The bone microenvironment increases phenotypic plasticity of ER+ breast cancer cells

doi:10.1016/j.devcel.2021.03.008

. 2021 Apr 19;56(8):1100-1117.e9.

doi: 10.1016/j.devcel.2021.03.008.

The bone microenvironment increases phenotypic plasticity of ER⁺ breast cancer cells

Igor L Bado¹, Weijie Zhang¹, Jingyuan Hu², Zhan Xu¹, Hai Wang¹, Poonam Sarkar³, Lucian Li⁴, Ying-Wooi Wan⁵, Jun Liu¹, William Wu⁶, Hin Ching Lo¹, Ik Sun Kim¹, Swarnima Singh⁷, Mahnaz Janghorban⁷, Aaron M Muscarella¹, Amit Goldstein¹, Purba Singh¹, Hyun-Hwan Jeong⁸, Chaozhong Liu⁴, Rachel Schiff⁹, Shixia Huang¹⁰, Matthew J Ellis¹, M Waleed Gaber³, Zbigniew Gugala¹¹, Zhandong Liu¹², Xiang H-F Zhang¹³

Affiliations

¹ Lester and Sue Smith Breast Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Dan L. Duncan Cancer Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
² Program in Quantitative and Computational Biosciences, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX 77030, USA.
³ Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
⁴ Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX 77030, USA.
⁵ Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX 77030, USA; Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
⁶ Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
⁷ Dan L. Duncan Cancer Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
⁸ Center for Precision Health, School of Biomedical Informatics, the University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
⁹ Lester and Sue Smith Breast Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Dan L. Duncan Cancer Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
¹⁰ Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
¹¹ Department of Orthopedic Surgery and Rehabilitation, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555, USA.
¹² Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX 77030, USA; Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
¹³ Lester and Sue Smith Breast Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Dan L. Duncan Cancer Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; McNair Medical Institute, Baylor College of Medicine, BCM600, One Baylor Plaza, Houston, TX 77030, USA. Electronic address: xiangz@bcm.edu.

PMID: 33878299
PMCID: PMC8062036
DOI: 10.1016/j.devcel.2021.03.008

The bone microenvironment increases phenotypic plasticity of ER⁺ breast cancer cells

Igor L Bado et al. Dev Cell. 2021.

. 2021 Apr 19;56(8):1100-1117.e9.

doi: 10.1016/j.devcel.2021.03.008.

Authors

Affiliations

¹ Lester and Sue Smith Breast Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Dan L. Duncan Cancer Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
² Program in Quantitative and Computational Biosciences, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX 77030, USA.
³ Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
⁴ Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX 77030, USA.
⁵ Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX 77030, USA; Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
⁶ Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
⁷ Dan L. Duncan Cancer Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
⁸ Center for Precision Health, School of Biomedical Informatics, the University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
⁹ Lester and Sue Smith Breast Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Dan L. Duncan Cancer Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
¹⁰ Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
¹¹ Department of Orthopedic Surgery and Rehabilitation, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555, USA.
¹² Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX 77030, USA; Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
¹³ Lester and Sue Smith Breast Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Dan L. Duncan Cancer Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; McNair Medical Institute, Baylor College of Medicine, BCM600, One Baylor Plaza, Houston, TX 77030, USA. Electronic address: xiangz@bcm.edu.

PMID: 33878299
PMCID: PMC8062036
DOI: 10.1016/j.devcel.2021.03.008

Abstract

Estrogen receptor-positive (ER⁺) breast cancer exhibits a strong bone tropism in metastasis. How the bone microenvironment (BME) impacts ER signaling and endocrine therapy remains poorly understood. Here, we discover that the osteogenic niche transiently and reversibly reduces ER expression and activities specifically in bone micrometastases (BMMs), leading to endocrine resistance. As BMMs progress, the ER reduction and endocrine resistance may partially recover in cancer cells away from the osteogenic niche, creating phenotypic heterogeneity in macrometastases. Using multiple approaches, including an evolving barcoding strategy, we demonstrated that this process is independent of clonal selection, and represents an EZH2-mediated epigenomic reprogramming. EZH2 drives ER⁺ BMMs toward a basal and stem-like state. EZH2 inhibition reverses endocrine resistance. These data exemplify how epigenomic adaptation to BME promotes phenotypic plasticity of metastatic seeds, fosters intra-metastatic heterogeneity, and alters therapeutic responses. Our study provides insights into the clinical enigma of ER+ metastatic recurrences despite endocrine therapies.

