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. 2018 Sep 10;34(3):466-482.e6.
doi: 10.1016/j.ccell.2018.08.001. Epub 2018 Aug 30.

Epigenetic and Transcriptomic Profiling of Mammary Gland Development and Tumor Models Disclose Regulators of Cell State Plasticity

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Free PMC article

Epigenetic and Transcriptomic Profiling of Mammary Gland Development and Tumor Models Disclose Regulators of Cell State Plasticity

Christopher Dravis et al. Cancer Cell. .
Free PMC article

Abstract

Cell state reprogramming during tumor progression complicates accurate diagnosis, compromises therapeutic effectiveness, and fuels metastatic dissemination. We used chromatin accessibility assays and transcriptional profiling during mammary development as an agnostic approach to identify factors that mediate cancer cell state interconversions. We show that fetal and adult basal cells share epigenetic features consistent with multi-lineage differentiation potential. We find that DNA-binding motifs for SOX transcription factors are enriched in chromatin that is accessible in stem/progenitor cells and inaccessible in differentiated cells. In both mouse and human tumors, SOX10 expression correlates with stem/progenitor identity, dedifferentiation, and invasive characteristics. Strikingly, we demonstrate that SOX10 binds to genes that regulate neural crest cell identity, and that SOX10-positive tumor cells exhibit neural crest cell features.

Keywords: breast cancer; cancer stem cells; cell state plasticity; intra-tumoral heterogeneity; mammary gland development; mammary stem cells; metastasis; neural crest cells; triple-negative breast cancer.

