Abstract
Tumour-initiating cells (TICs) are responsible for metastatic dissemination and clinical relapse in a variety of cancers1,2. Analogies between TICs and normal tissue stem cells have led to the proposal that activation of the normal stem-cell program within a tissue serves as the major mechanism for generating TICs3,4,5,6,7. Supporting this notion, we and others previously established that the Slug epithelial-to-mesenchymal transition-inducing transcription factor (EMT-TF), a member of the Snail family, serves as a master regulator of the gland-reconstituting activity of normal mammary stem cells, and that forced expression of Slug in collaboration with Sox9 in breast cancer cells can efficiently induce entrance into the TIC state8. However, these earlier studies focused on xenograft models with cultured cell lines and involved ectopic expression of EMT-TFs, often at non-physiological levels. Using genetically engineered knock-in reporter mouse lines, here we show that normal gland-reconstituting mammary stem cells9,10,11 residing in the basal layer of the mammary epithelium and breast TICs originating in the luminal layer exploit the paralogous EMT-TFs Slug and Snail, respectively, which induce distinct EMT programs. Broadly, our findings suggest that the seemingly similar stem-cell programs operating in TICs and normal stem cells of the corresponding normal tissue are likely to differ significantly in their details.
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Acknowledgements
The pBl.1 and pBl.3 murine PyMT tumour cell lines, from which pBl.1G and pBl.3G were derived, were gifts from the Harold L. Moses laboratory. We thank G. Bell for helping analysing the ChIP-seq data. We thank R. Bronson for help assessing the histopathology of the murine tumours. We thank A. Lambert and S. Thiagalingam for providing the MCF10A-Ras and MCF10A-Ras-C cells. We thank W. Israelsen and M. Vander Heiden for providing the MMTV-cre;p53+/−;BRCA1fl/fl murine mammary tumour samples. We thank the Keck Microscopy Facility at the Whitehead Institute for microscopy assistance and the Koch Institute Swanson Biotechnology Center (SBC) for technical support, especially the Histology and ES cell and Transgenics Cores. R.A.W. is an American Cancer Society and Ludwig Foundation professor. W.L.T. is supported by the National Research Foundation, Singapore (NRF-NRFF2015-04). This research was supported by the Breast Cancer Research Foundation, the Samuel Waxman Cancer Research Foundation, the Ludwig Center for Molecular Oncology at MIT, National Cancer Institute Program P01-CA080111, R01-CA078461, U01-CA184897 (to R.A.W.), K99-CA194160 (to X.Y.), the Wilshire Charitable Foundation/Andria and Paul Heafy Postdoctoral Fellowship (to X.Y.), the Mattina R. Proctor Foundation, and the Helen Hay Whitney Foundation (to X.Y.).
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X.Y. and R.A.W. conceived the project and prepared the manuscript. X.Y. designed and performed the experiments and analysed the data. W.L.T. contributed to the ChIP-seq experiments. T.S. generated the knock-in animals. Y.K. performed experiments and quantifications. F.R. performed the mammary fat pad injections. E.E. provided technical support.
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Competing financial interests: R.A.W. is a founder of and advisor for Verastem, Inc.
Extended data figures and tables
Extended Data Figure 1 Slug expression is associated with a partial EMT phenotype in normal MECs.
a, Validation of the Slug–YFP knock-in reporter. Mammary tumour sections from SlugYFP/+;MMTV-PyMT female mice were stained for YFP (green), Slug (red), cytokeratin (grey) and DAPI (blue). b, Validation of the Snail–YFP knock-in reporter. Mammary tumour sections from SnailYFP/+;MMTV-PyMT female mice were stained for YFP (green), Snail (red), cytokeratin (grey) and DAPI (blue). c, Lin− cells of normal mammary glands were separated into luminal MECs, basal MECs and stromal fibroblasts using CD24 and CD49f cell-surface markers. d, e, Representative FACS histogram showing relative expression levels of Slug–YFP and Snail–YFP reporters in the indicated cell populations in mammary glands during puberty (d) and during pregnancy (e). Note that luminal MECs from pregnant females exhibit higher levels of autofluorescence signals (grey dashed line in panel e). f, Normal human mammary tissue sections were stained for Slug or Zeb1 (green), CK14 (red), CK8 (grey), and DAPI (blue). Arrowheads indicate Slug+CK14+ cells. g, Representative FACS histogram showing expression level of the epithelial cell-surface marker EpCAM in the indicated populations of the normal mammary gland. Panels d, e, g, are representative of three independent experiments. All scale bars indicate 10 μm.