Keywords: FGFR/EZH2 axis; barcoding; bone metastasis; bone tropism; chromatin alteration; clonal evolution; endocrine resistance; epigenomic reprogramming; osteogenic cells; stemness.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

**Figure 1:. BME induces transient loss of ER expression in ER+ breast cancer cells.**
A. H&E staining of spontaneous metastases of HCI011 and WHM9 tumors to spine and hind limb, respectively. Scale bar: 100μm. B. Human-specific ER IHC staining are shown for spontaneous metastasis of HCI011 and WHIM9, respectively. Scale bar: 50μm. C. IF staining of ER, keratin 8 (k8), and DAPI in orthotopic (mammary) and IIA-induced BoM models of ER+ PDXs (HCI011 and WHIM9). Scale bars: 100μm. D. IF images of MCF7 cells following orthotopic and bone transplantation. Changes in ER expression are illustrated at different stages of tumor progression. Scale bars: 50μm. E. Dot plot depicting nuclear ER intensity of single cells (SCs) in orthotopic and BoM specimens from PDXs (HCI011, WHIM9) and cell lines (MCF7, ZR75-1), following IF staining as illustrated in 1C and 1D (n=3–6 mice per model). F. Dot plot depicting nuclear ER intensity of SCs in orthotopic and BoM specimens from M7-SCP2. Bone lesions were classified into “small” and “large” as defined in (E); (n=4 mice). G. Connected scatterplots showing the mean-normalized ER intensity of all cancer models used from Figure 1E to 1F. (n= 5 cell lines). H. Boxplot depicting changes in ESR1 early signature in matched BoM and primary specimens from BC patients (https://github.com/npriedig/). I. Scheme describing the PET-CT experiment on MCF7 orthotopic or IIA-induced BoM models. 18F-FES and 18F-FDG imaging were performed 48h apart, at week 1 and 7 post tumor transplantation. J. PET/CT scans showing the maximum intensity projection (MIP) of 18F-FDG and 18F-FES in bone as described in I. MCF7 BoMs were generated using IIA injection. Red arrows indicate tumor location. Scale: 0.2–0.5 SUV-bw (week 1) and 100–200 SUV-bw (week 7). K. Axial view of PET/CT scans depicting the uptake of 18F-FDG and 18F-FES in small and large lesions of MCF7 orthotopic tumors. Early time point (Week 1) and late time point (Week 7) were used to depict non palpable orthotopic tumor stage (small < 2mm) and the palpable tumor stage. Red arrows and scale: as in J. L. Relative quantification of radiolabeled 18F-FES uptake in small and large lesions of orthotopic and BoMs. Each dot represents the mean standard uptake values (mean SUV-bw) of 18F-FES normalized to the mean SUV of 18F-FDG for each mouse. Mann Whitney U-test is used for statistical analysis; n=5 mice per group. (**A–L**): P values were computed by two-tailed unpaired Student t-tests unless otherwise noted.

