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Supplementary Figure 1. Epigenomic Maps of Endocrine Therapy Sensitive and Resistant Cell Lines

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Supplementary Figure 1. Epigenomic Maps of Endocrine Therapy Sensitive and Resistant Cell Lines Supplementary Figure 1. Epigenomic maps of endocrine therapy sensitive and resistant cell lines. (A-C) ChIP-qPCR against H3K36me3 (A), H3K4me2 (B) or FAIRE-qPCR (C) was used to validate enrichment at randomly selected sites identified by ChIP-seq specific to MCF7 (left-side panel) or LTED (right-side panel) cells. Color code represents relative fold enrichment as calculated against an internal negative control. (D) Confocal analysis of immunofluorescence assay against ERα protein in MCF7-LTED HA (>12 mo) cells. Primary (prim.) and secondary (sec.) antibody controls are shown. (E) GSC analysis of the overlap between two previously published ERα cistromes and the epigenomic maps obtained in this study. Supplementary Figure 2. ERα prognostic target genes are coherently expressed in endocrine therapy resistant cell lines. (A) RT-qPCR analysis of ERα target genes across endocrine therapy-responsive or - resistant cell lines. Expression levels are normalized against MCF7 cells (dotted line). The average and SEM of replicates are shown. (B) RT-qPCR analysis of ERα expression across endocrine therapy-responsive or -resistant cell lines. Expression levels are normalized against MCF7 cells (dotted line). The average and standard error of the mean (SEM) of replicates are shown. Supplementary Figure 3. Reprogramming of the chromatin cascade increases Notch activity in endocrine therapy resistant cell lines. (A-B) RT-qPCR analysis of two γ-secretase complex subunits across endocrine therapy- responsive or -resistant cell lines. Expression levels are normalized against MCF7 cells (dotted line). The average and SEM of replicates are shown. (C-D-E) Kaplan-Meier plots of Luminal A, Luminal B or ET patients using Notch3 expression probe. Supplementary Figure 4. Targeted downregulation of Notch3 in ETR cancer cells. (A-B) Time-plot representing the distribution of recurrent and metastatic events in ERα- positive breast cancer patients. Percentage refers to the total number of patients. (C) RT- qPCR analysis of individual Notch member following specific siRNA in LTED cells. Data are reported as fold change vs. cells treated with a control siRNA. (D) Protein levels of LTED cells treated as in panel C. (E) Proliferation assays following silencing of individual Notch genes (mean and SEM of four replicates is plotted). Supplementary Figure 5. PBX1 is a bona fide Notch3 target and is activity is increased in endocrine therapy resistant breast cancer cells (A) RT-qPCR analysis of PBX1 in response to silencing of individual Notch family members. Data are reported as fold change vs. cells treated with a control siRNA. Three replicates are shown. (B) Protein level assessed by Immuno blot (IB) of Notch3, PBX1 and the negative control GAPDH in LTED cells treated with siRNA against Notch3 or control. (C) Confocal analysis of PBX1 protein distribution by immunofluorescence in MCF7-LTED HA (>12 mo) cells. Appropriate controls (primary antibody: prim; secondary antibody; sec.) are shown. (D) RT-qPCR analysis of PBX1 across endocrine therapy-responsive or -resistant cell lines. Expression levels are normalized against MCF7 cells (dotted line). The average and SEM of replicates are shown. (E) PBX1 expression in normal and ERα-positive breast cancer patients (TCGA dataset) (F) Expression profile of PBX1 in individual patient’s primary and metastatic lesions (Weigelt dataset). (G) DNA recognition motif search analysis at cell type specific PBX1 binding near (±20Kb) PBX1-dependent repressed genes commonly regulated in MCF7 and LTED cells. Supplementary Figure 6. PBX1 increases the expression of genes associated with poor outcome in endocrine therapy resistant cell lines (A) Microarray analysis and RT-qPCR-analysis of a breast cancer cell line panels against PBX1 expression. Growth of breast cancer cells with low PBX1 expression is not affected by the two independent siRNA against PBX1 used in this study. (B) PBX1 overexpression in MCF7 cells rescues growth inhibition induced by Notch3 silencing (C) RT-qPCR analysis of randomly selected PBX1-dependent expressed genes common to MCF7 and LTED cells is shown. Color code represents relative % reduction change calculated against a control siRNA negative. (D) Overlap of genes up-regulated in response to siPBX1 in MCF7 and LTED cells. Motif search analysis was performed using cell-type specific PBX1 binding in close proximity to the 167 genes whose expression is upregulated by siPBX1 in both cell lines. Supplementary Figure 7. PBX1 increases the expression of genes associated with poor outcome in endocrine therapy resistant cell lines (A) Kaplan-Meier curves using MCF7 or LTED specific