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Snail1-dependent p53 repression regulates expansion and activity of tumour-initiating cells in breast cancer

Nature Cell Biology volume 18, pages 1221–1232 (2016)Cite this article

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Abstract

The zinc-finger transcription factor Snail1 is inappropriately expressed in breast cancer and associated with poor prognosis. While interrogating human databases, we uncovered marked decreases in relapse-free survival of breast cancer patients expressing high Snail1 levels in tandem with wild-type, but not mutant, p53. Using a Snail1 conditional knockout model of mouse breast cancer that maintains wild-type p53, we find that Snail1 plays an essential role in tumour progression by controlling the expansion and activity of tumour-initiating cells in preneoplastic glands and established tumours, whereas it is not required for normal mammary development. Growth and survival of preneoplastic as well as neoplastic mammary epithelial cells is dependent on the formation of a Snail1/HDAC1/p53 tri-molecular complex that deacetylates active p53, thereby promoting its proteasomal degradation. Our findings identify Snail1 as a molecular bypass that suppresses the anti-proliferative and pro-apoptotic effects exerted by wild-type p53 in breast cancer.

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Figure 1: Snail1 expression levels correlate negatively with breast cancer patient survival harbouring wild-type, but not mutant, TP53 alleles.
Figure 2: Conditional deletion of Snail1 in MaECs restrains the formation and metastatic potential of MMTV-PyMT-induced mammary tumours.
Figure 3: Loss of Snail1 in MaECs inhibits expansion and activity of PyMT-induced TICs.
Figure 4: PyMT-cKO tumours retain differentiated status and exhibit impaired growth responses.
Figure 5: Snail1 maintains carcinoma cell de-differentiation.
Figure 6: Snail1 acts as a p53 repressor.
Figure 7: A Snail1/p53 axis regulates breast cancer tumorigenesis.

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Acknowledgements

This research was supported by a grant from the Breast Cancer Research Foundation (BCRF) to S.J.W. as well as the Startup Fund from China Pharmaceutical University, the National Natural Science Foundation of China (NSFC 81572745 and 91539115), and the Jiangsu Province Innovative Research Team and State Key Laboratory of Natural Medicines (SKLNMZZJQ201604) to Z.-Q.W. Additional support was provided by the Changjiang Scholar and Innovative Research Team (IRT1193) to Q.-L.G. and the US National Institutes of Health (U01 CA180980) to X.S.L. We thank M.-D. Lai (China Pharmaceutical University, China) for critically reviewing this manuscript, W. Gu (Columbia University, USA) and P.-C. Yang (Academia Sinica, Taiwan) for providing HA-tagged p53 WT and mutant plasmids, R. Weinberg (Massachusetts Institute of Technology, USA) for sharing human mammary epithelial cells (HMLEs), A. Cano (Universidad Autonoma de Madrid, Spain) for Snail1 mutants, Y. Kang and L. Wan (Princeton University, USA) for sharing protocols of mouse mammary epithelial cell isolation, B. Gyorffy (Hungarian Academy of Sciences, Hungary) for assistance with the human breast cancer databases and R. Kuick (University of Michigan, USA) for helpful discussions.

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Author notes

  1. Ting Ni, Xiao-Yan Li and Na Lu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Basic Medicine, State Key Laboratory of Natural Medicines, Jiangsu Provincial Key Laboratory of Carcinogenesis and Intervention, School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing 210009, China

    Ting Ni, Na Lu, Teng An, Rong Fu, Wen-Cong Lv, Yi-Wei Zhang, Xiao-Jun Xu, Qing-Long Guo & Zhao-Qiu Wu

  2. Division of Molecular Medicine and Genetics, Department of Internal Medicine, The Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, USA

    Xiao-Yan Li, R. Grant Rowe, Yong-Shun Lin, Amanda Scherer, Tamar Feinberg & Stephen J. Weiss

  3. Department of Biochemistry and Molecular Biology, School of Basic Medicine, Gannan Medical University, Ganzhou 341000, China