**Figure 2:. Tracing ER expression in bone metastases by evolving barcodes and ERE-reporter.**
A. Experimental design of BoM lineage tracing using an inducible CRISPR-Cas9 hgRNA evolving barcoding system. iCas9-expressing MCF7 cells were stably transduced with homing guide RNA A21 (hgRNA-A21) before transplantation to bone and induced weekly for four weeks before LCM and targeted sequencing. We collected 19 lesions from femur (#1–12) and tibia (#13–19). Barcoded parental cells were labelled as #20. B. Exact map of mouse femur before and after LCM of metastatic lesions #1–12. C. Heatmap showing hierarchical clustering of bone lesions #1–12, based on hgRNA-A21 mutations. D. IF quantification of SCs nuclear ER in lesions #1–12 (femur). (n=12 lesions); *P value*: Ordinary one-way ANOVA. E. Modular organization of lesions #1–12 following a high-dimensional undirected analysis of barcode mutations. Node sizes represent mean intensity of ER expression. F. Circus plot showing barcode deletions in bone lesions clustered in module 1 (see E). #20: pre-injected cells. G. Scatter plot showing Pearson correlation (r) between the Shannon index of bone lesions and their relative distance to lesion #1 or #7. P value: two-tailed t-test. H. IIA-induced BoM from ERE-GFP reporter MCF7 cells. ERE-GFP^Low, and ERE-GFP^high MCF7 cells were sorted based of their GFP expression. Tumor growth was measured by Bioluminescence. (n= 5–8 mice); *P value*: Two-Way ANOVA. I. IF staining of ER in BoM derived from ERE-GFP^Low MCF7 cells. The Gaussianized ER distribution is based on nuclear intensity of SCs; peak: mean ER expression per lesion. J. IF staining depicting a spatial distribution of ER based on the location of M7-SCP2 lesions relatively to the bone matrix. Scale bars: 25μm. Dot plots represent nuclear ER expression in cells proximal (≤ 2 cell distance) or distal (≥3 distance) to the bone matrix. *P value*s: two-tailed unpaired Student’s t-tests.

**Figure 3:. OGs promote the loss of ER expression and reduction of ER activities during early stages of bone colonization.**
A. IF of BoMs showing ER expression in PDXs (HCI011 and WHIM9) and MCF7 cells, relatively to Receptor activator of nuclear factor-KB (RANK) expression in osteoclasts. Scale bars: 100μm. Dot plots show ER quantification in SCs. (n=3–5 lesions). B. IF as in A. ER is co-stained with alpha smooth muscle actin (αSMA) and cluster of differentiation 31 (CD31) to depict endothelial cells. Dot plots show ER quantification in SCs. (n=3–5 lesions). C. IF as in A. ER is co-stained with alkaline phosphatase (ALP). Dot plots show ER quantification in SCs. (n=3–5 lesions). D. IF images of HCI011-derived primary cells and MCF7 cells in 3D monoculture and co-culture with FOBs and MSCs. Scale bars: 100μm. E. Heatmap showing the mean intensity of ER in primary cells (HCI011) and BC cell lines (MDA-MB-361, MCF7, ZR75-1, T47D, ZR75-30) in 3D monoculture (control) or co-culture with osteoclast precursors (U937), bone marrow stromal cells (Hs5), mouse pre-osteoblasts (MC3T3), human mesenchymal stem cells (MSC) and human pre-osteoblast (FOB). Histogram shows ER expression in monoculture versus co-culture of multiple cell lines with U937 or FOB cells; (n=3 biological replicates). Error bars: mean +/− SD. F. IF showing ER expression in M7-SCPs in 3D monoculture or co-culture with FOB cells. Vimentin (VIM). Scale bars: 50μm. G. IF quantification of ER expression in M7-SCP1 to 4; (n = 3 biological replicates). H. Graph representing ER expression in cancer cells alone or in co-cultured with FOB cells. Spearman correlation (r); (n = 5 cell lines); Error bars: mean +/− SE **(A–H):** P values were computed by two-tailed unpaired Student t-tests unless otherwise noted.

