Class I histone deacetylase inhibitor suppresses vasculogenic mimicry by enhancing the expression of tumor suppressor and anti-angiogenesis genes in aggressive human TNBC cells

Triple-negative breast cancer (TNBC) cells form angiogenesis-independent vessel-like structures to survive, known as vasculogenic mimicry (VM), contributing to a poor prognosis for cancer patients. Nuclear localized class I histone deacetylases (HDACs) enzymes, particularly HDACs 1, 2, 3 deacetylate chromatin histones, are overexpressed in cancers and epigenetically regulate the expression of genes involved in cancer initiation and progression. The specific HDAC inhibitor, entinostat, has been shown to attenuate tumor progression and metastasis in TNBC. In this study, we hypothesized that entinostat would enhance the expression of anti-angiogenic and tumor suppressor genes and would thus suppress VM structures in TNBC cells in a 3D Matrigel cell culture preclinical model. Our data indicated that invasive triple-negative MDA-MB-231, LM2-4 and BT-549 breast cancer cells, but not poorly invasive luminal MCF-7 cells, efficiently underwent matrix-associated VM formation. Approximately 80% of TNBC cells with the stem cell phenotype potential formed vessel-like structures when mixed with Matrigel and cultured in the low attachment tissue culture plate. The molecular mechanisms of VM formation are rather complex, while angiogenesis inhibitor genes are downregulated and pro-angiogenesis genes are upregulated in VM-forming cells. Our data revealed that treatment of the TNBC VM phenotype cells with entinostat epigenetically led to the re-expression of the anti-angiogenic genes, serpin family F member 1 (SERPINF1) and thrombospondin 2 (THBS2), and to that of the tumor suppressor genes, phosphatase and tensin homolog (PTEN) and p21, and reduced VM structures. We also found that treatment of the TNBC VM phenotype cells with entinostat downregulated the expression of vascular endothelial growth factor A (VEGF-A), and that of the epithelial-mesenchymal transition (EMT)-related genes, Vimentin and beta-catenin. METABIRC and TCGA breast cancer cohort mRNA expression data analysis revealed that a high expression of the anti-angiogenesis-associated genes, THBS2, SERPINF1 and serpin family B member 5 (SERPINB5), and of the tumor suppressor gene, PTEN, was associated with a better overall survival (OS) of breast cancer patients. Taken together, the findings of this study demonstrate that HDACs 1, 2, 3 partly contribute to VM formation in TNBC cells; thus, HDACs may be an important therapeutic target for TNBC.