Interferon-beta represses cancer stem cell properties in triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC), the deadliest form of this disease, lacks a targeted therapy. TNBC tumors that fail to respond to chemotherapy are characterized by a repressed IFN/signal transducer and activator of transcription (IFN/STAT) gene signature and are often enriched for cancer stem cells (CSCs). We have found that human mammary epithelial cells that undergo an epithelial-to-mesenchymal transition (EMT) following transformation acquire CSC properties. These mesenchymal/CSCs have a significantly repressed IFN/STAT gene expression signature and an enhanced ability to migrate and form tumor spheres. Treatment with IFN-beta (IFN-β) led to a less aggressive epithelial/non-CSC-like state, with repressed expression of mesenchymal proteins (VIMENTIN, SLUG), reduced migration and tumor sphere formation, and reexpression of CD24 (a surface marker for non-CSCs), concomitant with an epithelium-like morphology. The CSC-like properties were correlated with high levels of unphosphorylated IFN-stimulated gene factor 3 (U-ISGF3), which was previously linked to resistance to DNA damage. Inhibiting the expression of IRF9 (the DNA-binding component of U-ISGF3) reduced the migration of mesenchymal/CSCs. Here we report a positive translational role for IFN-β, as gene expression profiling of patient-derived TNBC tumors demonstrates that an IFN-β metagene signature correlates with improved patient survival, an immune response linked with tumor-infiltrating lymphocytes (TILs), and a repressed CSC metagene signature. Taken together, our findings indicate that repressed IFN signaling in TNBCs with CSC-like properties is due to high levels of U-ISGF3 and that treatment with IFN-β reduces CSC properties, suggesting a therapeutic strategy to treat drug-resistant, highly aggressive TNBC tumors.

Keywords: cancer stem cells; interferon-beta; triple-negative breast cancer; tumor microenvironment.

Fig. 1.

IFN-stimulated genes (ISGs) are repressed in mesenchymal/CSCs. (A) Transformed HMECs consist of two subpopulations, epithelial cells (epithelial morphology) and mesenchymal cells (mesenchymal morphology), as determined by bright field microscopy (10×). (B) Epithelial cells express E-CADHERIN, while mesenchymal cells express VIMENTIN, as determined by Western analysis. (C) Epithelial cells are characterized by a CD24^Lo/CD44^Hi cell surface profile, while mesenchymal cells are characterized by CD24^LO/CD44^HI profile, as determined by flow cytometry. (D) Mesenchymal cells express the pluripotent stem cell transcription factor, NANOG, relative to epithelial cells, as shown by Western analysis. Hs578t and MDA-MB-231 are TNBC cell lines used as positive controls. Mesenchymal cells (E) form robust tumor spheres at limiting dilution (stem cell frequency; P = 8.5e-23; n = 3) and (F) show enhanced migration in a cell motility assay (two-tailed t test, ***P < 0.001, n = 3). (G) An IFN-responsive gene signature derived from our previous publications (18, 19) shows ISGs (STAT1, STAT2, IRF9, IRF1, SOCS1, and OAS2) are repressed at least twofold in Mes/CSCs relative to Ep/non-CSCs, as determined by microarray analysis. (H–M) ISGs (STAT1, STAT2, IRF9, IRF1, SOCS1, and OAS2) are repressed in Mes/CSCs relative to Ep/non-CSCs, as determined by qRT-PCR. Data represent mean fold-changes ± SEM, n = 3 (**P < 0.01, ***P < 0.001).

Fig. 2.

