RUNX1 and RUNX2 transcription factors function in opposing roles to regulate breast cancer stem cells

Abstract

Breast cancer stem cells (BCSCs) are competent to initiate tumor formation and growth and refractory to conventional therapies. Consequently BCSCs are implicated in tumor recurrence. Many signaling cascades associated with BCSCs are critical for epithelial-to-mesenchymal transition (EMT). We developed a model system to mechanistically examine BCSCs in basal-like breast cancer using MCF10AT1 FACS sorted for CD24 (negative/low in BCSCs) and CD44 (positive/high in BCSCs). Ingenuity Pathway Analysis comparing RNA-seq on the CD24^-/low versus CD24^+/high MCF10AT1 indicates that the top activated upstream regulators include TWIST1, TGFβ1, OCT4, and other factors known to be increased in BCSCs and during EMT. The top inhibited upstream regulators include ESR1, TP63, and FAS. Consistent with our results, many genes previously demonstrated to be regulated by RUNX factors are altered in BCSCs. The RUNX2 interaction network is the top significant pathway altered between CD24^-/low and CD24^+/high MCF10AT1. RUNX1 is higher in expression at the RNA level than RUNX2. RUNX3 is not expressed. While, human-specific quantitative polymerase chain reaction primers demonstrate that RUNX1 and CDH1 decrease in human MCF10CA1a cells that have grown tumors within the murine mammary fat pad microenvironment, RUNX2 and VIM increase. Treatment with an inhibitor of RUNX binding to CBFβ for 5 days followed by a 7-day recovery period results in EMT suggesting that loss of RUNX1, rather than increase in RUNX2, is a driver of EMT in early stage breast cancer. Increased understanding of RUNX regulation on BCSCs and EMT will provide novel insight into therapeutic strategies to prevent recurrence.

Keywords: RUNX1; RUNX2; breast cancer; breast cancer stem cells; epithelial to mesenchymal transition.

Figure 1.

Analysis of differentially expressed genes in CD24^-/low/CD44^+/high BCSCs versus CD24^+/high/CD44^high premalignant basal MCF10AT1 breast cancer cells. (a) FACS was performed on MCF10AT1 using CD24 (PE-cy7) and CD44 (APC) antibodies to isolate BCSCs (CD24^−/low/CD44^+/high, yellow) and bulk non-BCSC (CD24^+/high/CD44⁺, pink). r1, r2, r3 refer to different replicates of FACS. (b) Principle component analysis demonstrated clustering of these two populations from each other. Parental cells were not subjected to FACS therefore suggesting an affect of FACS on gene expression. (c) A volcano plot demonstrating differentially expressed genes highlighting several genes that are well known to be altered in BCSCs including RUNX2, AR, ESR1, POSTN (Morra & Moch, 2011), CDH1, ESRP1, VIM, ZEB1, ZEB2, LTBP1, VCAN, WNT5A, and S1PR3 (Milara et al., 2012). BCSC, breast cancer stem cell; FACS, fluorescence-activated cell sorting; PCA, principal component analysis

Figure 2.

Pathway analysis of differentially expressed genes in BCSCs. (a) Ingenuity pathway analysis on differentially expressed genes in BCSC (CD24^−/low/CD44^+/high) versus bulk non-BCSC (CD24^+/high/CD44⁺) demonstrates increased cellular movement, invasion, migration, and EMT in BCSCs and decreased epithelial differentiation. (b) The activation score of the top 15 activated and top 15 inhibited upstream regulators are presented. The log2 fold change for these upstream regulators is also shown. (c) Some of the top most significant pathways are displayed (color scale represents the most significant being green and the least being red) as is their activation scores when IPA was able to calculate them (blue being inhibited and red being activated). The top 30 significant pathways that are altered are displayed in Figure S2. (d) The number of genes that are shared between these pathways is depicted with thicker lines indicating more genes in common between them. The circles are colored to indicate the degree of inhibition (blue) and activation (red). BCSC, breast cancer stem cell; EMT, epithelial-to-mesenchymal transition; IPA, Ingenuity Pathway Analysis

Figure 3.

RUNX regulation within BCSCs. (a) Normalized counts within the RNAseq demonstrate that RUNX1 is the predominant RUNX factor expressed within MCF10AT1, while RUNX2 is differentially expressed between BCSC (CD24^−/low/CD44^+/high, yellow) and bulk non-BCSC (CD24^+/high/CD44⁺, pink). RUNX3 is not expressed. (b) FACS for these subpopulations followed by western blot analysis demonstrated an increase of RUNX2 within BCSCs. (c) Densitometry of westerns previously published (Hong et al., 2018) demonstrate a decrease in RUNX1, CD24, and CDH1 and an increase in RUNX2, ZEB1, and VIM. (d) A heatmap of the RUNX2 interaction network is displayed. BCSC, breast cancer stem cell; FACS, fluorescence-activated cell sorting; RUNX, Runt-related transcription factor

Figure 4.

RUNX2 increases within MCF10CA1a in the murine mammary fat pad tumor microenvironment. (a) Western blot analysis of four replicates of parental MCF10CA1a and four tumor samples post-injection within the mammary fat pad demonstrate an increase in RUNX2 within the tumor sample. (b) Since RUNX2 may be higher in expression specifically within infiltrating mouse cells within the tumor, human-specific primers for RUNX2 were used to establish that RUNX2 increases specifically within the human MCF10CA1a in the tumor microenvironment. A control qPCR using these primers on mouse embryonic fibroblasts cells resulted in no detectable expression. qPCR, quantitative polymerase chain reaction; RUNX, Runt-related transcription factor

Figure 5.

Simultaneous RUNX1 and RUNX2 inhibition results in epithelial to mesenchymal transition. (a) MCF10AT1 were treated for 5 days with DMSO, an inhibitor that interferes with the interaction between CBFβ and RUNX factors, or an inactive version of this inhibitor that is chemically similar but does not interfere with these interactions. These were then allowed to recover for 7 days in fresh media and passaged as normal. (b) This treatment regimen results in slowed cell growth, cell death, and a more mesenchymal-like morphology. Bar in the upper left panel indicates 20 μm. (c) Western blot analysis demonstrates that the resulting cells are higher in RUNX2 and lower in RUNX1 and are more mesenchymal in their expression of CDH1 and VIM. CBFβ, core binding factor β; DMSO, dimethyl sulfoxide; RUNX, Runt-related transcription factor