Conserved role of FOXC1 in TNBC is parallel to FOXA1 in ER+ breast cancer

Summary Triple-negative breast cancer (TNBC) is characterized by lack of the estrogen (ER) receptor, progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2), and standard receptor-targeted therapies are ineffective. FOXC1, a transcription factor aberrantly overexpressed in many cancers, drives growth, metastasis, and stem-cell-like properties in TNBC. However, the molecular function of FOXC1 is unknown, partly due to heterogeneity of TNBC. Here, we show that although FOXC1 regulates many cancer hallmarks in TNBC, its function is varied in different cell lines, highlighted by the differential response to CDK4/6 inhibitors upon FOXC1 loss. Despite this functional heterogeneity, we show that FOXC1 regulates key oncogenes and tumor suppressors and identify a set of core FOXC1 peaks conserved across TNBC cell lines. We identify the ER-associated and drug-targetable nuclear receptor NR2F2 as a cofactor of FOXC1. Finally, we show that core FOXC1 targets in TNBC are regulated in parallel by the pioneer factor FOXA1 and the nuclear receptor NR2F2 in ER + breast cancer.


Fig. S4. Tumor suppressors and Oncogenes among the conserved targets of FOXC1 (Related to
Figure 2).ChIP-seq traces and rlog normalized mRNA values from RNA-seq of selected FOXC1 conserved targets (A) THRB, Thyroid hormone receptor TRβ, a known tumor suppressor in TNBC, whose high expression correlates with better survival 3 , forms heterodimers with RXR upon binding the agonist T3, and promotes tumor suppressive pathways 4 .TRβ also inhibits the expression of RUNX2, an oncogene in TNBC 5 .In our data, FOXC1 directly regulates the expression of THRB, RXRA, and RUNX2 in most cell lines (B) ARHGAP24, a tumor suppressor in many cancers suppresses cell invasion via regulating small GTPase RAC1 signaling in TNBC 6 .(C) INPP4B, is commonly inactivated in TNBC and promotes tumorigenesis by modulating degradation of EGFR and MET receptors 7 .Inactivation of INPP4B leads to higher tumor volume, sensitizing tumors to PI3K and MEK inhibitors, which is a potential targeted therapy approach for TNBC (D) SLC7A5, FOXC1 upregulates oncogeneSLC7A5 (LAT1), which is typically over-expressed in TNBC, and mediates uptake of Leucine in exchange for glutamine 8 .As Leucine is a regulator of mTORC1, SLC7A5 is speculated to promote proliferation via the Akt/mTORC1 pathway and is a target in preclinical trials.FOXC1 activates expression of SLC7A5 nearly 2-fold in all four cell lines via binding site within the first intron.(E) FOXC1 was seen to activate expression of PDE7B, which inhibits cell growth and tumor development by regulating cAMP concentration in TNBC 9 .Bar plots showing the normalised expression of mRNA in parental (Blue, WT) and FOXC1_KO (Red, KO) in TNBC cell lines.Statistical significance and p-value determined using multiple unpaired t tests using Graphpad Prism (Asterisk represents * = p < 0.05, ** = p < 0.005, ***= p < 0.0005, **** = p < 0.00005).5).ChIP-seq traces and rlog normalized mRNA values from RNA-seq of FOXC1 core targets whose function is as yet unknown in TNBC (A) CHI3L2: FOXC1 binds the promoter and directly activates expression of CHI3L2 (Chitinase-3-Like Protein 2), and its expression strongly correlates with that of FOXC1 in patient tumor samples (Fig. 3f).Furthermore, the expression of CHI3L2 is associated with poor outcomes in gliomas 11 .The function of this ligand in breast cancer is unknown, although CHI3L1, a closely associated ligand, is well-studied for its oncogenic role in many cancers 12 .(B) LONRF1: FOXC1 also activates expression of LONRF1 (LON Peptidase N-Terminal Domain and Ring Finger 1) via binding at intragenic sites, a binding site that is conserved in Fujioka cell line as well.Although the role of LONRF1 in breast cancer is yet to be studied, LONRF1 is downregulated in prostate cancer patient samples and is speculated to be part of the core set of genes for estimating recurrence and prognosis of prostate cancer 13 .(C) DDIT4: Another prognostic factor, DDIT4 (REDD1), is a key protein in cancer cachexia and hypoxia involved in Hif1 signalling whose expression correlates with chemoresistance in TNBC 14 .(D) SYNPO: SYNPO (Synaptopodin) is regulated by FOXA1 in MCF-7 as well.Although there are no studies for the role of SYNPO in breast cancer, SYNPO2, a related protein, is a well-studied oncogene that promotes metastasis in breast cancer via PI3K/Akt/mTOR pathway 15 .Bar plots show the normalised expression of mRNA in parental (Blue, WT) and FOXC1_KO (Red, KO) in TNBC cell lines.Statistical significance and p-value determined using multiple unpaired t tests using Graphpad Prism (ns = not significant, * = p < 0.05, ** = p < 0.005, *** = p < 0.0005).