IFN-β–mediated canonical signaling reactivates ISG expression in Mes/CSCs. (A and B) Single-dose IFN-β (100 IU/mL) induces biphasic signaling kinetics in both Ep/non-CSCs and Mes/CSCs, with rapid, transient induction of phosphorylated STAT1 (P-STAT1) and P-ISGF3 (0.5–8 h), followed by induction and sustained expression of unphosphorylated STATs 1 and 2, IRF9 (U-STAT1, U-STAT2, IRF9), and U-ISGF3 (24–120 h), as determined by Western analysis. (C and D) Single-dose IFN-β (100 IU/mL) induces ISG transcripts by at least twofold (4 and 96 h) in both Mes/CSCs and Ep/non-CSCs relative to untreated controls, as determined by microarray analysis using an IFN-responsive gene signature derived from our previous publications (18, 19). (E and F) ISG transcripts (P-ISGF3; IFI16, IRF1, U-ISGF3; MX1, OAS2) are induced in both Mes/CSCs and Ep/non-CSCs (4 h), with less total gene expression in Mes/CSCs, as determined by qRT-PCR. Data represent mean fold-changes ± SEM, n = 3 (**P < 0.01, ***P < 0.001).

Fig. 3.

Sustained exposure to IFN-β represses CSC properties and inhibits migration. Sustained exposure to IFN-β (100 IU/mL, 6 wk) induces (A) CD24 expression, as shown by flow cytometry, and (B) an epithelium-like morphology, as determined by bright field microscopy (10×). (C) IFN-β removal (1 wk) results in loss of CD24, as determined by flow cytometry. (D) Sustained IFN-β (100 IU/mL, 2–6 wk) represses mesenchymal markers (VIMENTIN, SLUG) and removal for 1 wk and reestablishes expression (VIMENTIN and SLUG), as determined by Western analysis (line indicates separate blots). (E) Sustained IFN-β (100 IU/mL, 4 wk) represses AIG (two-tailed t test, ****P < 0.0001, ±SD, n = 3). (F) Sustained IFN-β (100 IU/mL, 2–6 wk), followed by removal for 5 d, partially represses tumor sphere formation at limiting dilution (stem cell frequency; P = 1.22e-08, n = 3). (G) Sustained IFN-β (100 IU/mL, 2–6 wk), followed by removal for 2 d represses cell migration in Mes/CSCs (one-way ANOVA, ***P = 0.0004, ±SD, n = 3) without altering repressed migration in Ep/non-CSCs (one-way ANOVA, ±SD, ns; n = 3).

Fig. 4.

Mes/CSCs have elevated, stable U-ISGF3 expression critical for cell migration. (A) U-ISGF3 (STAT1, STAT2, and IRF9) expression is elevated in Mes/CSCs relative to Ep/non-CSCs, along with mesenchymal markers, as determined by Western analysis. (B) Mes/CSCs have elevated U-ISGF3 expression independent of IFN-β in the presence of the JAK1/2 inhibitor, Ruxolitinib (line indicates separate blots). (C) The stability of U-ISGF3 is increased in Mes/CSCs relative to Ep/non-CSCs following cycloheximide treatment, as determined by Western analysis. (D) Efficient knockdown of IRF9 in Mes/CSCs as determined by Western analysis (1 wk after lentiviral transduction). All samples were on the same blot; vertical line indicates where the blot was cut. (E) IRF9 KD represses cell migration in Mes/CSCs over time (0–70 h). Data represent means ± SD (one-way Anova, ***P = 0.0009, n = 3), (two-tailed t test, ns; n = 3).

Fig. 5.

Clinical relevance of IFN-β in TNBC. (A) IFN-β mRNA is significantly elevated in breast tumor stroma of invasive ductal carcinomas relative to the normal noncancerous stroma (two-tailed t test, P < 0.0001). (B) An elevated, experimentally derived IFN-β metagene signature significantly correlates with improved recurrence-free survival in basal/TNBC patients (P = 3e-05). (C) TNBC subtypes are characterized by differential expression of a CSC metagene signature; the mesenchymal (M) subtype has an elevated CSC metagene signature, and the immune-modulatory (IM) subtype has a repressed CSC metagene signature (Wilcox P = 9.13e-05). (D) An elevated IFN-β metagene signature significantly correlates with the presence of TILs (Wilcox P = 0.000628). (E) An elevated IFN-β metagene signature significantly correlates with a repressed CSC metagene signature (R = 0.587, P = 1.27e-06).