Abstract
Approximately 75% of all breast cancers express the nuclear hormone receptor estrogen receptor α (ERα). However, the majority of mammary tumors from genetically engineered mouse models (GEMMs) are ERα-negative. To model ERα-positive breast cancer in mice, we exogenously introduced expression of mouse and human ERα in an existing GEMM of p53-deficient breast cancer. After initial ERα expression during mammary gland development, expression was reduced or lost in adult glands and p53-deficient mammary tumors. Chromatin immunoprecipitation (ChIP)-sequencing analysis of primary mouse mammary epithelial cells (MMECs) derived from these models, in which expression of the ERα constructs was induced in vitro, confirmed interaction of ERα with the DNA. In human breast and endometrial cancer, and also in healthy breast tissue, DNA binding of ERα is facilitated by the pioneer factor FOXA1. Surprisingly, the ERα binding sites identified in primary MMECs, but also in mouse mammary gland and uterus, showed an high enrichment of ERE motifs, but were devoid of Forkhead motifs. Furthermore, exogenous introduction of FOXA1 and GATA3 in ERα-expressing MMECs was not sufficient to promote ERα-responsiveness of these cells. Together, this suggests that species-specific differences in pioneer factor usage between mouse and human are dictated by the DNA sequence, resulting in ERα-dependencies in mice that are not FOXA1 driven. These species-specific differences in ERα-biology may limit the utility of mice for in vivo modeling of ERα-positive breast cancer.
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Acknowledgements
We are grateful to Eline van der Burg, Ute Boon, Renske de Korte-Grimmerink, Micha Nethe and Yongsoo Kim for their technical support with the experiments. We thank the Netherlands Cancer Institute Genomics Core Facility, Mouse Clinic for Cancer and Aging, Animal Facility, and Animal Pathology Facility for their expert technical support.
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GSE127863.
Funding
This work was financially supported by the Oncode Institute; the Center for Translational Molecular Medicine (CTMM) Breast Care Project; the Netherlands Organization for Scientific Research (NWO: Cancer Genomics Netherlands (CGCNL), Cancer Systems Biology Center (CSBC), Zenith 93,512,009, Vici 91,814,643); the EU Seventh Framework Program (EurocanPlatform project 260,791); the European Research Council (ERC Synergy project CombatCancer); and a National Roadmap grant for Large-Scale Research facilities from NWO.
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LMC, LH, WZ and JJ designed research. LMC, LH, AD and ES performed research. LMC, LH, RB, SK, WZ and JJ analyzed data. LMC, WZ and JJ wrote the paper.
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Supplementary Fig. S1
Exogenously-introduced ERα is localized to the nucleus. a PCR analysis of primary MMECs derived from Trp53F/F, Trp53F/F;mERα, Trp53F/F;hERα, Trp53F/F;HA-mERα and Trp53F/F;HA-hERα mice to detect AdCre-induced recombination of the Trp53 locus (Trp53F depicts not-recombined, Trp53Δ depicts recombined DNA) and the Col1a1 locus (lower band depicts not-recombined, upper band depicts recombined DNA). FwT1, FwT10, RvT10 refer to primers used for PCR at the Trp53 locus. FwC1, RvC1, RvC2 refer to primers used for PCR at the Col1a1 locus. Locations of the primers are indicated in Fig. 1a. b Subcellular fractionation of AdCre-induced recombined primary MMECs derived from Trp53F/F, Trp53F/F;mERα and Trp53F/F;HA-mERα (top) and Trp53F/F, Trp53F/F;hERα and Trp53F/F;HA-hERα mice (bottom) shows enrichment of mouse and human ERα mostly in the soluble-nuclear fraction and some in the chromatin-bound fraction in full medium conditions. Tubulin was used as a marker for the cytoplasmic fraction, Topoisomerase 1 (TOP1) for the soluble-nuclear fraction and histone H3 for the chromatin-bound fraction. (PNG 825 kb)
Supplementary Fig. S2
Tumor latency of WP-HA-mERα and WP-HA-hERα mice is affected to a similar extent as WP control mice by 17β-estradiol stimulation due to methylation-mediated silencing. a Kaplan-Meier analysis of mammary tumor-specific survival reflecting modest differences between the ERα overexpressing models compared to WP, split between WP-mERα and WPHA-mERα (left) and WP-hERα and WP-HA-hERα (right) compared to WP. Mantel-Cox: WP (n = 11, 214 days) versus WP-mERα (n = 20, 228 days, p = 0.0053); WP-HA-mERα (n = 20, 233 days, p = 0.0008); WP-hERα (n = 20, 240 days, p = 0.0029); WP-HA-hERα (n = 20, 215 days, p = 0.1610). *** p < 0.001, ** p < 0.01, ns p > 0.05. b Distribution of histological subtypes of tumors derived from the different models (left) and the luciferase positive tumors that are either ERα-positive or ERα-negative (right). The tumors are either >90% sarcomatoid, >90% carcinomatoid or consists of both histological subtypes (mixed). The absolute number of