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

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.

Figure 1. miR-199a is enriched in MaSCs and is functionally critical for MaSC activity

(a) Heat map representing miRNAs with >2-fold differential expression between P4 and P5 cells. (b) Table of selected miRNAs used for in vitro mammosphere (MS) and in vivo cleared fat pad (CFP) reconstitution analyses. (c) qRT-PCR analysis of the expression levels of the 3′ and 5′ arms (3p and 5p) of miR-199a in P4 compared to P5. n=4 biologically independent samples; data represented mean ± SEM. (d) In situ hybridization analysis (ISH) of miR-199a-5p in the terminal end buds (TEBs). miR-199a is stained blue and nuclei are stained in red. (e) P4 and (f) P5 cells transduced with the indicated constructs are used for limiting dilution cleared fat pad reconstitution assay. 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. (g) Krt14 (K14-green) and Krt8 (K8-red) staining with reconstituted mammary outgrowths from control and miR-199a-OE P4 cells. (h) Number of P5 mammospheres formed after 3 generations of in vitro passage, and the ratio of sphere number between miR-199-OE group vs. control. 5,000 cells in the indicated conditions were seeded (n=3 biologically independent samples; data represents mean ± SEM). (i) Confocal K14+K8 staining images of mammospheres from control and miR-199-OE P5 cells. (j) Left: Flow cytometry isolation of P4-Lgr5⁺ and P4-Lgr5⁻ cells from the Lgr5-EGFP knockin reporter mouse glands. Lgr5⁺ cells are represented in blue dots. Right: qRT-PCR analysis of miR-199a expression in Lgr5⁺ and Lgr5⁻ P4 cells (n=3 biologically independent samples; data represents mean ± SEM). Scale bars: 20 μm (upper panel) and 5 μm (lower panel) in d, 2 mm in e and f, 25 μm in g, and 40 μm in i. * P<0.05, ** P<0.01, *** P<0.005 by two-tailed Student’s t-test in bar graphs.

Figure 2. miR-199a induces stem cell-like gene signatures and is enriched in cancer stem cells

(a) GSEA demonstrating the enrichment of gene sets related to MaSC, CSC, undifferentiated tumor cells, and Claudin-Low tumors in the ranked gene list of miR-199a-OE vs. control HMLE cells. (b) Heat map of HMLE-miR-199a-OE microarray data representing fold change expression of EMT markers, stem cell transcription factors (SC-TFs), and stem cell (SC) markers. Fold change is represented as Log2 ratio. (c) Western blot analysis of epithelial (blue) and mesenchymal (red) markers. (d) In vitro quantification of mammospheres formed by 2,000 control or miR-199a-OE HMLE cells seeded. (e) qRT-PCR of mRNA extracted from 5 day HMLE control or miR-199a-OE mammospheres. (f-h) qRT-PCR of miR-199a levels in HMLE-Neu-Twist1-ER-OE tumor initiating cells (TICs) (f), CD24⁺/Thy1⁺ TICs isolated from early and late stage spontaneous MMTV-Wnt-1 tumors (g), CD24⁻/CD44⁺ TICs isolated from HCI-002 human breast cancer PDX (h) as compared to the non-TIC counterparts (n=3 biologically independent samples; data represents mean ± SEM) in d–h. *P<0.05, **P<0.01, ***P<0.005 by two-tailed Student’s t-test in d–h.

Figure 3. Identification of LCOR as a direct target gene of miR-199a

(a) Heat map showing the differential expression of predicted miR-199a target genes (TargetScan v7.0) in P4 vs P5 cells. Fold change is represented as Log2 ratio. (b) qRT-PCR analysis of top candidate genes in mouse NMuMG and human HMLE cells after transfection with the indicated miRNA mimics. (c) Summary of the knockdown effect of the selected candidate miR-199a targets in functional assays using mouse MECs. MS: mammosphere formation assay; CFP: cleared fat pad mammary reconsititution assay. (d) qRT-PCR analysis of Lcor expression in different lineages of mouse MECs. (e) Immunohistochemistry (IHC) (left panel) analysis of Lcor and immunofluorescence (right panel) of Lcor and K14 localization in the mammary ducts. (f) qRT-PCR analysis of LCOR after 48h transfection of miR-199a-3p and miR-199a-5p in HMLE cells. (g) Western blot analysis of LCOR in control and miR-199a-OE HMLE cells. (h) Schematic diagram of the miR-199a binding sites on the LCOR 3′UTR. (i) Normalized activity of the luciferase reporter containing the WT mouse Lcor 3′ UTR or various miR-199a seed sequence mutants, after co-transfection with miR-199a-3p or miR-199a-5p, or control miRNAs in Hela cells. The reporters were divided in two groups, UP and DOWN, containing upper and lower halves of the Lcor 3′-UTR, respectively. Scale bars: 20 μm in e. n=3 biologically independent samples; data represents mean ± SEM in b, d, f and i.* P<0.05, ** P<0.01, *** P<0.005 by two-tailed Student’s t-test in bar graphs.

