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Supplemental Digital Content is available in the text., Rationale: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. Objective: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. Methods and Results: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. Conclusions: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system.

Anaplastic thyroid carcinoma (ATC) is an extremely aggressive thyroid cancer subtype, refractory to the current medical treatment. Among various epigenetic anticancer drugs, bromodomain and extra-terminal inhibitors (BETis) are considered to be an appealing novel class of compounds. BETi target the bromodomain and extra-terminal of BET proteins that act as regulators of gene transcription, interacting with histone acetyl groups. The goal of this study is to delineate which pathway underlies the biological effects derived from BET inhibition, in order to find new potential therapeutic targets in ATC. We investigated the effects of BET inhibition on two human anaplastic thyroid cancer-derived cell lines (FRO and SW1736). The treatment with two BETis, JQ1 and I-BET762, decreased cell viability, reduced cell cycle S-phase, and determined cell death. In order to find BETi effectors, FRO and SW1736 were subjected to a global transcriptome analysis after JQ1 treatment. A significant portion of deregulated genes belongs to cell cycle regulators. Among them, MCM5 was decreased at both mRNA and protein levels in both tested cell lines. Chromatin immunoprecipitation (ChIP) experiments indicate thatMCM5is directly bound by the BET protein BRD4.MCM5silencing reduced cell proliferation, thus underlining its involvement in the block of proliferation induced by BETis. Furthermore, MCM5 immunohistochemical evaluation in human thyroid tumor tissues demonstrated its overexpression in several papillary thyroid carcinomas and in all ATCs. MCM5 was also overexpressed in a murine model of ATC, and JQ1 treatment reducedMcm5mRNA expression in two murine ATC cell lines. Thus, MCM5 could represent a new target in the therapeutic approach against ATC.

Abnormal expression of non-coding micro RNA (miRNA) has been described in medullary thyroid carcinoma (MTC). Expression of genes encoding factors involved in miRNA biogenesis results often deregulated in human cancer and correlates with aggressive clinical behavior. In this study, expression of four genes involved in miRNA biogenesis (DICER, DROSHA, DCGR8, and XPO5) was investigated in 54 specimens of MTC. Among them, 33 and 13 harbored RET and RAS mutations, respectively. DICER, DGCR8, and XPO5 mRNA levels were significantly overexpressed in MTC harboring RET mutations, in particular, in the presence of RET634 mutation. When MTCs with RET and RAS mutations were compared, only DGCR8 displayed a significant difference, while MTCs with RAS mutations did not show significant differences with respect to non-mutated tumors. We then attempted to correlate expression of miRNA biogenesis genes with tumor aggressiveness. According to the TNM status, MTCs were divided in two groups and compared (N0 M0 vs. N1 and/or M1): for all four genes no significant difference was detected. Cell line experiments, in which expression of a RET mutation is silenced by siRNA, suggest the existence of a causal relationship between RET mutation and overexpression of DICER, DGCR8, and XPO5 genes. These findings demonstrate that RET- but not RAS-driven tumorigenic alterations include abnormalities in the expression of some important genes involved in miRNA biogenesis that could represent new potential markers for targeted therapies in the treatment of RET-mutated MTCs aimed to restore the normal miRNA expression profile.