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Normal and cancerous mammary stem cells evade interferon-induced constraint through the miR-199a–LCOR axis

Nature Cell Biology volume 19, pages 711–723 (2017)Cite this article

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Abstract

Tumour-initiating cells, or cancer stem cells (CSCs), possess stem-cell-like properties observed in normal adult tissue stem cells. Normal and cancerous stem cells may therefore share regulatory mechanisms for maintaining self-renewing capacity and resisting differentiation elicited by cell-intrinsic or microenvironmental cues. Here, we show that miR-199a promotes stem cell properties in mammary stem cells and breast CSCs by directly repressing nuclear receptor corepressor LCOR, which primes interferon (IFN) responses. Elevated miR-199a expression in stem-cell-enriched populations protects normal and malignant stem-like cells from differentiation and senescence induced by IFNs that are produced by epithelial and immune cells in the mammary gland. Importantly, the miR-199a–LCOR–IFN axis is activated in poorly differentiated ER− breast tumours, functionally promotes tumour initiation and metastasis, and is associated with poor clinical outcome. Our study therefore reveals a common mechanism shared by normal and malignant stem cells to protect them from suppressive immune cytokine signalling.

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Figure 1: miR-199a is enriched in MaSCs and is functionally critical for MaSC activity.
Figure 2: miR-199a induces stem-cell-like gene signatures and is enriched in cancer stem cells.
Figure 3: Identification of LCOR as a direct target gene of miR-199a.
Figure 4: LCOR suppresses MaSC function and is downregulated in stem cell populations.
Figure 5: miR-199a and LCOR functionally influence the initiation of ER− breast tumours in vivo.
Figure 6: LCOR primes the IFN-α response.
Figure 7: Stem cells and differentiated cells respond differently to the IFN-α signalling.
Figure 8: Immune and autocrine IFN-related effects on mammary gland and tumour cells.

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Acknowledgements

We thank A. Welm for providing the PDX and A. Prat for technical advice for CL subtype classification. This work was supported by a Susan G. Komen Fellowship to T.C.-T. (PDF15332075), and grants from the Brewster Foundation, the Breast Cancer Research Foundation, Department of Defense (BC123187), and the National Institutes of Health (R01CA141062) to Y.K. This research was also supported by the Genomic Editing and Flow Cytometry Shared Resources of the Cancer Institute of New Jersey (P30CA072720).

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Authors and Affiliations

  1. Department of Molecular Biology, Princeton University, Princeton, New Jersey, 08544, USA

    Toni Celià-Terrassa, Daniel D. Liu, Abrar Choudhury, Xiang Hang, Yong Wei, Jose Zamalloa, Raymundo Alfaro-Aco, Rumela Chakrabarti, Bong Ihn Koh, Heath A. Smith, Christina DeCoste & Yibin Kang

  2. Lewis-Sigler Institute, Princeton University, Princeton, New Jersey, 08544, USA

    Jose Zamalloa

  3. Department of Breast Surgery, Fudan University Shanghai Cancer Center, Shanghai, 200032, China

    Yi-Zhou Jiang, Jun-Jing Li & Zhi-Ming Shao

  4. Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China

    Yi-Zhou Jiang, Jun-Jing Li & Zhi-Ming Shao

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  1. Toni Celià-Terrassa
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Contributions

T.C.-T. and Y.K. designed experiments. T.C.-T., D.D.L., A.C., X.H., Y.W., R.A.-A., R.C., B.I.K. and H.A.S. performed the experiments. J.Z., Y.-Z.J., C.D., J.-J.L. and Z.-M.S. provided crucial samples and technical advice. T.C.-T. and Y.K. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Yibin Kang.

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Integrated supplementary information

Supplementary Figure 1 miR-199a promotes MaSC activity and basal-like features.