Figure 4. LCOR suppresses MaSC function and is downregulated in stem cell populations

(a) Quantification of mammosphere formation of 20,000 P4 cells in the indicated conditions (n=3 biologically independent samples; data represents mean ± SEM). (b–c) Limited dilution cleared fat pad reconstitution assay of P4 (b) and (c) P5 cells after transduction with the indicated constructs. Representative images show the outgrowth. Each pie chart represents a mammary gland with blackened area showing 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. (d) qRT-PCR of mRNA extracted from mammospheres formed by HMLE cells after transduction with the indicated constructs (n=3 biologically independent samples; data represents mean ± SEM). (e) GSEA demonstrating the enrichment of gene sets related to MaSC and Claudin-low tumors in the ranked gene list of LCOR-KD vs control HMLE cells. (f–g) qRT-PCR analysis of Lcor expression in Lgr5⁺ MaSC-enriched P4 cells (f), CD24⁺/Thy1⁺ MMTV-Wnt-1 TICs (g) and CD24⁻/CD44⁺ TICs isolated from HCI-002 PDX (h) as compared to their non-stem cell counterparts (n=3 biologically independent samples; data represents mean ± SEM). Scale bars: 2 mm in b and c. * P<0.05, ** P<0.01 by two-tailed Student’s t-test in bar graphs.

Figure 5. miR-199a and LCOR functionally influence the initiation of ER⁻ breast tumors in vivo

(a–b) Kaplan-Meier distant relapse-free survival (DRFS) curve of breast cancer patients with higher or lower than median RNA expression levels of (a) miR-199a and (b) LCOR in their tumors. (c) miR-199a and LCOR protein expression levels in TNBC (n=59 patient samples) and non-TNBC tumors (n=150 patient samples). Each sample was scored as weak (low expression) or strong (high expression) according to staining intensities of miR-199a by ISH and LCOR by IHC. (d) Quantification of tumorspheres formed by 10,000 cells from multiple human breast cancer PDXs in different tumor subtypes with the indicated conditions (n=3 biologically independent samples; data represents mean ± SEM). (e) Tumor take rate of HCI-001, HCI-002 and HCI-010 upon MFP injection of indicated cells. n=number of MFP injections as indicated in the table. Tumor initiating cell (TIC) frequency calculated by the ELDA software is indicated in red. (f–g) Tumor growth of HCI-001 (f) and HCI-002 (g) upon MFP injection of 20,000 cells in the indicated conditions (n=10 mouse mammary glands). (h) Metastatic nodule counts in the indicated organs 10 days after intracardiac (I.C.) injection of 100,000 4TO7 cells in Balb/c mice (n=10 mice). Each dot represents a value and the lines represent the mean and SD. (i) Bioluminescence imaging (BLI) quantification of the metastatic growth of the control and LCOR-KD MDA-MB-231 cells after intracardiac (I.C.) injection of 100,000 cells in Ncr-nu/nu mice (n=10 mice). Scale bar: 100 μm in c. P-value by log-rank test in a and b, Fisher’s exact test in c. P value by Pearson’s Chi-squared test in e. * P<0.05, ** P<0.01, *** P<0.005 by two-tailed Student’s t-test in d, f, g, h and i. ^# P>0.05 in d.

Figure 6. LCOR primes the IFN-α response

(a) Schematic representation of LCOR mutants. (b) Quantification of mammospheres formed by 5,000 HMLE cells with ectopic expression of the indicated LCOR constructs (n=3 biologically independent samples; data represents mean ± SEM). (c) Unsupervised hierarchical clustering of the HMLE expressing various LCOR constructs based on transcriptomic profiles. (d) GSEA of the IFN-α response gene-set (M5911) in the ranked gene list of LCOR, LSKAA and ΔHTH vs. control HMLE cells. (e) GSEA of the IFN-α response gene set in the ranked gene list of the LCOR-KD or miR-199a-OE vs. control HMLE and MDA-MB-231 cells. (f) GSEA of the IFN-α response gene set in the indicated gene list from the current study. (g) GSEA of the IFN-α response gene set in the gene list of CD24⁻/CD44⁺ CSC vs. non-CSCs in ER⁻ breast cancer. (h) Quantification of mammospheres formed by P4 (20,000) and P5 (10,000) cells treated with different doses of IFN-α, from 10 to 1000 U/ml in 8 days (n=3 biologically independent samples; data represented mean ± SEM). * P<0.05, ** P<0.01 by two-tailed Student’s t-test in b and h.