    Zhi-Ping Liu

  4. Department of Mathematics, Shanghai Normal University, Shanghai 200234, China

    Xiao-Qi Zheng

  5. Department of Hematology and Oncology, The Affiliated Zhongda Hospital, Southeast University Medical School, Nanjing 210009, China

    Bao-An Chen

  6. Department of Biostatistics and Computational Biology, The Dana-Farber Cancer Institute, Harvard School of Public Health, Harvard University, Boston, Massachusetts 02115, USA

    X. Shirley Liu

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  1. Ting Ni
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Contributions

Z.-Q.W. and S.J.W. designed and supervised the project, analysed data, wrote the manuscript and approved the final version; T.N., X.-Y.L. and N.L. conducted experiments and analysed data. T.A., R.F., W.-C.L., Y.-W.Z., R.G.R., Y.-S.L., A.S. and T.F. conducted experiments. Z.-P.L., X.-J.X., X.-Q.Z., B.-A.C., X.S.L. and Q.-L.G. analysed data and provided relevant advice.

Corresponding authors

Correspondence to Zhao-Qiu Wu or Stephen J. Weiss.

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Integrated supplementary information

Supplementary Figure 1 A relationship between relapse-free survival rate and Snail1 expression level in different molecular subtypes of breast cancer patients.

(a,b) Kaplan–Meier survival analysis of the relationship between relapse-free survival (RFS) rates and Snail1 expression level in HER2+ (a) or basal (b) subtype of breast cancer patients bearing wild-type or mutant TP53 alleles. Survival data are fitted by the ‘survfit’ function. Kaplan-Meier curves are drawn by the ‘ggsurv’ function in R package ‘survival’. Differences between two survival curves are measured by the G-rho family of tests. n represents number of patients.

Supplementary Figure 2 Snail1 excision in MaECs impairs PyMT-induced tumour progression without affecting mammary gland development or lactation.

(a) Cross-sections of X-gal/LacZ staining in 7 wk-old preneoplastic glands (left panel) and 19 wk-old primary tumours (right panel) derived from PyMT-cKO;ROSA-LacZ mice (the image is representative of images from 5 mice). Strong X-gal/LacZ staining is detected in the epithelium, but not stromal compartment. (b) Western blot analysis of Lin−pNECs or Lin−NECs derived from preneoplastic glands (7 wks of age) and neoplastic tumours (19 wks of age) of PyMT-WT and PyMT-cKO;ROSA-LacZ mice (results are representatives of five independent experiments). (c) Gross view of primary tumours isolated from 15 wk-old PyMT-WT and PyMT-cKO mice (the image is representative of images from 5 mice). Dashed areas outline the primary tumours developing from the proximal to distal sites of the mammary glands. (d) Whole-mount Carmine red staining of mammary glands from WT and cKO mice at 5, 9 and 15 weeks of age as well as lactation day 3 (the image is representative of images from 5 mice). Unprocessed blots are shown in Supplementary Fig. 8.

Supplementary Figure 3 Flow cytometry analysis of preneoplastic glands from PyMT-WT and -cKO mice.

(a) Quantification of Lin−CD29loCD24+ basal subpopulation in normal glands from WT and cKO mice as well as preneoplastic glands from PyMT-WT and-cKO mice. Data are presented as mean ± s.e.m. (n = 6 independent experiments). ∗∗P < 0.01, two-sided Student’s t test. (b) Flow cytometry of Lin−CD29+CD90+ subpopulation in preneoplastic glands from PyMT-WT and-cKO mice (the image is representative of images from 6 mice). (c) Quantification of Lin−CD29+CD90+ subpopulation in preneoplastic glands as described in (b). Data are presented as mean ± s.e.m. (n = 6 independent experiments). N.S., not significant, two-sided Student’s t test. (d) Size of tumourspheres formed by PyMT-WT or-cKO pNECs dissected from mammary glands of 6 wk-old mice. 1°, 2° and 3° denote the three successive generations of pNECs used in the assay, respectively. Data are presented as mean ± s.e.m. (n = 6 independent experiments). ∗∗P < 0.01, two-sided Student’s t test.

