Induction of the TEAD Coactivator VGLL1 by Estrogen Receptor-Targeted Therapy Drives Resistance in Breast Cancer

Abstract

Resistance to endocrine therapies (ET) is common in estrogen receptor (ER)-positive breast cancer, and most relapsed patients die with ET-resistant disease. Although genetic mutations provide explanations for some relapses, mechanisms of resistance remain undefined in many cases. Drug-induced epigenetic reprogramming has been shown to provide possible routes to resistance. By analyzing histone H3 lysine 27 acetylation profiles and transcriptional reprogramming in models of ET resistance, we discovered that selective ER degraders, such as fulvestrant, promote expression of vestigial-like 1 (VGLL1), a coactivator for TEF-1 and AbaA domain (TEAD) transcription factors. VGLL1, acting via TEADs, promoted the expression of genes that drive the growth of fulvestrant-resistant breast cancer cells. Pharmacological disruption of VGLL1-TEAD4 interaction inhibited VGLL1/TEAD-induced transcriptional programs to prevent the growth of resistant cells. EGFR was among the VGLL1/TEAD-regulated genes, and VGLL1-directed EGFR upregulation sensitized fulvestrant-resistant breast cancer cells to EGFR inhibitors. Taken together, these findings identify VGLL1 as a transcriptional driver in ET resistance and advance therapeutic possibilities for relapsed ER+ breast cancer patients. Significance: Transcriptional reprogramming mediated by the upregulation of the TEAD coactivator VGLL1 confers resistance to estrogen receptor degraders in breast cancer but provides alternative therapeutic options for this clinically important patient group.

Figure 1. Mapping altered epigenetic landscapes in fulvestrant-resistant breast cancer identifies VGLL1.

A, Ratio of H3K27ac in MCF7-FULVR versus MCF7 cells at gene promoters in a window of ± 1.5 kb centred on the transcriptional start site. B, Genome browser view of H3K27ac ChIP-seq signal at the VGLL1, VGLL3 and GREB1 genes. C, RNA-seq data showing upregulated genes (red, Padj < 0.01, log2 FC > 2) and downregulated genes (blue, Padj < 0.01, log2 FC < -2) in FULVR relative to MCF7 cells. D, Protein lysates prepared from MCF7 and FULVR cells were immunoblotted for the indicated proteins. Protein lysates were prepared from FULVR cells cultured in the presence of 100 nM fulvestrant. E, RT-qPCR using RNA prepared from MCF7 and T47D cells following addition of 100 nM fulvestrant for 24 hours (* = p<0.05). F, Cells were transfected with ESR1 siRNA or a non-targeting siRNA. RNAs prepared 48 hours after transfection were used for RT-qPCR. *P<0.05 (Student’s t-test, two-tailed). Data are presented as mean + s.e.m. *P<0.05 (Student’s t-test, two-tailed). G-H, RNA-scope was performed using a probe for VGLL1 in matched pre- and post-fulvestrant treated patient samples. Representative images show the results for matched samples from one patient. Nuclei were visualised with DAPI, and individual VGLL1 mRNA molecules detected with Cy5-labelled probes. P=0.03 (one-tailed Wilcoxon signed rank test). Scale bar, 10µm.

Figure 2. VGLL1 is recruited to TEAD4 binding regions in fulvestrant-resistant MCF7 cells.

A, Venn diagram showing overlap between TEAD4 ChIP-seq peaks in MCF7 and FULVR cells. Top-most enriched transcription factor motifs are shown. B, Correlation between VGLL1 and TEAD4 occupancy at TEAD4 peaks in FULVR cells. P-value was calculated using the Spearman test; r_s, Spearman’s correlation coefficient. C, Heatmap showing TEAD4 and VGLL1 binding at TEAD4 peaks common to MCF7 and FULVR (shared) and unique peaks in each cell line, in a window of ± 2kb around the peak centre. D, VGLL1 binding is enriched at TEAD4 peaks in FULVR cells. Binding enrichment was calculated as VGLL1-TEAD4 co-binding over the mean expected value after generating random permutations of the TEAD4 peaks (Chi-squared test p-value <0.0001). E, Average normalized ChIP-seq signal of TEAD4, VGLL1 and H3K27ac centred at TEAD4 peaks in FULVR cells. F, Average ChIP-seq signal of H3K27ac on VGLL1 peaks divided in quartiles based on the peak coverage in FULVR cells. G, Genome browser view of VGLL1, TEAD4 and H3K27ac ChIP-seq signal, together with the RNA-seq signal in MCF7 and FULVR cells at TEAD target genes.

Figure 3. VGLL1 facilitates development of resistance to fulvestrant.