**Figure 4:. OGs confer endocrine resistance.**
A. Relative mRNA expression of ESR1 in 3D monoculture or co-culture of MCF7 with FOB. Data result from FACS-sorted MCF7. (n=3 technical replicates). B. Dot plot representing ER transcriptional activity in MCF7 cells expressing pGL2 ERE-luciferase reporter. MCF7 cells were cultured in 3D with or without OGs (FOB and MSC) for 7 days. (n=10 technical replicates); Error bars: mean +/− SD. C. Confocal images showing the expression of progesterone receptor (PR) in IIA-induced BoM from MCF7 and HCI011. Dot plots show nuclear PR IF intensity. (n=3 lesions). D. Dot plots depicting ER intensity in 3D mono- and co-culture of MCF7 cells with FOB following 24 hours treatment with 10nM 17β-estradiol, 20nM fulvestrant and 100nM 4-Hydroxytamoxifen (tamoxifen); n=5 fields. E. Violin plot showing the response of luciferase-labelled MCF7 and ZR75-1 cells to 100nM of 4-Hydroxytamoxifen (Tamoxifen) and 20nM of fulvestrant in 3D mono- or co-culture with OGs (FOB). Bioluminescence was acquired 72 hours post-treatment. (n=12 and 10 technical replicates for and ZR75-1). F. Graphs representing the proliferation of MCF7 cells and M7-SCP1-4 in monoculture and MSC co-culture following 1 week of treatment with 20nM fulvestrant or 100nM tamoxifen; n= 5 cell lines. G. Time course experiment depicting growth kinetics of un-entrained and bone-entrained M7-SCP2 cells in vehicle or 20nM fulvestrant conditions; n=6 technical replicates. H. Growth curve showing response of IIA-induced MCF7 BoMs to estrogen depletion. Ovariectomized (OV) mice were additionally treated with Letrozole (OV+AI), daily. Results are based on BLI. (n=10 mice); *P value*: Two-Way ANOVA. I. Dot plot showing BoM growth in wild-type (WT) and OV+AI mice at week 2 and week 5 post tumor transplantation; n=10 mice. P value: two-tailed unpaired Student’s t-test. J. H&E staining showing MCF7 metastatic lesions in wild-type (WT) and “OV+AI” groups. K. Growth curve depicting the response of ZR75-1-derived BoM as in H. (n=9–10 mice); P value: Two-Way ANOVA. L. Dot plot showing statistical growth differences in ZR75-1 as in I. M. H&E staining of ZR75-1 metastatic lesions as in J. (**A–M**): P values were computed by two-tailed unpaired Student t-tests unless otherwise noted.

**Figure 5:. BME drives a global phenotypic shift involving multiple pathways.**
A. Experimental summary diagram to evaluate molecular in cancer cells exposed to BME. TRAP-seq was performed on 3D co-culture of MCF7 and OGs (MSCs), RPPA on un-entrained (MCF7 and M7-SCP2) and bone-entrained cells (MCF7-Bo and M7-SCP2-Bo), and ATAC-seq on un-entrained (M7-SCP2) or bone-entrained (M7-SCP2-Bo) cells. B. Box plot depicting gene signature alternations in MCF7 monoculture (MSC-) and co-cultures (MSC) from TRAP-seq. Analysis was performed using a non-parametric and unsupervised GSVA (Hänzelmann et al., 2013). Specific colors represent different treatment conditions. (n= 4 biological replicates each with 3 technical replicates); P value: two-tailed unpaired Student’s t-test. C. Waterfall plot showing the gene ontology analysis of TRAP-seq data from PANTHER classification system, based on false discovery rate (FDR). D. Heatmap depicting expression changes in luminal and stemness-related markers from RPPA data. Parental cells (MCF7 and M7-SCP2), and bone-entrained BC cells (MCF7-Bo and M7-SCP2-Bo) are compared; n: 4 biological replicates and 3 technical replicates. E. Heatmap depicting EMT/MET markers from RPPA data as described in D. F. Heatmap depicting receptor tyrosine kinases from RPPA data as described in D. G. IF showing vimentin, E-cadherin, and Keratin 8 expression in IIA-induced bone MCF7 and ZR75-1 BoMs. H. Volcano plot showing epigenetic reprogramming of bone-entrained M7-SCP2 cells based on differentially enriched peaks from ATAC sequencing analysis. 2644 peaks were significantly altered in bone-entrained M7-SCP2 (FDR ≤0.05) and highlighted in pink. I. Volcano plot based on opened promotors identified by ATAC-seq analysis. FDR < 0.05 is highlighted in pink. J. Pie chart depicting the genomic distribution of differentially altered peaks between un-entrained and bone-entrained M7-SCP2 cells. K. Heatmaps and summary plots showing chromatin opening near the transcription start site TSS. 5000 bp before and after TSS are represented. L. Genomic track showing peak variations in the ESR1 gene of un-entrained (M7-SCP2) and bone-derived (M7-SCP2-Bo) cells in blue and red color, respectively. 3 major peaks are highlighted epigenetic reversibility in M7-SCP2-Bo *in vitro*.

