HDAC inhibitors modulate Hippo pathway signaling in hormone positive breast cancer

Abstract

Breast cancer has constantly been the leading causes of death in women, and hormone receptor (HR) positive, HER2 negative is the majority subtype. Histone deacetylase (HDAC) inhibitors (HDACi) have shown clinical benefit in HR ( +) breast cancer patients. The Hippo pathway is an important cellular pathway involving proliferation, cell contact, and cancer. Hippo pathway proteins YAP/TAZ are often viewed as pro-tumorigenic; however, recent studies support a role of YAP as a tumor suppressor in HR ( +) breast cancer. Few studies have investigated the link between HDACi and the Hippo pathway. In our study, we demonstrate that HDACi induces transcriptional downregulation of YAP expression, while conversely activating a TEAD-mediated transcriptional program with upregulation of canonical Hippo pathway genes. We subsequently identified four Hippo canonical genes (CCDC80, GADD45A, F3, and TGFB2) that were upregulated by HDACi and associated with significantly improved survival in a HR ( +) breast cancer cohort. We further validated experimentally that HR ( +) breast cancer cells treated with HDACi resulted in upregulation of CCDC80 and GADD45A. A pan-cancer analysis of TCGA database demonstrated lower CCDC80 and GADD45A expression in tumor tissue compared to non-tumor samples in BRCA (breast cancer), LAML (acute myeloid leukemia), and UCS (uterine carcinosarcoma). Further analysis of HR ( +) breast cancer patients in the METABRIC dataset revealed high CCDC80 and/or GADD45A expression associated with significantly better survival outcomes compared to patients with low expression. Our study provides evidence for a novel mechanism of HDACi clinical activity, as well as a potential role for CCDC80 and GADD45A in HR ( +) breast cancer.

Fig. 1

RNA-seq of HR( +) breast cancer cells treated with romidepsin reveals pathway enrichment including cell cycle and Hippo pathways. A Venn diagram of differentially expressed genes (DEG) from RNA-seq data performed on MCF-7 or T47D cells, treated with either romidepsin 50 nM or DMSO for 24 h. B–D Gene ontology (GO) analysis (B), dot plot (C), and ridge plot (D) of DEGs from the RNA-seq data. E GSEA analysis for cell cycle pathway from the RNA-seq data. F Leading enriched pathways from DAVID analysis of the RNA-seq data. G, H Heat map of mRNA expression from the RNA-seq dataset of Hippo pathway signature genes [49] in T47D (G) and MCF-7 (H)

Fig. 2

HDACi upregulates Hippo pathway targets CYR61, CTGF, and TEAD luciferase reporter activity, while downregulating YAP expression. A MCF-7 and T47D cells were treated with romidepsin 50 nM for 24 h. qRT-PCR was performed for quantification of CYR61 and CTGF mRNA. Fold change (Y-axis) represents fold change of romidepsin-treated cells compared to DMSO-treated cells. Each dot represents a biological replicate (i.e., this experiment repeated 3 times). Blue: MCF-7. Red: T47D. *sign denotes p < 0.05. B A549, MCF-7, and T47D treated with romidepsin 50 nM and measured for luciferase activity by the 8xGTIIC-TEAD luciferase reporter. Activity fold changes normalized to DMSO-treated cells. Each dot represents a biological replicate (3 repeats for each cell line). (C) Heat map of mRNA expression of CTGF and CYR61 from the transcriptome datasets (see methods for full list). Abbreviations for cell lines: MCF: MCF-7 HR ( +) breast cancer cell line. T47: T47D HR ( +) breast cancer cell line. 231: MDA-MB-231 (TNBC). CUB: VM-CUB1 urothelial cancer cell line. UC: UM-UC-3 urothelial cancer cell line. SAHA: Vorinostat. siHDAC1_2: treated with combination of siRNAs against HDAC1 and HDAC2. Color palette denotes fold change compared to individual controls. D Multiple cell lines treated with romidepsin 30 nM for 24 h and blotted for YAP expression. Plus sign denotes treatment with romidepsin, minus sign denotes treatment with DMSO. (Romidepsin 30 nM was chosen for immunoblotting studies as this concentration demonstrated consistent changes across cell lines tested) E T47D cells treated with romidepsin 30 nM at various time points and blotted for YAP expression. F MCF-7 treated with romidepsin 30 nM for 24 h and blotted for YAP, phosphorylated YAP (p-YAP). G MCF-7 and T47D cells treated by romidepsin 30 nM for 24 h. qRT-PCR was performed for quantification of YAP1, TAZ, LATS1, and LATS2 mRNA. Fold change (Y-axis) represents fold change of romidepsin-treated cells compared to DMSO-treated cells. Each dot represents a biological replicate (Total 3 biological repeats). Blue: MCF-7. Red: T47D. *sign denotes p < 0.05. H Heat map of mRNA expression of TAZ and YAP from the transcriptome datasets (see methods for full list). Cell line denotation described in legend for Fig. 2C. Color palette denotes fold change compared to individual controls

Fig. 3

Hippo pathway downstream genes CCDC80 and GADD45A are associated with survival in HR ( +) breast cancer. A Overall survival analysis for genes GADD45A, CCDC80, F3, and TGFB2 from the TCGA METABRIC dataset, filtered for HR ( +) HER2(-) breast cancer cases. mRNA expression levels were downloaded for each patient sample and categorized into 1:1 ratio of high expression versus low expression, followed by comparison of overall survival. B Overall survival (OS) curves between patients with high expression of CCDC80 or GADD45A (High C-G) versus low expression (Low C-G). C Three-gene classifier for intrinsic subtype distribution between high C-G patients versus low C-G patients. D Recurrence free survival (RFS) curves between patients with high expression of CCDC80 or GADD45A (High C-G) versus low expression (Low C-G). E Heat map of mRNA expression of CCDC80 and GADD45A from the transcriptome datasets (see methods for full list). Cell line denotation described in legend for Fig. 2C. Color palette denotes fold change compared to individual controls. F, G MCF-7 and T47D treated with romidepsin 50 nM for 24 h. qRT-PCR was performed for quantification of GADD45A and CCDC80 mRNA. Fold change (Y-axis) represents fold change of romidepsin-treated cells compared to DMSO-treated cells. H MCF-7 and T47D treated with verteporfin 4 μM for 24 h. In Fig. 3F–H, each dot represents a biological replicate (i.e., repeated 3 times), blue bars and red bars are MCF-7 and T47D, respectively. *sign denotes p < 0.05

Fig. 4

Pan-cancer transcriptomic analysis reveals upregulation of CCDC80 and GADD45A in tumor tissue in breast cancer patient samples. A, B Comparison of tumor and normal samples for CCDC80 expression (A) or GADD45A expression (B), from 31 TCGA datasets, analyzed by GEPIA2 web browser database. Datasets showing decreased CCDC80 expression in tumor versus normal are shown here. C Comparison of Hippo pathway genes between HDAC1 high versus HDAC1 low patient samples analyzed from the METABRIC database. D Comparison of Hippo pathway genes between HDAC2 high versus HDAC2 low patient samples analyzed from the METABRIC database. E Proposed mechanism for this study. In our current working model, HDAC inhibitors result in downregulation of YAP and ESR1 expression transcriptionally, while activating TEAD-based transcriptional activation of Hippo pathway downstream genes including CCDC80 and GADD45A. Higher expression of CCDC080 and GADD45A is associated with improved outcomes in HR( +) HER2(−) breast cancer