A, MCF7-FULVR cells were transfected with two independent VGLL1 siRNAs. Growth was determined with the SRB assay five days after transfection. Data are mean ± s.e.m of n=6 independent wells. Results of one representative experiment are shown; similar results were obtained in two additional independent experiments. * P<0.05 (Mann-Whitney test, two tailed). Also shown are expression levels of VGLL1 following siVGLL1 transfection (n=3, *p<0.05 (Student’s t-test, two-tailed)). B, The synergistic activation mediator (SAM) uses a modified, catalytically dead Cas9 (dCas9), together with a sgRNA targeting to a specific gene promoter, for transcriptional activation of endogenous genes(57). A sgRNA targeted to bp -156 to -134 of the VGLL1 gene, was identified from the sgRNA list in ref.(57). C, MCF7-ActCas9-VGLL1 cells and MCF7-ActCas9-Vector cells were cultured with 100 nM fulvestrant and cell confluency was measured using Incucyte live cell imaging. Data show mean ± sem of n=9 representative images. D, RNA prepared from the indicated cells was used for RT-qPCR (mean ± sem; n=3). E, Immunoblotting of cell lysates prepared from the indicated cell lines. VGLL1-FULVR-100 and VGLL1-FULVR-1000 are fulvestrant resistant cell lines derived from the parental MCF7 ActCas9-VGLL1 cell line after continuous culturing in the presence of either 100 nM or 1000 nM fulvestrant, respectively. F, Growth of the indicated cell lines treated with increasing concentrations of fulvestrant to a maximum of 1 µM, for five days. Cell growth was estimated using the SRB assay and is shown as percentage relative to vehicle (n=6). Half maximum inhibitory concentration (IC₅₀) is indicated. G, MCF7 ActCas9-VGLL1-FULVR cells were transfected with siVGLL1 and growth assessed as in A. Data are mean ± s.e.m of n=6. One representative experiment is shown; similar results were obtained in two additional independent experiments. * P<0.05 (Mann-Whitney test, two tailed). RT-qPCR for VGLL1 is also shown (n=3, *p<0.05 (Student’s t-test, two-tailed)). H, Venn diagram comparing differentially expressed genes in MCF7-FULVR and MCF7-ActCas9-VGLL1-FULVR cells. 5,749 genes differentially expressed (padj<0.05) between MCF-FULVR and fulvestrant-sensitive MCF7 cells. 2,241 differential genes were identified in MCF7-ActCas9-VGLL1-FULVR-1 versus MCF7-ActCas9-VGLL1. There were 6,923 differential genes in MCF7-ActCas9-VGLL1-FULVR-2 relative to MCF7-ActCas9-VGLL1 cells.

Figure 4. Genes upregulated in fulvestrant-resistant breast cancer cells depend on VGLL1 transcriptional activity.

A, Genes predicted as VGLL1 targets in FULVR cells are more highly expressed in FULVR cells than in MCF7 cells. Genes were segregated into those with no VGLL1 peaks, and those with 2-4 and ≥5 VGLL1 peaks. The y-axis shows the log2 fold change in gene expression determined from RNA-seq in FULVR cells versus the parental MCF7 cells. ****P<0.0001 (Mann-Whitney test, two tailed). B, Genes activated by VGLL1 (n = 762) are over-expressed in FULVR cells relative to MCF7 cells, compared to not-VGLL1 targets (n = 8,932). VGLL1-activated genes and not-VGLL1 targets were determined by RNA-seq in FULVR cells transfected with VGLL1 siRNAs. The VGLL1-activated genes were defined as genes downregulated by VGLL1 siRNAs (P<0.0001 (Mann-Whitney test, two tailed)). C, Normalized average H3K27ac signal on the promoters of VGLL1 activated genes in FULVR cells and MCF7 cells. D, VGLL1-activated genes (n = 762) are more highly expressed in FULVR cells than the not-VGLL1 targets (n = 8,932). The y-axis shows normalized gene expression values from RNA-seq. P<0.0001 (Mann-Whitney test, two tailed). E, GO molecular function sets enriched in VGLL1-activated genes. F, RNA-seq data represented as a volcano plot for the comparison between FULVR vs MCF7 cells. G, RT-qPCR was performed using RNA prepared from the indicated cell lines. Gene expression was normalized to GAPDH expression and is shown as log2 fold difference relative to expression in MCF7 ActCas9-Vector cells.

Figure 5. VGLL1 induces EGFR expression and sensitivity to EGFR inhibition in FULVR cells.

