Histone modifiers driving Notch context-dependent activity in cancer

Deregulated Notch signaling is linked to onset and progression in various cancers. Epigenetic machinery writing or erasing chromatin status of NOTCH genes has been vigorously studied but not fully described yet. In particular, methylation status of H3K27 and availability of corresponding DNA loci for transcription is based on balanced work of antagonizing chromatin modifiers including the EZH2 methyltransferase component of Polycomb-Repressive Complex 2 (PCR2) and demethylases like JMJD3 or UTX. In the current work, we aimed to contribute to the discovery of additional mechanisms regulating transcription of NOTCH1 and NOTCH3 genes in contrasting cancer contexts: T-cell acute lymphoblastic leukemia (T-ALL) where Notch is a well-known oncogene and cervical cancer where Notch proteins are supposed to act as oncosuppressors, concentrating on roles of demethylase JMJD3 and methyltransferase EZH2 favoring chromatin opening and condensation, respectively. In case of T-ALL where low methylation status of H3K27 at NOTCH-gene corresponding loci favors transcription of NOTCH genes and their targets, we found that pharmacological inhibition of JMJD3 by GSKJ4 decreased the expression levels of NOTCH3, NOTCH1, the target gene DELTEX1, and c-Myc, and abrogated cell viability in both Notch1- and Notch3-dependent T-cell contexts, as confirmed with the accumulation of anti-proliferative factor p27 and apoptosis-associated cleaved form of PARP. Anti-growth effects of GSKJ4 were partially rescued by exogenous Notch1, Notch3, and c-Myc expression indicating a possible involvement of Notch/c-Myc regulatory axis. Specularly, in cervical cancer, EZH2 is responsible for the epigenetic silencing of tumor-suppressor genes. Supporting the anti-cancerous role of Notch1 protein in this context, we found that EZH2 inhibition upregulated the levels of the Notch receptors, ligands, and target genes, potently suppressed the growth of cervical cancer cells in vitro, and was associated with upregulation of cell cycle blockers p21cip, p27kip, and p53 and reduced expression of c-Myc. Similar effects on cell viability and expression of cell cycle regulators were achieved through transient ectopic introduction of Notch1 in model cell lines. Confirming the partial dependence of observed anti-growth effects of EZH2 inhibition on Notch activation, combined treatment with GSK126 and γ-secretase 2 inhibitor (GSI) abrogated GSK126 effects on cell viability by restoring the number of viable cells and p21cip, p27kip, and p53 levels. Furthermore, EZH2 inhibition suppressed the motility of human cervical cancer cells and upregulated epithelial phenotype marker E-cadherin (E-Cadh) and/or reduced the expression of promesenchymal vimentin in cell-line dependent fashion. Proving the relevance of Notch signaling in the epithelial phenotype maintenance, the combination of GSK126 with GSI restored E-Cadh and/or vimentin levels and partially rescued cell migratory capacity. The results of our work contribute to the increasing amount of evidence that pharmacological inhibition of histone methyltransferases and demethylases might be a promising strategy for controlling oncogene activation, malignant cell growth and metastatic capacity thus opening new roads for creation of novel targeted cancer therapies.