tumors analyzed are indicated. c Representative images of bioluminescence imaging of luciferase expression in cell lines derived from WP-mERα, WP-hERα and WP-HA-hERα luciferase-negative tumors, treated with DMSO or 5-Aza-2’-deoxycytidine. d Kaplan-Meier analysis of mammary tumor-specific survival of WP, WP-HA-mERα and WP-HA-hERα mice treated with 17β-estradiol slow-release pellets (E2; left) and with vehicle (veh; right). Mantel-Cox: E2: WP (n = 11, 165 days) versus WP-HA-mERα (n = 10, 200.5 days, p = 0.0090); WP-HA-hERα (n = 11, 190 days, p = 0.0671). Veh: WP (n = 9, 224 days) versus WP-HA-mERα (n = 9, 244 days, p = 0.0324); WP-HA-hERα (n = 9, 243 days, p = 0.0067). ** p < 0.01, * p < 0.05, ns p > 0.05. e Distribution of all palpable tumors, identified in vehicle- and E2-treated mice, separated in luciferase signal being negative, weakly positive and positive. WP-HA-mERα veh, 18 tumors; WP-HA-mERα E2, 10 tumors; WP-HA-hERα veh, 14 tumors; WP-HA-hERα E2, 10 tumors. (PNG 719 kb)
Supplementary Fig. S3
The FOXA1 motif is absent at ERα binding sites in mouse cells, while present in 820 human cells. Expression levels of mouse Esr1 and human ESR1 transcripts in primary MMECs derived from AdCre-induced recominbed Trp53F/F, Trp53F/F;HA-mERα and Trp53F/F;HA-hERα primary MMECs upon DMSO or E2 stimulation, derived from RNA-seq data. b Heatmap illustrating raw peak intensity of HA-tag ChIP-seq in primary MMECs derived from Trp53F/F, Trp53F/F;HA-mERα and Trp53F/F;HA-hERα mice upon AdCre induced recombination, separated in sites shared by both HA-mERα and HA-hERα, sites specific for HA-hERα and sites specific for HA-mERα. A window of 5kb around the peak is shown. c Average read count profiles of the HA-tag ChIP-seq peak signal at shared sites, HA-hERα specific sites and HA-mERα specific sites. d Spider plot representing the differential enrichment of the fraction of DNA motifs identified in ERα binding sites (present in at least 10% of binding sites) of human MCF7 and mouse HA-mERα and HA-hERα MMECs (as shown in Fig. 4b.) compared to publicly available datasets. ERα binding motifs in human MCF7 are compared to motifs identified in human breast tumors (GSE40867), human endometrial tumors (GSE94524), and human healthy breast tissue (GSE99680) (left). ERα binding motifs in mouse HA-mERα and HA-hERα MMECs are compared to motifs identified in mouse mammary gland (GSE43415) and E2 stimulated mouse uterus (GSE36455) (right). e mRNA expression levels of ERα and the transcription factors FOXA1 and GATA3, that are expressed relatively high in MCF7 and low in primary MMECs, derived from RNA-seq data. f mRNA expression levels of three additional co-factors; SRC1, SRC2 and SRC3. SRC3 also shows relatively high expression in MCF7 compared to MMECs. Data derived from RNA-seq data. (PNG 2612 kb)
Supplementary Fig. S4
Expression of human ERα in combination with additional transcription factors does not sensitize MMECs to E2 stimulation. a RT-qPCR analysis using primers specific for human ERα, Flag-tagged SRC1, Flag-tagged SRC2, SRC3 and FOXA1 and primers recognizing both mouse and human GATA3 in MMECs transduced with lentiviral constructs containing the indicated cDNAs. Data represent mean + SD, n = 3. b RT-qPCR analysis using primers specific for mouse Areg (left) and mouse Ccnd1 (right) in MMECs transduced with lentiviral constructs containing the indicated cDNAs after stimulation with DMSO, E2 or ICI. Data represent mean + SD, n = 2. Two-way ANOVA: Areg: Empty vector DMSO versus ICI, p = 0.0052; hERα DMSO versus E2, p = 0.0156; hERα/hSRC1/2/3/hFOXA1 DMSO versus ICI, p = 0.0264; hERα/hSRC1/2/3/hFOXA1/HA-hGATA3 DMSO versus ICI, p = 0.0065. Ccnd1: hERα DMSO versus E2, p = 0.0025; hERα/hSRC1/2/3/hFOXA1/HA-hGATA3 DMSO versus ICI, p = 0.0103. ** p < 0.01, * p < 0.05, ns p > 0.05. c,d Representative images (c) and quantification (d) of clonogenic assays of MMECs transduced with lentiviral constructs containing the indicated cDNAs and treated with DMSO, E2 or ICI. Data represent mean + SD, n = 2. Two-way ANOVA: hERα DMSO versus E2, p = 0.0001; hERα/hSRC1/2/3 DMSO versus E2, p = 0.0114; Empty vector DMSO versus hERα/hSRC1/2/3 DMSO, p = 0.0020; Empty vector DMSO versus hERα/hSRC1/2/3/hFOXA1 DMSO, p = 0.0001; Empty vector DMSO versus hERα/hSRC1/2/3/hFOXA1/HA-hGATA3 DMSO, p = 0.0001. *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05. (PNG 892 kb)
Supplementary Table S1
Primer sequences. (DOCX 21 kb)
Supplementary Table S2
Antibodies. (DOCX 16 kb)
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Cornelissen, L.M., Henneman, L., Drenth, A.P. et al. Exogenous ERα Expression in the Mammary Epithelium Decreases Over Time and Does Not Contribute to p53-Deficient Mammary Tumor Formation in Mice. J Mammary Gland Biol Neoplasia 24, 305–321 (2019). https://doi.org/10.1007/s10911-019-09437-z
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DOI: https://doi.org/10.1007/s10911-019-09437-z