A, Genome browser view of VGLL1 and TEAD4 ChIP-seq signal at the EGFR gene and EGFR enhancer (highlighted). B, ChIP-qPCR for TEAD4, VGLL1 and H3K27ac in MCF7 ActCas9-Vector, MCF7 ActCas9-VGLL1 and MCF7 ActCas9-VGLL1-FULVR cells showing VGLL1 and TEAD4 binding at the EGFR enhancer together with EGFR enhancer activation exclusively in FULVR cells. The CTGF -8.3kb region was used as a negative control for VGLL1/TEAD4 binding. C, RT-qPCR for EGFR in the indicated MCF7-ActCas9 cells (n=3, *p<0.05 (Student’s t-test, two-tailed)). D, Immunoblotting for EGFR and downstream EGFR signalling proteins showing EGFR up-regulation and activation of the EGFR pathway in MCF7 ActCas9-VGLL1-FULVR cells. E, RT-qPCR for dCAS9, VGLL1 and EGFR in MCF7 ActCas9-VGLL1-FULVR cells transfected with two independent dCAS9 siRNAs show reduction in VGLL1 and EGFR expression. F, Growth of the indicated MCF7-ActCas9 cells treated with increasing concentrations of the EGFR inhibitor erlotinib (0.05 µM to 12.5 µM) for 5 days. Growth is shown as percentage relative to vehicle treatment. G, Western blot of EGFR and the downstream EGFR signalling proteins in the indicated MCF7 ActCas9 cell lines treated with erlotinib at the indicated concentrations (24 h). H, Model for a mechanism by which inhibition of ER activity and concomitant VGLL1 induction drive EGFR expression in fulvestrant-resistant breast cancer cells, to promote cell survival and growth. I, EGFR ranks as the most significantly co-expressed gene with VGLL1 in breast cancer patients from METABRIC and is also the highest ranked protein correlated with VGLL1 in breast cancer from TCGA Firehose legacy cohort. In each case the top 3 highest ranked genes are shown. The ranking and correlations were generated from cBioportal (accessed 15/12/2022).

Figure 6. Verteporfin inhibits VGLL1 transcriptional activity and resensitises breast cancer cells to fulvestrant.

A, VP impairs the growth of FULVR cells. Growth in the indicated FULVR cells treated with increasing concentrations of VP is shown as percentage of growth relative to vehicle. B, ChIP-qPCR for VGLL1 and TEAD4 in MCF7-FULVR cells showing reduced VGLL1 binding at the target genes in the presence of VP (2µM, 24 h). The y-axis shows DNA enrichment calculated as the percentage of input. C, Volcano plot of RNA-seq data (from n=4 biological replicates) showing upregulated genes (red, adjusted P<0.01, log2 (fold change) > 1) and downregulated genes (blue, adjusted P<0.01, log2 (fold change) < -1) in MCF7-FULVR cells treated with VP, (2µM, 24 h) compared to vehicle (DMSO). D, RT-qPCR in MCF7-FULVR cells treated with VP (2µM, 24 h) showing that VP selectively downregulates the expression VGLL1 targets, while the expression of the not-VGLL1 target (APEX1) is not affected by VP. E, Genes downregulated by VP are highly enriched in VGLL1 peaks. The y-axis shows the number of VGLL1 peaks over the expected value after generating random permutations of the VGLL1 peaks showing that the top-most downregulated genes after VP treatment in FULVR cells (n = 631) have significantly higher number of VGLL1 peaks compared to the bottom genes not differentially expressed by VP (n = 631). Data are presented as box-and-whiskers plots (whiskers extend from the 5th to the 95th percentile; the box extends from the 25th to the 75th percentile; the line within the box represents the median). **** P<0.0001 (Mann-Whitney test, two tailed). F, Genome browser view of VGLL1 and TEAD4 normalized ChIP-seq signal and normalized RNA-seq signal in FULVR cells showing representative examples of VGLL1 activated genes (direct VGLL1 targets) and not-VGLL1 targets. G, VGLL1 activated genes (n = 762) are preferentially downregulated by VP compared to not-VGLL1 targets (n = 8,932). The y-axis shows fold change in gene expression from RNA-seq between VP versus DMSO treatment in MCF7-FULVR cells. ****P<0.0001 (Mann-Whitney test, two tailed). H, Breast cancer patients with higher VGLL1 expression display increased levels of expression of the VGLL1 activated genes compared to patients with lower VGLL1 expression. Patients from the METABRIC breast cancer dataset were stratified according to high (top quantile, n=495) or low (bottom quantile, n=495) VGLL1 expression levels. Data are presented as in E. ****P<0.0001 (Mann-Whitney test, two tailed). I, Kaplan–Meier plot representing the percentage of metastasis-free survival in patients with ER+ breast cancer patients treated with endocrine therapies showing that patients with higher expression of the VGLL1 activated genes signature display lower survival rates (log-rank Mantel-Cox test). J, Tumor growth curves for mice bearing the KCC_P_3837 FulvR clone 2 (VGLL1 high) PDX model, treated as shown. Simple linear regression shows that slopes in the combination arm is significantly (*** = p <0.0001) different from each of those in vehicle, fulvestrant or verteporfin treatment arms. K, Mean tumor volumes at 6 weeks; dots show sizes of the individual tumors. * = two-tailed unpaired T test, P < 0.05; ** = p <0.005.