , Zhang X , Nagi C , Ando' S 
Published: March 2017

Background: Estrogen receptor (ESR1) mutations occur at a high frequency in metastatic breast tumors in patients treated with hormonal therapy in the metastatic setting. We do not know if these mutations are involved in metastasis.

Experimental design and methods: We generated ESR1 Y537S homozygous mutations using CRISPR Casp-9 technology. Treatment synergy was evaluated using Compusyn. Athymic mice were used in tumor xenograft studies. ChIP-Seq and transcriptome analyses were performed.

Results: We generated CRISPR ESR1 Y537S mutation homozygous knock-in clones and lentiviral stable pools in MCF-7 cells. Transcriptome profiling revealed elevated expression of Hallmark pathways, including EMT and estrogen-regulated gene expression. The EMT in mutant cells was associated with a switch from E-cadherin to vimentin, and increased expression of SNAIL and TWIST. Mutant cell growth was resistant to tamoxifen, but responsive to fulvestrant treatment. Synergistic treatment effects were observed with fulvestrant and everolimus or palbociclib. CRISPR Y537S mutant knock-in cells grown in the mammary fat-pad of athymic mice spontaneously metastasized to distant organs including the lung, intestine, and kidneys. In the presence of estrogen, there was no difference in the frequency of distant macrometastases between parental wild-type ER and CRISPR Y537S mutant ER mice. However, in the absence of estrogen, 80% of CRISPR Y537S mutant ER mice displayed overt distant macrometastases, but none were observed in parental wild-type ER mice (p=0.04). Interestingly, although CRISPR Y537S mutant ER tumors grown in the mammary fat-pad were unresponsive to tamoxifen treatment, tamoxifen significantly inhibited the growth of mutant tumors at the distant microenvironment (8-fold). Distant tumors retained ER expression and hormone sensitivity. Comparison of residual tamoxifen-treated metastatic tumors with tumors grown at the primary mammary fat-pad site using immunoblot analysis demonstrated significant reduction in estrogen-regulated gene expression, but no effect on the expression of biomarkers associated with EMT, suggesting a disconnect between EMT and distant metastasis in mutant cells. EMT genes were also identified as direct binding site targets in Y537S mutant cells compared with wide-type ER using ChIPSeq. We discovered that expression of the Y537S mutant was dominant, driving the growth of distant metastatic tumors when co-expressed with wild-type ER cells. A Y537S ER mutant-specific gene expression signature predicted poor disease-free survival of ER-positive patients using the METABRIC database, and lung-specific metastasis-free survival in a Memorial Sloan Kettering dataset.

Conclusion: The Y537S ER mutation is a driver of distant metastasis in ER-positive breast cancer cells. Although tamoxifen treatment was ineffective at reducing the growth of mutant cells grown at the primary site, it was effective at reducing distant metastasis. A Y537S ER mutant-specific gene expression signature predicted poor disease-free, and distant lung metastasis in ER-positive patients. Mutation status is a potential new predictive factor for hormone therapy of metastatic breast cancer patients on maintenance hormonal therapy.

Citation Format: Fuqua SAW, Gu G, Rechoum Y, Gelsomino L, Dustin DJ, Corona-Rodriguez A, Beyer AR, Pejerrey SM, Gao M, Tsimelzon A, Tian L, Zhang X, Nagi C, Ando' S. The Y537S ESR1 mutation is a dominant driver of distant ER-positive breast cancer metastasis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S4-02.