Extended Data Figure 2 Differential expression of Snail and Slug in mammary tumours.
a, b, Quantifications of the frequencies of Slug–YFP+ and Snail–YFP+ tumour cells (a) and quantifications of Slug versus Snail expression (b) at different stages of mammary tumour development by immunofluorescence staining. For each stage, tumours from six animals were analysed for the quantifications. c, Individual channels of the stained image in Fig. 2e. d, Quantifications of E-cadherin and Zeb1 positivity (high-grade carcinomas from six animals were quantified). e, f, Quantification of CK8 and CK14 expression profile of Snail–YFP-positive and Slug–YFP-positive tumour cells. For each stage, tumours from six animals were analysed for the quantifications. b, d–f, n is number of cells.
Extended Data Figure 3 Snail activation is associated with invasive changes in mammary tumour cells ex vivo.
a, Freshly isolated tumour organoids stained for YFP (green), CK14 (red), CK8 (grey) and DAPI (blue). Note that only background staining was detected for YFP and CK14. Scale bar, 20 μm. b, Tumour organoids from animals of the indicated genotypes were cultured in type I collagen gel for 48 h and stained for YFP (green), phalloidin (red) and DAPI (blue). Scale bar, 10 μm. c, Frequency of CK8+CK14+ leader cells expressing Slug–YFP and Snail–YFP (n, number of cells). Tumour organoids from five different animals were analysed for each genotype. d, Schematic diagram summarizing expression patterns of Snail and Slug in the normal mammary gland and at different stages of mammary tumour development in the MMTV-PyMT model.
Extended Data Figure 4 Differential expression of Snail and Slug in MMTV-Neu and BRCA-1/p53-minus models of mammary tumours.
a–c, Representative immunofluorescence images of sections of aggressive MMTV-Neu tumours stained for DAPI (blue), cytokeratin (red) and Slug (green, a), Snail (green, b) or Zeb1 (green, c). Scale bar, 10 μm. d, H&E staining showing representative histology of differentiated area in MMTV-cre;p53+/−;BRCA1fl/fl tumours. Scale bar, 50 μm. e, Representative immunofluorescence images of the differentiated areas in MMTV-cre;p53+/−;BRCA1fl/fl tumours stained for the indicated proteins. Five tumours were analysed, and quantifications are shown in f (n, number of cells). Scale bar, 10 μm. g, H&E staining showing representative histology of differentiated area in MMTV-cre;p53+/−;BRCA1fl/fl tumours. Scale bar, 50 μm. h, Representative immunofluorescence images of the invasive areas in MMTV-cre;p53+/−;BRCA1fl/fl tumours stained for the indicated proteins. Five tumours were analysed, and quantifications are shown in i (n, number of cells).
Extended Data Figure 5 Differential expression of Snail and Slug in human breast cancer cell lines.
a, Representative immunofluorescence images of indicated human breast cancer cell lines stained for DAPI (blue), SNAIL (green) and SLUG (red). Scale bar, 10 μm. b, Quantification of SLUG versus SNAIL expression in indicated human breast cancer cell lines (n, number of cells). Five fields were counted for each cell line. c, Representative image showing the morphologies of the series of MCF10A cell lines in culture. Scale bar, 50 μm. d, Western blot showing expression of SLUG and SNAIL in the indicated MCF10A cell lines. Panels a–d represent two independent experiments. Uncropped western blots are available in Supplementary Information.
Extended Data Figure 6 Isolation of tumour cell subpopulations with differential Snail and Slug expression by FACS.
a, b, Representative whole-mount images showing tumour progression in the transplantation model of mammary tumours illustrated in Fig. 3a. The implanted cells initially formed rudimentary gland-like structures (a) and eventually progressed to become high-grade carcinomas that spontaneously metastasize to the lungs. The RFP marker allows detection of pulmonary metastases as shown in b. Scale bars, 500 μm. Images represent five independent experiments. c, d, FACS profiles of RFP+ tumour cells in the pulmonary metastases corresponding to the primary tumours shown in Fig. 3b, c. Major populations are outlined with dashed circles. e, SnailYFP/+;MMTV-PyMT tumour cells were separated into indicated populations by FACS. The morphologies of the unfractionated cells and the purified populations are shown. Scale bar, 50 μm. f, Western blots showing expression of EMT markers in the indicated cell populations. g, SlugYFP/+;MMTV-PyMT tumour cells were separated into indicated populations by FACS. The morphologies of the unfractionated cells and the purified populations are shown. Scale bar, 50 μm. h, Western blots showing expression of Slug, YFP and Snail in the indicated cell populations. Uncropped western blots are available in Supplementary Information. e–h, Data represent three independent experiments.
Extended Data Figure 7 Fractionation of primary mammary tumours.
a, Experimental scheme for Fig. 3f, g and Extended Data Fig. 8a–g. b, c, Tumour cell subpopulations from SnailYFP/+;MMTV-PyMT tumour cell line (b) and SlugYFP/+;MMTV-PyMT tumour cell line (c) were injected subcutaneously at limiting dilutions to score primary tumour formation. Tumour-initiation cell frequencies were evaluated by ELDA. b, c, Tumour initiation was scored and presented as (number of tumour incidences/number of injections).