Fig. S2 .
Fig. S2.RNA-seq analysis of TNBC cell lines and FOXC1_KO clones (Related to Figure 1).(A)Principal component analysis of TNBC cell lines "WT" and the FOXC1_KO "KO" using rlog transformed data for the top 500 variable genes reveals greater similarity between gene expression signatures of Hs578t and BT-549 cell lines, as compared to MDA-MB-231.(B) Correlation plot of biological replicates in each sample shows clustering of KO with respective parental cell lines.(C) Number of significantly (adjusted p-value < 0.05) differentially expressed genes (DEGs) identified in each WT vs FOXC1_KO pair.(D) Heatmap of log fold change (Log2FC) values from normalized mRNA expression of the 172 genes that were significantly (padj value <0.05) differentially expressed between WT and FOXC1_KO of the four TNBC cell lines in RNA-seq analysis.(E) Representation of the eight states for identifying patterns gene expression changes across the WT and FOXC1_KO in the four cell lines revealing 15 patterns of expression.(F) Heatmap depicting change in gene expression within 15 unsupervised k-means clusters in the previous panel.(G) Enrichment of GO BP terms within each identified K-means cluster from panel D. Note that clusters 12 and 13 did not have any significantly enriched GO terms.(H) Bar plots showing the normalised expression of ESR1 mRNA in parental (Blue, WT) and FOXC1_KO (Red, KO) in TNBC cell lines.Statistical significance determined using DESeq2 (ns = not significant).

Fig. S3 .
Fig. S3.Analysis of FOXC1 binding sites (Related to Figure 2).(A) Pearson's correlation of normalized reads in peaks in individual biological replicates of ChIP-seq from four different TNBC cell lines.(B) Distribution of FOXC1 peaks in features in the respective cell

Fig. S7 .
Fig. S7.Conservation of FOXC1 peaks in AML and TNBC (Related to Figure 3).Overlap of FOXC1 ChIP-seq signal in Fujioka (AML) cell lines with the five FOXC1 peak clusters in TNBC from Fig. 3a.

Fig. S8 .
Fig. S8.FOXC1 and crucial Erα-associated transcription factors in ER+ breast cancer bind to similar sites (Related to Figure 5).(A) Normalized gene expression from RNA-seq data (top panel) or protein expression from 10 of FOXC1 and crucial Erα-associated transcription factors (NR2F2, FOXA1, GATA3 and ESR1) in the four TNBC cell lines used in this study.(B) Average intensity of FOXC1 peaks in in Bt-549, Hs578t and

Fig. S9 .
Fig. S9.Genes regulated by FOXC1 in TNBC as well as FOXA1 in luminal breast cancer (Related to Figure 5).(A) Heatmap of log fold change (Log2FC) of normalized mRNA expression of the 164 core targets of FOXC1 in the four TNBC cell lines used in this study.(B) Enrichment of pathways from the hallmark gene set molecular signatures database (MSigDB) using the 164 core targets of FOXC1.(C) Selected genes from among the 164 core targets of FOXC1 that have an important role in cancer.(D) ChIP-seq traces and rlog normalized mRNA values from RNA-seq of FOXC1 core targets (RUNX2, TGFBI and CDK6) that have overlapping FOXA1 peaks in MCF-7.Statistical significance and p-value determined using multiple unpaired t tests using Graphpad Prism (ns = not significant, * = p < 0.05, ** = p < 0.005, **** = p < 0.00005).

Fig. S10 .
Fig. S10.Novel targets of FOXC1 (Related to Figure5).ChIP-seq traces and rlog normalized mRNA values from RNA-seq of FOXC1 core targets whose function is as yet unknown in TNBC (A) CHI3L2: FOXC1 binds the promoter and directly activates expression of CHI3L2 (Chitinase-3-Like Protein 2), and its expression strongly correlates with that of FOXC1 in patient tumor samples (Fig.3f).Furthermore, the expression of CHI3L2 is associated with S1: Full uncropped Western Blot image, related to Figure 1A.Protein expression levels of FOXC1 and β-actin in four TNBC cell lines and their CRISPR FOXC1_KO clones (BT549, MDA-MB-468, Hs578t, MDA-MB-231).After transfer, the nitrocellulose membrane was cut into two strips, and the top strip was incubated with anti-FOXC1 (ab227977) and the bottom strip was incubated with anti-β-actin (ab6276).A total of 75 µg of protein was loaded to ensure no FOXC1 protein expression is visible in the FOXC1-KO clones.Data S2/Methods S2: Full uncropped Western Blot image, related to Figure 4B.Protein expression of FOXC1 and NR2F2 in co-immunoprecipitation assays performed as described in the Methods section.After transfer, the nitrocellulose membrane was cut into two strips, and the top strip was incubated with anti-FOXC1 (ab227977) and the bottom strip was incubated with anti-NR2F2 (ab211777).Molecular weight marker on the left-most and right-most lane shows 80 KDa (top band) and 30 KDa (bottom band).