Figure 7. Stem cells and differentiated cells respond differently to the IFN-α signaling

(a) qRT-PCR result showing the fold change of stem cell-related genes (red) and luminal differentiation-related genes (blue) in mammospheres formed by P4 and P5 with or without IFN-α 1000 U/ml treatment for 6 days. (n=3 biologically independent samples; data represented mean ± SEM of fold change in IFN-α vs Ctrl). (b) GSEA of the MaSC and luminal upregulated gene sets generated in this study, as well as Nanog-Oct4-Sox2 transcriptional factors (NOS-TFs) targets gene set and undifferentiated downregulated genes in the ranked gene list of IFN-α-treated P5 and P4 cells cells vs. control. (c–d) Cleared fat pad reconstitution assay of 200 P4 (c) and 3,000 P5 (d) cells after trice a week treatment of 100,000 U IFN-α for 3 weeks. Representative images show the outgrowth. Each pie chart represents a mammary gland with blackened area showing the percentage of mammary gland outgrowth (n=10 mammary glands injected); P value by two-tailed Student’s t–test). (e–f) Quantification of mammospheres formed by 10,000 P5 (e) and P4 (f) cells with or without IFN-α treatment after transduction of the indicated constructs (n=3 biologically independent samples; data represents mean ± SEM). Plots represent the enhanced difference of treated conditions vs. the control conditions (without treatment). (g) Tumor take rate upon MFP injection of 10,000 control and miR-199a-OE HCI-001 cells, with or without treatment with 100,000 U IFN-α as indicated in the schematics. P value calculated by one-way ANOVA of the tumor incidence. (h–i) Quantification of PDX cell tumorspheres formed by 10,000 HCI-001 cells, with the indicated conditions (n=3 biologically independent samples; data represents mean ± SEM). (j) Senescence-associated β-galactosidase (SA-β gal) assay of MDA-MB-231 cells comparing control and LCOR-OE cells, and with or without IFN-α treatment for 72 hours (n=3 biologically independent samples; data represents mean ± SEM; source of data in Supplementary Table 4). Scale bars: 2 mm in c, d, and 100 μm in j. * P<0.05, ** P<0.01, *** P<0.005 by two-tailed Student’s t-test in a, e, f, h and i.

Figure 8. Immune and autocrine IFN related effects on mammary gland and tumor cells

(a) Percentage of total IFN-α-expressing cells from digested mammary glands and tumors at the indicated stages, analyzed by flow cytometry after intracellular IFN-α staining of single cell suspensions. (b) Relative percentage of IFN-α positive cell types of the mammary gland, analyzed by flow cytometry after co-staining intracellular IFN-α with CD3e (T-cells), CD11c (dendritic cells), F4/80 (macrophages) and Lin⁻CD24⁺ (epithelial cells). (c) Flow cytometry analysis showing the percentage of the F4/80 (macrophages) positive cells in the mammary gland at different stages. (d) Flow cytometry analysis of the percentage of IFN-α positive cells within the total mcarophage population. In a–d n=4 biologically independent samples; data represents mean ± SEM. (e) Quantification of IFN-α levels in the CM of the indicated cells, detected by ELISA (n=3 biologically independent samples; data represents mean ± SEM). (f) Quantification of mammospheres formed by P4 (20,000 cells), P5 (10,000 cells) and (g) P5-miR-199a-OE (10,000 cells) cells treated 1:3 with CM from involution macrophages and neutralizing antibodies (NAb) against IFN-α/β (2.5 μg/ml) (n=3 biologically independent samples; data represents mean ± SEM). (h) Quantification of mammospheres formed by 10,000 P5 and P4-Lcor cells with or without treatment with NAb against IFN-α/β (2.5 μg/ml) (n=5 biologically independent samples; data represents mean ± SEM). (i) Quantification of PDX cell tumorspheres formed by 10,000 HCI-001 treated 1:3 with CM from involution macrophages and conditions indicated (n=3 biologically independent samples; data represents mean ± SEM). (j) Schematic diagram showing the compilation of the 27-gene ISDS. See Methods for details. (k–m) Kaplan-Meier relapse-free survival (RFS) (k), distant metastasis-free survival (DMFS) (l), and overall survival (OS) (m) analysis of the ISDS gene signature in ER⁻ breast cancer using the KM plotter. (n) Schematic model for the conserved function of the miR-199a-LCOR axis in allowing the evasion of normal mammary gland and breast cancer cells from macrophage-derived and autocrine IFN-α. * P<0.05 by Student’s t-test in a, c and d respect to the virgin 9 week condition. * P<0.05, ** P<0.01, *** P<0.005 by Student’s t-test in e–i. P-value by log-rank tests in k–m.