Extended Data Figure 8 Breast TICs express Snail.
a, H&E staining showing the histology of the donor primary tumour where cells used in Fig. 3f were isolated from. Scale bar, 200 μm. b, The original pulmonary metastases spawned by the primary tumour (left panel), and pulmonary metastases formed by the indicated tumour cell populations following tail-vein injection. Scale bar, 500 μm. c, Higher magnification images of H&E-stained lung sections showing histology of the original pulmonary metastases in the donor animal (left panel), and pulmonary metastases formed by the Slug–YFPlowEpCAMlow tumour cells following tail-vein injection. Scale bar, 200 μm. d, Representative immunofluorescence staining image of sections of pulmonary metastases formed by the Slug–YFPlowEpCAMlow tumour cells were stained for DAPI (blue), Slug (green), CK14 (red) and CK8 (grey). Arrowheads indicate Slug-positive cells. Scale bar, 20 μm. Images represent four independent experiments. e, H&E staining of the donor primary tumour where cells used in Fig. 3g were isolated from (left panel) and H&E staining of primary tumours formed by the indicated populations following subcutaneous implantation (with 25% Matrigel). Scale bar, 200 μm. f, Primary tumour burdens formed by the indicated populations after subcutaneous implantation (for EpCAMlowSluglow cells 1 × 104 cells were injected, for the other two groups 1 × 105 cells were injected). Primary tumours and lungs were analysed 10 weeks post injection (n = 10 sites of injection for each group). Open circle indicates failure of tumour initiation. Source Data is associated with this figure. g, H&E staining of lung sections showing metastatic outgrowths spawned by the indicated cell populations following subcutaneous implantation. Scale bar, 500 μm.
Extended Data Figure 9 Snail and Slug are differentially employed by normal MaSCs and breast TICs.
a, Kaplan–Meier plots showing survival of patients with the indicated subtypes of breast cancers. Patient groups were separated based on SLUG (top row) or SNAIL (bottom row) mRNA expression. b, Western blot confirming Slug and Snail knockdown in established PyMT tumour cell line transduced with the indicated shRNA expression vectors. The shLuciferase (shLuc) shRNA was used as a control. c, Western blot confirming SLUG and SNAIL knockdown in MDA-MB-231 cells transduced with the indicated shRNA expression vectors. shLuc was used as a control. Uncropped western blots are available in Supplementary Information. d, Tumour-sphere formation efficiencies (no. tumour spheres/1,000 cells for MDA-MB-361 cells, and no. tumour spheres/200 cells for all the other cell lines) of the indicated human breast cancer cells transduced with shSLUG#2, shSNAIL#2 and the shLuc control (mean + s.d., n = 5 technical replicates per group). Data represent two independent experiments. e, f, SUM159 (e) and SUM149 (f) cells transduced with the indicated shRNAs were injected subcutaneously at limiting dilutions to score primary tumour formation. Tumour initiation was scored and presented as (no. of tumour incidences/no. of injections). Data represent two independent experiments. g, The organoid forming efficiencies of normal MECs transduced with the indicated shRNA expression vectors (mean ± s.d., n = 6 technical replicates per group, *P < 0.001, NS, not significant.). Scale bar, 100 μm. Data represent three independent experiments.
Extended Data Figure 10 Slug and Snail occupy different genomic regions.
a, Western blots showing expression of EMT-TFs and EMT markers in the PyMT tumour cell lines used for the ChIP-seq analyses. Uncropped western blots are available in Supplementary Information. Data represent three independent experiments. b, Pulmonary metastases formed by 100,000 cells of the indicated cell lines following tail-vein injection (n = 9 animals per group). Source Data is associated with this figure. c, Box plot showing distributions of fold enrichment of all peaks identified in Snail ChIP and Slug ChIP. Horizontal bar indicates the median and whiskers indicate the top and bottom tertiles. d, Sample top motifs enriched around the summits of the anti-Snail and anti-Slug ChIP peaks. e, Sample ChIP-seq signals for Slug and Snail are shown. Left column shows promoters bound by Slug only. Right column shows promoters bound by Snail only. Arrows indicate the directions of transcription. f, MCF10A human mammary epithelial cells were transduced with rtTA and SNAIL driven by a tet-on promoter, untreated (left panel) or treated with 2 μg ml−1 doxycycline (dox) for 48 h (right panel), and stained for E-cadherin (green) and ZEB1 (red). Scale bar, 20 μm. Data represent five independent experiments.
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Ye, X., Tam, W., Shibue, T. et al. Distinct EMT programs control normal mammary stem cells and tumour-initiating cells. Nature 525, 256–260 (2015). https://doi.org/10.1038/nature14897
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DOI: https://doi.org/10.1038/nature14897
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