Conflict of interest statement

Declaration of Interests

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Multi-lineage potential is present in fetal and adult basal mammary cells.
(A) Experimental strategy for epigenetic and transcriptional profiling of mammary cells. (B) Representative ATAC-seq profiles of biological replicates from fMaSCs, adult basal cells, luminal progenitors (LP), and mature luminal cells (ML). (C) Total numbers of ATAC-seq peaks in mammary cells, separated into promoter (<±3 kb Transcription Start Site (TSS)) and distal regions (>±3 kb TSS). ***p<0.001 (LP and ML vs. both fMaSC and basal). (D) RNA-seq expression of basal and luminal genes across mammary subpopulations, from two averaged biological replicates. The thick horizontal middle line is the media; height of the box is the interquartile range (IQR); dotted vertical line is 1.5X IQR; dots are the outliers. (E) ATAC-seq and RNA-seq of basal and luminal genes across mammary cell subpopulations. Mean ± SEM (n=2). See also Figures S1 and S2 and Table S1.
Figure 2.
Figure 2.. Chromatin features associate SOX10 with the mammary stem cell state.
(A, B) ATAC-seq signal at UARs (A) and corresponding H3K27ac signal (B) specific to the indicated mammary cell type; each row represents a specific genomic locus. (C) Shannon entropy of UARs vs. all ATAC-seq peaks. (D) Percentage of UARs and URRs located at distal (>±3kb TSS) or promoter (<±3kb TSS) regions. (E) GREAT analysis of genes associated with cell type specific UARs. (F) Enrichment of transcription factor motifs at UAR/URR across mammary cell subpopulations. (G) Transcript level of Sox factors from F. Mean ± SEM (n=2). See also Figure S3 and Table S2.
Figure 3.
Figure 3.. SOX10 is expressed in mammary tumors.
(A) Strategy to modify the Sox10 locus and characterize SOX10high and SOX10low tumor cells. (B) tdTomato fluorescence (y-axis) from control and Sox10tdTomato tumor cells grown in 2-D (top) and sorting strategy to isolate luminal-like and basal-like mammary tumor cells and evaluate tdTomato fluorescence (bottom). (C) ATAC-seq of the Sox10 locus in PY230 tumor cells grown in 2-D or from orthotopic tumors. (D) Wholemount view of Sox10 expression in C3–1 and Trp53;Brca1 mammary tumors with a Sox10-H2BVenus reporter. (E) Sox10 transcript levels in tumor cells sorted by Sox10 fluorescent reporter signal. Mean ± SEM (PY230: n=2; C3–1 SOX10high: n=10; C3–1 SOX10low: n=4). (F) SOX10 expression in human breast cancers from 2012 TCGA (n=508). The thick horizontal middle line is the media; height of the box is the interquartile range (IQR); dotted vertical line is 1.5X IQR; dots are the outliers. (G) Tissue section from ERPRHER2 breast tumor immunostained for SOX10, K8, and K14. See also Figure S4 and Table S3.
Figure 4.
Figure 4.. SOX10+ tumor cells exhibit mammary stem/progenitor features.
(A) GSEA of fMaSC genes in SOX10high vs. SOX10low tumor cells. (B) Relative expression of LP and ML genes in PY230 tumor cells. Mean ± SEM (n=2). (C) TF motifs enriched in chromatin regions differentially accessible between SOX10high vs. SOX10low PY230 tumor cells. (D) ATAC-seq of PY230 tumor cells at stem/progenitor- and ML-associated loci. (E) PCA of normal and tumor mammary cell types using ATAC-seq signal (left) and the interpretation of projected tumor PC scores shown as heatmaps (right). (F) Correlation of chromatin accessibility in PY230 tumor cells with UARs and URRs in normal mammary cells. (G) GSEA of UAR or URR associated gene sets, and genes upregulated following Sox10OE in SOX10high vs. SOX10low tumor cells. (H) Expression levels of stem/progenitor or ML genes in SOX10high (upper 50%) and SOX10low (lower 50%) human breast tumors, taken from RNA-seq of 2012 TCGA breast tumors (n=528). The thick horizontal middle line is the media; height of the box is the interquartile range (IQR); dotted vertical line is 1.5X IQR; dots are the outliers. See also Figure S5 and Table S4.
Figure 5.
Figure 5.. SOX10+ tumor cells exhibit de-differentiation and mesenchymal features.
(A, B) Low (A) and high (B) magnification image of C3–1; Sox10-H2BVenus mammary tumors immunostained for K8, K14, and GFP (SOX10). (C) Single cell dissociation of a C3–1; Sox10-H2BVenus mammary tumor immunostained for K8 and K14. (D) Quantification of keratin status in four C3–1; Sox10-H2BVenus mammary tumors. Average percentage and 95% confidence interval from two images for each tumor are shown. (E) Tumorsphere grown from C3–1; Sox10-H2BVenus mammary cells plated in 3-D culture, immunostained for K8 and K14. (F) Relative expression of differentiation and mesenchymal genes in PY230 tumor cells. Mean ± SEM (n=2). (G) PY230 Sox10tdTomato tumor showing SOX10+ cells (red) in the primary tumor margin and near vasculature (white). PY230 Sox10tdTomato tumor cells were labeled with a LV-GFP to visualize tumor cells not expressing SOX10 (green). (H) Rank order list of SOX10 co-expression genes in human breast tumors with epithelial (blue) and EMT-associated (red) genes highlighted. (I) Tissue section from an ERPRHER2 human breast tumor immunostained for SOX10 and VIM. See also Figure S5 and Movie S1.
Figure 6.
Figure 6.. Neural crest cell features are present in SOX10+ tumor cells.
(A) GSEA of NCC-related genes in SOX10high vs. SOX10low tumor cells. (B) Heatmap of NCC-related genes that are >1.5 fold up-regulated in SOX10high cell fractions of PY230 tumors (n=2). (C) ATAC-seq of NCC-specifier genes in SOX10high PY230 tumor cells compared to LP and ML. (D) Venn diagram showing overlap of NCC-related genes with genes showing more accessible chromatin in SOX10high PY230 tumor cells. (E) GSEA of SOX10 co-expression genes from the TCGA with GO NCC-migratory (GO:0001755) and NCC-differentiation (GO:0014033) genes. See also Figure S6 and Table S5.
Figure 7.
Figure 7.. SOX10 correlates with differentiation state and functionally contributes to tumor development.
(A) Representative profiles of ChIP-seq from SOX10-biotinylated (two biological replicates) and control PY230 tumors. All ChIP-seq signals are shown as RPM over input. (B) SOX10 and control ChIP-seq signal at all SOX10 binding sites (FDR<1×10−100). (C) Average SOX10 ChIP-seq signal with 95% confidence interval (CI) at UARs of each mammary cell type. (D) BETA summary of SOX10 function as a transcriptional activator and repressor in PY230 cells. (E) Specific activated and repressed targets of SOX10 binding from BETA. (F) SOX10 ChIP-seq profiles at stem/progenitor, EMT, and NCC genes. (G) GREAT analysis with genes positively or negatively regulated by SOX10 binding. (H) Tumor formation following orthotopic transplantation of wild-type and Sox10Null PY230 cells. (I) Kaplan-Meier survival curve for C3–1 Sox10WTor Sox10WT/Null animals. See also Figure S7 and Table S6.

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