Supplementary Figure 4 Preneoplastic glands and advanced tumours from PyMT-cKO mice exhibit defects in collective invasion, proliferation and survival.

(a) Immunohistochemical staining of K14 expression at the tumour-stromal borders in primary tumours isolated from 13 wk-old PyMT-WT and-cKO mice (the image is representative of images from 5 mice). Magnified images of boxed areas in red and brown (at left) are shown in the middle and right panels, respectively. Arrows mark invasive strands that are characteristic of K14+ invading leader cells at the tumour-stromal borders. (b) Immunohistochemical staining of Ki67, phospho-Histone H3 and cleaved caspase 3 in the preneoplastic mammary glands of 4 wk-old PyMT-WT and-cKO females (the image is representative of images from 5 mice). Magnified areas of boxed sections are shown in the bottom panels. Green arrows in middle panels denote phospho-Histone H3-positive cells (that is, proliferative cells). Red arrows in right panels denote cleaved caspase 3-positive cells (that is, apoptotic cells). (c–e) Quantification of Ki67 (c), phospho-Histone H3 (p-H3; d) and cleaved caspase 3 (C-casp3; e) positive cells (%) as shown in (b). One thousand to two thousand cells were counted in 10 random fields of each slide. Data are presented as mean ± s.e.m. (n = 5 independent experiments). ∗P < 0.05, ∗∗P < 0.01, two-sided Student’s t test.

Supplementary Figure 5 Snail1 maintains TIC function in established adenocarcinomas.

(a) Flow cytometry analysis (CD24/CD29/CD61 profiling) of LIN− mammary epithelial cells isolated from transplanted tumours (n = 5, each) that were derived from adeno-βGal or-Cre transduced Snail1fl/fl adenocarcinoma cells. Tumours were retrieved 9 wks post-transduction. (b) Quantification of LIN−CD29loCD24+/LIN−CD29loCD61+ subpopulations in control and Snail1-deleted orthotopic tumours. Data are presented as mean ± s.e.m. (n = 5 independent experiments). NS, not significant, two-sided Student’s t test. (c) Flow cytometry analysis (CD24/CD90 profiling) of LIN− mammary epithelial cells isolated from transplanted tumours (the image is representative of images from 5 mice) as described in (a). (d) Quantification of LIN−CD24+CD90+ subpopulations in control and Snail1-deleted orthotopic tumours. Data are presented as mean ± s.e.m. (n = 5 independent experiments). NS, not significant, two-sided Student’s t test. (e) Flow cytometry analysis of ALDH activity of LIN− mammary epithelial cells isolated from transplanted tumours (the image is representative of images from 5 tumours) that were derived from Snail1fl/fl adenocarcinoma cells. Tumours were retrieved 9 wks post-transduction. DEAB-treated samples were used as gating control (left panel). (f,g) Sorted Lin−ALDH− and Lin−ALDH+ cells from transplanted tumours as described in (e) were subjected to tumoursphere formation assay. Representative tumourspheres are shown (f) with quantification of tumourshphere formation (number and size) (g). Data are presented as mean ± s.e.m. (n = 5 independent experiments). ∗∗P < 0.01, two-sided Student’s t test. (h) Flow cytometry analysis (CD24/CD61/CD29/ALDH profiling) of Lin− mammary epithelial cells isolated from transplanted tumours (the image is representative of images from 5 tumorus) as described in (e). (i) Quantification of Lin−ALDH+ TICs isolated from control and Snail1-deleted orthotopic tumours. Data are presented as mean ± s.e.m. (n = 5 independent experiments). ∗P < 0.05, two-sided Student’s t test. (j) Quantification of tumoursphere formation (number and size) by adeno-βGal or-Cre transduced Lin−ALDH+ cells sorted from transplanted tumours as described in (e). Data are presented as mean ± s.e.m. (n = 5 independent experiments). ∗P < 0.05, ∗∗P < 0.01, two-sided Student’s t test. Source data are provided in Supplementary Table 1 (h).

Supplementary Figure 6 Deletion of Snail1 represses tumour proliferation and survival but does not affect Rb activity or induce DNA damage in PyMT-induced tumours.

