The opposing effects of interferon-beta and oncostatin-M as regulators of cancer stem cell plasticity in triple-negative breast cancer

Background Highly aggressive, metastatic and therapeutically resistant triple-negative breast cancers (TNBCs) are often enriched for cancer stem cells (CSC). Cytokines within the breast tumor microenvironment (TME) influence the CSC state by regulating tumor cell differentiation programs. Two prevalent breast TME cytokines are oncostatin-M (OSM) and interferon-β (IFN-β). OSM is a member of the IL-6 family of cytokines and can drive the de-differentiation of TNBC cells to a highly aggressive CSC state. Conversely, IFN-β induces the differentiation of TNBC, resulting in the repression of CSC properties. Here, we assess how these breast TME cytokines influence CSC plasticity and clinical outcome. Methods Using transformed human mammary epithelial cell (HMEC) and TNBC cell models, we assessed the CSC markers and properties following exposure to OSM and/or IFN-β. CSC markers included CD24, CD44, and SNAIL; CSC properties included tumor sphere formation, migratory capacity, and tumor initiation. Results There are three major findings from our study. First, exposure of purified, non-CSC to IFN-β prevents OSM-mediated CD44 and SNAIL expression and represses tumor sphere formation and migratory capacity. Second, during OSM-induced de-differentiation, OSM represses endogenous IFN-β mRNA expression and autocrine/paracrine IFN-β signaling. Restoring IFN-β signaling to OSM-driven CSC re-engages IFN-β-mediated differentiation by repressing OSM/STAT3/SMAD3-mediated SNAIL expression, tumor initiation, and growth. Finally, the therapeutic use of IFN-β to treat OSM-driven tumors significantly suppresses tumor growth. Conclusions Our findings suggest that the levels of IFN-β and OSM in TNBC dictate the abundance of cells with a CSC phenotype. Indeed, TNBCs with elevated IFN-β signaling have repressed CSC properties and a better clinical outcome. Conversely, TNBCs with elevated OSM signaling have a worse clinical outcome. Likewise, since OSM suppresses IFN-β expression and signaling, our studies suggest that strategies to limit OSM signaling or activate IFN-β signaling will disengage the de-differentiation programs responsible for the aggressiveness of TNBCs. Electronic supplementary material The online version of this article (10.1186/s13058-019-1136-x) contains supplementary material, which is available to authorized users.


OSM and IFN- Gene Signatures and TNBC Patient Survival Analysis
The list of top 20 OSM-induced target genes (SI Table 1) were derived from our previously published microarray dataset (1) in which transformed HMEC-Ep/non-CSC were treated  OSM (10 ng/mL) every 48h for 3 weeks. The list of top 20 IFN--induced target genes (SI Table 2) were derived from our previously published microarray dataset (3) in which transformed HMEC-Ep/non-CSC were treated  IFN- (100 IU/mL) for 96h. Gene expression is represented as Log2 Fold-change (cytokine-treated relative to their respective untreated controls). These experimentally-derived gene signatures were applied to publicly available TCGA and EGA TNBC patient tumor datasets through Breast Kaplan Meier Plotter (2) to assess the probability of recurrence free survival ( Fig 3G). For the analysis, IFN- target genes were inverted relative to OSM target genes.

Cell Migration Assays
Transformed HMECs (500 cells/well) were seeded into 96-well ClearView plates with 8-mm pores (#4852 Essen Bioscience). Plates were incubated and imaged at 10 x magnification at the indicated time points. Analysis was performed using live cell Incucyte Zoom imaging software (Essen BioScience). Cell migration through the pores to the bottom chambers was imaged and quantified.

In vivo tumor initiation studies Mice
Athymic nu/nu female mice (6-8 weeks old) were purchased from the Athymic Animal and Xenograft Core of Case Western Reserve University. All procedures were performed in compliance with the Case Western Reserve University Institutional Animal Care and Use Committee (IACUC).

Bioluminescence Imaging (BLI)
Bioluminescence imaging was performed using IVIS Spectrum In Vivo Imaging System (Perkin Elmer). Mice were anesthetized with 3% Isoflurane in 2l/min oxygen and then injected Intraperitoneally (IP) with 150 L Luciferin (15 mg/mL, GoldBio) 5 min prior to imaging. Mice were then transferred into the IVIS Spectrum where they were kept under general anesthesia throughout the procedure. Imaging was performed bi-weekly for 3 weeks.

Sub-cutaneous injection of OSM and/or IFN--overexpressing cells
BT549 cells transduced with GFP followed by control vector (vec) (BT549-GFP-vec), or vectors encoding human OSM, human OSM and IFN-, or human IFN- were subcutaneously injected into previously anesthetized nude mice, as described. 2 different cell numbers were injected into the same mouse: 20,000 in the right flank and 200,000 in the left one. Cells were re-suspended in a solution containing 40% culture medium, 50% Matrigel and 10% Luciferin at a final concentration of 400,000 and 4x10 6 /mL respectively so that 50 L were injected each time. Tumors were measured with a caliper twice weekly.

Animal sacrifice and tumor excision
On day 21 post-engraftment, mice were sacrificed by CO2 asphyxiation. Tumors were removed, measured by caliper and weighted.  The list of top 20 OSM-induced target genes was derived from our previously published microarray dataset (1) in which transformed HMEC-Ep/non-CSC were treated  OSM (10 ng/mL) every 48h for 3 weeks. Gene expression is represented as Log2 fold-change (OSM treated relative to untreated control).