(a) Mammary gland dissociation of different MEC (P3: Lin− epithelial cells) populations and the lentiviral strategy to conduct gain- and loss-of function experiments. P4 denotes the MaSC-enriched Lin−CD24+CD29high basal population and P5 the Lin−CD24+CD29low luminal population. (b) qRT-PCR test of the miR-199a ectopic expression after lentiviral transduction of pLEX-miR-199a expression construct in different mammary epithelial cells (MECs) P3: total MECs; P4: basal/MaSC-enrich MECs; and P5: luminal MECs. The test was performed at least in 3 independent experiments for each cell population, and the figure shows one representative test. Source of data in Supplementary Table 4. (c) P3 cells were isolated and transduced with the control or miR-199a lentiviruses and subjected to limited dilution cleared fat pad reconstitution assay. (d) P4 cells were isolated and transduced with the indicated miRZIP constructs and subjected to limited dilution cleared fat pad reconstitution assay. In c and d Representative images show outgrowth. Each pie chart represents a mammary gland with the blackened area denoting the percentage of mammary gland outgrowth. Tables below represent serial dilution injections with the corresponding take rate. n = number of mammary fat pad injections as indicated in the table. Shown in red are the repopulation frequencies for each condition and P value by Pearson’s Chi-squared test, obtained with the ELDA software. (e) Keratin-14 (K14-green) and Keratin-8 (K8-red) staining with reconstituted mammary outgrowths from control and miR-199a-OE P5 cells. (f) Quantification of the K14+ basal cells of mammary outgrowth from control and miR-199a-OE P3, P4 and P5 cells. n = 10 ducts and terminal end buds (TEB) sections scored from 3 mammary outgrowths. (g) Table representing the mammary gland take-rate after cleared fat pad injection of unsorted MECs overexpressing miR-199a or control vector after 3 generations. Injected cell numbers are indicated. Scale bars: 2 mm in c and d; 25 μm in e. ∗P < 0.05, ∗∗P < 0.01 by two-tailed Student’s t-test in f.

Supplementary Figure 2 miR-199a enhances stem cell-like properties without inducing epithelial-mesenchymal transition (EMT).

(a) Venn diagram representation of the overlap of up-regulated miRNAs in P4-MaSCs with the up-regulated miRNAs in Claudin-low (CL) tumors based on the TCGA dataset (2015 release). The common core represents the top-5 common miRNAs in the CL subtype. (b) qRT-PCR quantification of miR-199a levels in control, TGF-β-treated and Twist1-ER-OE HMLE cells. (c) qRT-PCR analysis of EMT markers and transcription factors in miR-199a-OE and control HMLE cells. (d) GSEA of the reported transcriptional factors Nanog-Oct4-Sox2 (NOS-TFs) targets gene set1 in the ranked gene list of miR-199a-OE HMLE cells versus control cells. n = 3 biologically independent samples; data represents mean ± s.e.m. in b and c. ∗P < 0.05, ∗∗P < 0.01 by two-tailed Student’s t-test in b and c.

Supplementary Figure 3 Evaluation of LCOR as a miR-199a target.

(a) Western blot analysis of LCOR protein level in P4 (MaSCs) and P5 (Luminal) cells. (b) qRT-PCR analysis of Lcor in MaSC-enriched P4 cells, luminal progenitor cells (P5-CD61+) and luminal mature cells (P5-CD61−); n = 3 biologically independent samples; data represents mean ± s.e.m. (c,d) qRT-PCR analysis of LCOR expression upon miR-199a ectopic expression in multiple mammary epithelial cell lines (c) and breast cancer cell lines (d). ∗P < 0.05, ∗∗P < 0.01 by two-tailed Student’s t-test in b and P value by two-tailed Student’s t-test in c,d.

Supplementary Figure 4 LCOR is a potent MaSC suppressor.

(a,b) qRT-PCR analysis of Lcor mRNA level in P4 transduced with pLEX vector or pLEX-Lcor (a) and P5 transduced with Lcor shRNA (b). (c) P4 cells were isolated and transduced with the indicated constructs, followed by cleared fat pad injections of 1,000 cells (n = 9 mouse mammary glands). Representative images and pie charts show the outgrowth and mammary gland filling percentage. Two-tailed Student’s t-test showed non-significance (n.s.). (d) qRT-PCR analysis of LCOR expression in control and LCOR-KD HMLE cells. (e) Quantification of mammospheres formed by 5,000 control and LCOR-KD HMLE cells. (f) Quantification of mammosphere formation of 10,000 control or LCOR-OE HMLE cells; counted at 8 days in e and f. Scale bars: 2 mm in c. n = 3 biologically independent samples; data represents mean ± s.e.m. and ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005 by two-tailed Student’s t-test in a,b,d,e and f.

Supplementary Figure 5 miR-199a and LCOR clinical analysis and in vivo functional validation.