**Figure 6:. Osteogenic cell-secreted FGFs promote endocrine resistance.**
A. Network depicting functional protein association between FGFR1, PDGFRB and ER using the STRING database. Kmeans clustering (k=3) was used to represent 3 major centroids (depicted as red, green, and cyan spheres) and their most closely associated proteins based on unsupervised data mining. B. Graph showing the expression of all human 22 fibroblast growth factor (FGF) family proteins (FGF1–23) in OGs (FOB) RNA sequencing dataset (n=3 technical triplicates). C. IF images of Alkaline Phosphatase (ALP) and basic fibroblast growth factor (FGF2/bFGF) in normal bone tissue. Nuclei is shown in blue (DAPI). Scale bar: 50μm D. IF images showing decreased ER expression in tumors established in FGF2 enriched BME. E. The scatter dot plot represents ER quantification from tumors according to FGF2 enrichment (Low and High) in adjacent stromal cells (n=3–4 lesions). Mean expression is represented in blue. The Gaussian curve simulates ER distribution based on nuclear intensity. Peaks: mean expression of ER. F. Immunoblots depicting the effect of recombinant FGF2 (20ng/ml) on ER expression in multiple BC models including PDX HCI011. Cells were treated for 24h. ER expression was summarized as connected dot plot graphs; n = 3 and 4 cell lines. *P value*s: one-tailed unpaired Student t-tests. G. Histogram showing effect of recombinant FGF2 (20ng/ml) on MCF7 and ZR75-1 cell growth in 3D; n=6 technical replicates. H. Bone-In-Culture-Array (BICA) assay showing synergistic effects between 2.5μM pan FGFR inhibitor (BGJ398) and 20nM fulvestrant in MCF7 and ZR75-1 models. n=6 technical replicates. I. Annotated bar plot showing the association of histone modifications with basic FGF (FGF2) gene signatures using the Enrichr platform (https://amp.pharm.mssm.edu/Enrichr/). Processed ChIP-sequencing data was obtained from epigenomic roadmap project (Roadmap Epigenomics Consortium et al., 2015). Histograms represent the association score with FGF2 signaling. Signatures are sorted based on P value ranking. Only p values < 0.05 and <0.01 were shown for the top and bottom panel, respectively. J. Immunoblotting showing alteration of EZH2 expression in multiple cells following a 24h treatment with 1μM pan FGFR inhibitor (BGJ398) or 20nM FGF2 recombinant (FGF2r). Primary cells generated from HCI011 (ER+ PDX) were cultured in 3D and treated with 1μM pan FGFR inhibitor (BGJ398) or vehicle for 24h. Normalized EZH2 expression (/β-actin) was shown as dot plots. (n=4 cell lines); *P value*: one-tailed unpaired Student t-test. K. Dot plots depicting the effect of 20nM recombinant FGF2 on H3k27me3 of multiple ER+ BC 3D models, based on quantified IF images (n=3 technical replicates). *P value*s: One-tailed paired Student t-tests. L. IF quantification of EZH2 expression in paired metastases and orthotopic tumor. MCF7 cells were transplanted to bone and to mammary gland of nude mice, which led to tumor formation at multiples sites including lung, ovary, bone and mammary gland. Metastatic tissues were harvested for IF quantification and shown as a dot plot graph. (n=3 images); Mean expression of EZH2 is indicated in blue. M. IF showing co-expression of ER and EZH2 in 3D monocultures and co-cultures (+FOB) models of MCF7 and PDX HCI011. Scale bars: 50μm. N. Heatmap showing relative expression of nuclear ER and EZH2 in MCF7 and PDX HCI011 SCs as depicted in M. O. Effect of the EZH2 inhibitor EPZ011989 on ESR1 mRNA expression after 24 hours of treatment; n=4 cell lines. P. Dot plots showing IF quantification of EZH2 and ER in MCF7 BMMs and macrometastases (n= 3–5 lesions). Q. Reversibility of epigenetic changes based on post-translational modification (PTM) analysis. The percent changes in H3k27me3 between parental cells (MCF7), M7-SCP2 and bone-derived (MCF7-Bo and M7-SCP2-Bo) after multiple passages *in vitro* (Passages: P0 and P10). (n=4 biological replicates); *P value*s are relatively to parental cells. The right panel depicts the temporal epigenetic changes (H3k27me3/H3) (**A–Q**): P values were computed by two-tailed unpaired Student t-tests unless otherwise noted.