PBX1 dependent expressed genes in endocrine treated breast cancer patients. (B) Kaplan-Meier curves using PBX1 dependent expressed genes common to MCF7 and LTED cells in endocrine treated breast cancer patients. (C). Genome-wide analysis of PBX1-bound regions from MCF7 and LTED. Results are represented as in figure 1B Supplementary Figure 8. pKNOX1 expression is increased and Notch-PBX1 signaling is essential for resistant cells proliferation. (A) PKNOX1 expression in normal and ERα-positive breast cancer patients (TCGA dataset). (B) Expression profile of PKNOX1 in individual patient’s primary and metastatic lesions (Weigelt dataset). (C) RT-qPCR analysis of PKNOX1 across endocrine therapy-responsive or -resistant cell lines. Expression levels are normalized against MCF7 cells (dotted line). The average and SEM of replicates are shown. (D) The γ- secretase inhibitors (GSI) MRK003 can efficiently downregulate PBX1 expression at the mRNA (left panel) and protein (righ panel) level in LTED cells. Supplementary Figure 9. Notch-PBX1 regulated genes are enriched in transcriptional signature associated with breast cancer outcome. (A) Oncomine concepts map analysis reveals that genes down-regulated by MRK003 or siPBX1 are highly expressed in datasets representing poor-outcome or high-grade breast cancer signatures. (B) Oncomine concepts map analysis reveals that genes down- regulated by both MRK003 and siPBX1 are highly expressed in high-grade breast cancer, (C) ERBB2+ or triple negative breast cancer and (D) are significantly underexpressed in ERα-positive breast cancer. Supplementary Figure 10 NOTCH-PBX1 driven transcriptional program discriminate high-grade patients. (A) Kaplan-Meier curves comparing the genomic grade index (GGI) signature against Notch-PBX1 signature in ERα positive breast cancer patients (B) Kaplan-Meier curves comparing the genomic grade index (GGI) signature against Notch-PBX1 signature in high-grade (3) ERα positive breast cancer patients Supplementary Figure 11. NOTCH-PBX1 driven transcriptional program is specific to ERα breast cancer. (A) Kaplan-Meier curves testing Notch-PBX1-signature in ERα negative or triple negative breast cancer patients subsets. (B) Kaplan-Meier analysis using PBX1 and the 24 genes in non-breast solid tumors. Patients are split using the median value of an average of the selected probes. (C) Enrichment analysis for Notch3 and PBX1 mRNA probes in a panel of human cancers. Enrichment maps are obtained from the Gene Enrichment Profiler tool (http://xavierlab2.mgh.harvard.edu/EnrichmentProfiler/enrichmentMaps.html). Supplementary Figure 12. Model of ET-resistance in breast cancer. ET-responsive tumor’s proliferation is supported mainly by ERα signaling and to a lesser extent by NOTCH signaling. At this stage tumors are responsive to several drugs regimen that target ERα but can develop resistance in response to drug treatment. After the tumor acquire resistance, the importance of ERα signaling is over-ridden by an addiction to NOTCH/PBX1/PKNOX1 signaling. Based on the Kaplan-Meier analysis, primary breast tumors with no NOTCH signaling are unlikely to acquire resistance. Changes in ERα and NOTCH signaling are paralleled by epigenetic remodeling of the chromatin landscape. Increased NOTCH activity promotes PBX1 expression and over-expression of PKNOX1 changes its DNA binding specificity guiding the over-expression of genes promoting ETR. Supplementary Table 1. Transcriptional profile of of MCF7 and LTED cells. Differentially expressed genes in MCF7 and LTED cells cultured in stripped media. Microarray data from 3 biological replicate were analyzed, P value and fold difference (as a ratio LTED/MCF7 expression) are reported. Supplementary Table 2. Transcriptional profile of PBX1 depleted MCF7 and LTED cells. Microarray analysis of MCF7 and LTED cells depleted of PBX1 in stripped media. Three biological replicates were analyzed and averaged and P value and fold difference (as a ratio siPBX1/siCTRL) are reported. Supplementary Table 3. siPBX1 and MRK003 regulated genes. The gene list was obtained by comparing microarrays of LTED cells depleted of PBX1 or treated by MRK003. Only genes significantly regulated by both treatment are reported. Supplementary Table 4. PBX1 derived prognostic signature. The probe list used to calculate Kaplan-Meier curve is provided. Supplementary Table 5. List of primer used in the study A ChIP-qPCR: H3K36me3 B ChIP-qPCR: H3K4me2 S1 MK36me3 1 LK36me3 1 MK4me2 1 LK4me2 1 MK36me3 2 LK36me3 2 MK4me2 2 LK4me2 2 MK36me3 3 LK36me3 3 MK4me2 3 LK4me2 3 MK36me3 4 LK36me3 4 MK4me2 4 LK4me2 4 MK36me3 5 LK36me3 5 MK4me2 5 LK4me2 5 MK36me3 6 LK36me3 6 MK4me2 6 LK4me2 6 MK36me3 7 LK36me3 7 MK4me2 7 LK4me2 7 MK36me3 8 LK36me3 8 MK4me2 8 LK4me2 8 MK36me3 9 LK36me3 9 MK4me2 9 LK4me2 9 MK36me3 10 LK36me3 10 MK4me2 10 LK4me2 10 C ChIP-qPCR: FAIRE MFAIRE 1 LFAIRE 1 enrichment MFAIRE 2 LFAIRE
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