(a) Immunohistochemical staining of Ki67, phospho-Histone H3 and cleaved caspase 3 in control and Snail1-deleted orthotopic tumours (the image is representative of images from 5 tumours). Red arrows in panels to the right indicate cleaved caspase 3-positive cells. (b) Quantification of Ki67, phospho-Histone H3 (p-H3) and cleaved caspase 3 (C-casp3) positive cells (%) as shown in (a). One thousand to two thousand cells were counted in 10 random fields of each slide. Data are presented as mean ± s.e.m. (n = 5 independent experiments). ∗∗P < 0.01, two-sided Student’s t test. (c-e) Immunohistochemical staining of p-Rb (c), p-ATM (d) and γ-H2AX (e) in preneoplastic glands of 7 wk-old PyMT-WT or PyMT-cKO mice (left panels) or formed from neoplastic Snail1fl/fl mammary epithelial cells that were transduced with adeno-βGal or-Cre prior to a 9 wk transplantation period (right panels). The image is representative of images from 5 mice or orthotopic tumours. (f) Quantification of p-Rb, p-ATM and γ-H2AX positive cells (%) as shown in c–e. One thousand to two thousand cells were counted in 10 random fields of each slide. Data are presented as mean ± s.e.m. (n = 5 independent experiments). NS, not significant, two-sided Student’s t test. (g) Immunoblot analysis of Lin−NECs isolated from primary tumours of 12 wk-old PyMT-WT and PyMT-cKO mice (the image is representative of images from 5 mice). Arrows denote the specific bands with their expected molecular weights. Unprocessed blots are shown in Supplementary Fig. 8.

Supplementary Figure 7 Snail1 binds and negatively regulates p53 protein levels.

(a) pNECs or NECs were recovered from 7 wk-old or 19 wk-old PyMT-WT mice, respectively. Cells were transduced with adeno-βGal or-Cre, and subjected to RT-PCR analysis. Data are presented as mean ± s.e.m. (n = 5 independent experiments). ∗∗P < 0.01, one-way ANOVA test. (b) Cycloheximide (CHX; 100 μg ml−1) pulse-chase analysis of p53 protein levels in control or Snail1-deleted pNECs as shown in a. Quantification of p53 protein expression levels are shown in bottom panel. (c) 293T cells were co-transfected with the indicated plasmids at a ratio of 1:10 and treated with MG132 (10 μM) for 6 h. Cell lysates collected for IP) analysis. (d) Immunofluorescent staining of p53 and Pan-AcK in NECs as described in (c). Arrows denote the cells expressing comparable levels of p53 between adeno-βGal and-Cre transduced cells. (e) GST control or GST-p53 protein bound to GST beads was co-incubated with FLAG-tagged Snail1 protein purified from rabbit reticulocyte lysates, and the mixture subjected to GST pull-down assays. (f) Lysates form MDA-MB-231 cells were subjected to IP assay. (g) 293T cells were co-transfected with equal amount of HA-p53-WT and FLAG-Snail1, and the cell lysates were prepared and subjected to IP analysis. (h) 293T cells co-transfected with GFP-p53 and FLAG-Snail1 at a ratio of 1:10, and then treated with 1 μM trichostatin A (TSA) for 24 h. Cell lysates were prepared for immunoblotting. (i) 293T cells were co-transfected with the indicated plasmids at a ratio of 1:1:10 and treated with DMSO or MG132 (10 μM) for 6 h. Cell lysates collected for IP analysis. The numbers shown in the blots are the ratios of MDM2/p53 in the IP’ed products. Blots are representatives of three independent experiments. Asterisks and arrows denote IgGs and specific bands with their expected molecular weights, respectively. Results are representatives of three (c,e–i) or five (b,d) independent experiments. Unprocessed blots are shown in Supplementary Fig. 8. Source data are provided in Supplementary Table 1 (b,i).

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Ni, T., Li, XY., Lu, N. et al. Snail1-dependent p53 repression regulates expansion and activity of tumour-initiating cells in breast cancer. Nat Cell Biol 18, 1221–1232 (2016). https://doi.org/10.1038/ncb3425

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