**Figure 7:. EZH2 integrates multiple signals from BME and drives the phenotypic shift of ER+ breast cancer cells.**
A. Scheme to evaluate ER loss in syngeneic murine models. AT3 cells were pre-treated with EZH2 inhibitor (EPZ011989) for 2 weeks before being transplanted to bone of wild-type or osterix-depleted C57BL/6 mice (Osx-cre^ERT2 ROSA-LoxP-DTR). B. IHC staining depicting ER expression in orthotopic tumors derived from EZP011989 pretreated AT3 cells. C. IHC staining of ER in BoM models presented in A. D. Dot plots showing ER expression in IIA-induced AT3 BoM in control (WT-mice) and osteoprogenitor-depleted (Osx- cre^ERT2 ROSA-LoxP-DTR) mice at SC level. E. Progression free survival (PFS) curve of IIA-induced BoMs following treatment with the EZH2 inhibitor EPZ011989 or the ER inhibitor fulvestrant in combination or as single agents. BL images show the effect of combination treatment on IIA-induced BoMs. (n=9–10 mice); Log-rank test was used for survival analysis. * p<0.05. F. microCT and H&E images depicting tumor burden after pre-clinical experiment described in E. All groups revealed BoM formation except for combination treatment group (EPZ > Fulvestrant). G. Growth curve showing the effect of EPZ011989 pretreatment on the fulvestrant response of endocrine resistant ZR75-1 BoMs. Single agent and combination treatment groups are shown in blue and red, respectively. Multiple ANOVA was used for statistical analysis. BLI of metastatic burden at week 8 was shown as dot plot. (n= 6 and 8 mice for Fulvestrant and combination group, respectively. H. PET-CT images showing 18F-FDG uptake in spontaneous BoMs (Hind limbs) following single agent (fulvestrant) or combination (EPZ > fulvestrant) treatment. (n=4 mice). **(A-H):** P values were computed by two-tailed unpaired Student t-tests unless otherwise noted.

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Comment in

Evolving barcodes shed light into evolving metastases.
Kang Y, Kuperwasser C. Kang Y, et al. Dev Cell. 2021 Apr 19;56(8):1077-1079. doi: 10.1016/j.devcel.2021.03.029. Dev Cell. 2021. PMID: 33878297

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[1] Afgan E, Baker D, Batut B, Van Den Beek M, Bouvier D, Ech M, Chilton J, Clements D, Coraor N, Grüning BA, et al. (2018). The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 46, W537–W577 - PMC - PubMed

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The bone microenvironment increases phenotypic plasticity of ER⁺ breast cancer cells

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The bone microenvironment increases phenotypic plasticity of ER⁺ breast cancer cells

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