(a) Kaplan–Meier relapse free-survival (RFS) curve of triple-negative breast cancer (TNBC) patients stratified by higher or lower than the median miR-199a (n = 65 patients) and LCOR expression (n = 168 patients) in a previously described TNBC patient cohort2. (b) Multivariate analysis adjusted for age, tumor size, lymph nodes, tumor grade, miR-199a and LCOR expression in the TNBC samples2 (n = 168 patients). (c) Oncomine analysis of LCOR log2 median centered expression in triple-negative breast cancer (TN; n = 49 patients) compared to non-triple-negative breast cancer (n = 300 patients) (TCGA dataset). The box represents 75th, 50th and 25th percentile of values, and the whiskers represent maximum and minimum data points. (d) Inverse correlation of the expression of miR-199a and LCOR in TNBC, as analyzed by by ISH (miR-199a) and IHC (LCOR). Samples (n = 59 tumors) were scored as weak (low expression) or strong (high expression) according to staining intensities. (e) Kaplan-Meier distant metastasis-free survival (DMFS) curve of breast cancer patients stratified by higher or lower than the median LCOR expression using the NKI295 data-set3 (n = 147 patients). (f) Schematic diagram illustrating the procedure of patient-derived xenograft (PDX) maintenance in NSG mice, transduction and functional assays. (g) Tumor incidence of HCI-003 (ER+PR+) and HCI-009 (TNBC) upon mammary fat pad injection of indicated cells. n = number of mammary fat pad injections as indicated in the table. Tumor-initiating cell (TIC) frequency calculated by the ELDA software is indicated in red and P value by Pearson’s Chi-squared test. (h) Hematoxylin-eosin and LCOR IHC analysis of mammary tumors formed by mammary fat pad injection of the indicated PDX cells with or without the overexpression of miR-199a or LCOR. Scale bars: 50 μm in h. Log-rank test in a,e; Cox proportional hazard in b, Wilcoxon un-paired test in c, and Chi-square test in d.

Supplementary Figure 6 LCOR mutant functionality and response to interferons.

(a) Co-immunoprecipitation of co-transfected estrogen receptor (ER) and wild-type or LSKAA mutant LCOR in HMLE cells. LCOR and LSKAA are HA tagged and were immunoprecipitated with anti-HA antibody and inmmunoblotted for ER. Lanes correspond to: 10% Input and anti-HA pull-down. (b) Immunofluoresence analysis of WT and HTH mutant HA-LCOR using anti-HA antibody in HMLE cells. (c) Quantification of mammospheres formed by P4 (20,000 cells) and P5 (10,000 cells) mammospheres treated with 1000 U/ml IFN-α or IFN-γ (n = 5 biologically independent samples; data represented mean ± s.e.m.). Scale bars: 20 μm in b. ∗P < 0.05 by two-tailed Student’s t-test in c.

Supplementary Figure 7 LCOR induces an interferon response and senescent-differentiation state.

(a) K14 (green) and K8 (red) immunofluorescence staining of mammospheres formed by control or Lcor-OE P4 cells with or without IFN-α (1,000 U ml−1) treatment. (b) Quantification of mammospheres formed by HMLE after transduction with the indicated expression constructs, and with or without treatment with 1,000 U ml−1 IFN-α (n = 3 biologically independent samples; data represents mean ± s.e.m.), 10,000 cells were seeded and mammospheres counted at 8 days. (c,d) Quantification of PDX cell tumorspheres formed by 10,000 HCI-010 cells, with the indicated conditions (n = 3 biologically independent samples; data represents mean ± s.e.m.). (e) Senescence-associated β-galactosidase (SA-β gal) assay of HMLE cells with or without wt and ΔHTH mutant LCOR expression, and with or without IFN-α treatment for 48 h (n = 3 biologically independent samples; data represents mean ± s.e.m.; source of data in Supplementary Table 4). (f) GSEA of the senescence up-regulated gene set (M9143; ref. 4) in the ranked gene list of LCOR, LCOR-ΔHTH or miR-199a overexpressing HMLE cells versus control. (f) K14 (green) and K8 (red) immunofluorescence staining of mammospheres formed by control or Lcor-OE P4 cells with or without IFN-α (1000 U/ml) treatment. Scale bars: 25 μm in a, and 100 μm in e. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005 by two-tailed Student’s t-test in b,c and d.

Supplementary Figure 8 IFN-α secretion and clinical significance of the Interferon-Stem Cell Down Signature (ISDS).

(a) Flow cytometry plots representative of Fig. 8a, b data showing Isotype control (FITC Rat IgG1), IFN-α positive cells and percentage of macrophages within the IFN-α positive population in the indicated conditions. Numbers in red are the percentage of positive cells. (b) qRT-PCR analysis of Ifn-α and Ifn-β genes in virgin mammary gland macrophages and peritoneal macrophages (n = 3 biologically independent samples; data represents mean ± s.e.m.). (c) Kaplan–Meier relapse free survival (RFS) curves of individual ISDS genes in ER− breast cancer using the KM plotter5. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005 by two-tailed Student’s t-test in b. P-value by log-rank tests in c.

Supplementary Figure 9 Western blot scanned films.

Boxes highlight lanes used in figures.

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Celià-Terrassa, T., Liu, D., Choudhury, A. et al. Normal and cancerous mammary stem cells evade interferon-induced constraint through the miR-199a–LCOR axis. Nat Cell Biol 19, 711–723 (2017). https://doi.org/10.1038/ncb3533

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