THE REPRESSION OF MEF2 TRANSCRIPTION FACTORS EXERTED BY CLASS IIA HDACS AND THEIR DEGRADATION STIMULATED BY CDK4 DETERMINE THE ACQUISITION OF HALLMARKS OF TRANSFORMATION IN FIBROBLASTS.

MEF2 transcription factors (TFs) are well known regulators of differenziative and adaptive responses, with predominant roles in muscular, cerebral and immune districts. However, literature concerning the contribution of MEF2 TFs in processes of transformation and oncogenesis is scattered and contradictory; class IIa HDACs (HDAC4, HDAC5, HDAC7, HDAC9) are well-established repressors of MEF2 activity and increasing numbers of selective class IIa HDACs inhibitors are under preclinical screening for various diseases, including cancer. However, a clear demonstration of the oncogenic functions of these proteins is still missing. The aim of this work was to clarify the possible involvement of the HDAC-MEF2 axis in carcinogenesis using as a model different mesenchymal cell lines with varying degrees of immortalization. Here, we incontrovertibly demonstrate a pro-oncogenic role of a nuclear resident form of HDAC4/HDAC7 in NIH-3T3 and BALB/c fibroblasts. Through a DNA microarray experiment we identified the signature of HDAC4 and, as expected, among the genes directly repressed by HDAC4 many are MEF2 targets. We demonstrated that most of the transforming potential of HDAC4 is due to the repression of MEF2 transcriptional activity and that the MEF2-HDAC axis is particularly active in Soft-tissue Sarcomas; in these tumors the binding between HDAC4 and MEF2 could be an effective therapeutic target, as proved by us in vitro. We also demonstrated that the repression of MEF2 activity could also be exerted by common oncogenes, such as RAS and AKT, which act independently from class IIa HDACs by inducing a decrease in the half-life of MEF2C and MEF2D proteins. We reported that MEF2C/D are subjected to a cyclic degradation during cell-cycle with peaks of dysregulation concomitant with S phase entry. The signal that controls the cyclic degradation of MEF2 is the phosphorylation by CDK4/CyclinD1 on two serine residues, conserved among the MEF2 family members, except for MEF2B and a transcriptional variant expressed in skeletal muscles. As a consequence of this phosphorylation, MEF2C/D are bound by the E3-ligase SKP2 that mediates their poly-ubiquitylation and degradation in the proteasome. The cyclic degradation of MEF2 proteins is required for the correct progression of the cell-cycle, as any interference in this degradation process causes an arrest in G1 because of MEF2-mediated transcription of p21/CDKN1A; on the contrary, any increase in MEF2 degradation causes an aberrant progression in the cell-cycle, a common feature of cancer cells. In summary, we demonstrated that in fibroblasts MEF2 activity could be alternatively repressed by class IIa HDACs or through a cell-cycle based degradation process; in both the cases MEF2 repression results in an increase in cell proliferation and in the acquisition of hallmarks of transformation

Regulation of class IIa HDAC activities: It is not only matter of subcellular localization

In response to environmental cues, enzymes that influence the functions of proteins, through reversible post-translational modifications supervise the coordination of cell behavior like orchestral conductors. Class IIa histone deacetylases (HDACs) belong to this category. Even though in vertebrates these deacetylases have discarded the core enzymatic activity, class IIa HDACs can assemble into multiprotein complexes devoted to transcriptional reprogramming, including but not limited to epigenetic changes. Class IIa HDACs are subjected to variegated and interconnected layers of regulation, which reflect the wide range of biological responses under the scrutiny of this gene family. Here, we discuss about the key mechanisms that fine tune class IIa HDACs activities

The Histone Code of Senescence

Senescence is the end point of a complex cellular response that proceeds through a set of highly regulated steps. Initially, the permanent cell-cycle arrest that characterizes senescence is a pro-survival response to irreparable DNA damage. The maintenance of this prolonged condition requires the adaptation of the cells to an unfavorable, demanding and stressful microenvironment. This adaptation is orchestrated through a deep epigenetic resetting. A first wave of epigenetic changes builds a dam on irreparable DNA damage and sustains the pro-survival response and the cell-cycle arrest. Later on, a second wave of epigenetic modifications allows the genomic reorganization to sustain the transcription of pro-inflammatory genes. The balanced epigenetic dynamism of senescent cells influences physiological processes, such as differentiation, embryogenesis and aging, while its alteration leads to cancer, neurodegeneration and premature aging. Here we provide an overview of the most relevant histone modifications, which characterize senescence, aging and the activation of a prolonged DNA damage response

MEF2 and the tumorigenic process, hic sunt leones

While MEF2 transcription factors are well known to cooperate in orchestrating cell fate and adaptive responses during development and adult life, additional studies over the last decade have identified a wide spectrum of genetic alterations of MEF2 in different cancers. The consequences of these alterations, including triggering and maintaining the tumorigenic process, are not entirely clear. A deeper knowledge of the molecular pathways that regulate MEF2 expression and function, as well as the nature and consequences of MEF2 mutations are necessary to fully understand the many roles of MEF2 in malignant cells. This review discusses the current knowledge of MEF2 transcription factors in cancer

Transformation by different oncogenes relies on specific metabolic adaptations

Metabolic adaptations are emerging as common traits of cancer cells and tumor progression. In vitro transformation of NIH 3T3 cells allows the analysis of the metabolic changes triggered by a single oncogene. In this work, we have compared the metabolic changes induced by H-RAS and by the nuclear resident mutant of histone deacetylase 4 (HDAC4). RAS-transformed cells exhibit a dominant aerobic glycolytic phenotype characterized by up-regulation of glycolytic enzymes, reduced oxygen consumption and a defect in complex I activity. In this model of transformation, glycolysis is strictly required for sustaining the ATP levels and the robust cellular proliferation. By contrast, in HDAC4/TM transformed cells, glycolysis is only modestly up-regulated, lactate secretion is not augmented and, instead, mitochondrial oxygen consumption is increased. Our results demonstrate that cellular transformation can be accomplished through different metabolic adaptations and HDAC4/TM cells can represent a useful model to investigate oncogene-driven metabolic changes besides the Warburg effect

Photosensitization of pancreatic cancer cells by cationic alkyl-porphyrins in free form or engrafted into POPC liposomes: The relationship between delivery mode and mechanism of cell death

Cationic porphyrins bearing an alkyl side chain of 14 (2b) or 18 (2d) carbons dramatically inhibit proliferation of pancreatic cancer cells following treatment with light. We have compared two different ways of delivering porphyrin 2d: either in free form or engrafted into palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes (L-2d). Cell cytometry shows that while free 2d is taken up by pancreatic cancer cells by active (endocytosis) and passive (membrane fusion) transports, L-2d is internalized solely by endocytosis. Confocal microscopy showed that free 2d co-localizes with the cell membrane and lysosomes, whereas L-2d partly co-localizes with lysosomes and ER. It is found that free 2d inhibits the KRAS-Nrf2-GPX4 axis and strongly triggers lipid peroxidation, resulting in cell death by ferroptosis. By contrast, L-2d does not affect the KRAS-Nrf2-GPX4 axis and activates cell death mainly through apoptosis. Overall, our study demonstrates for the first time that cationic alkyl porphyrins, which have a IC50 ~ 23 nM, activate a dual mechanism of cell death, ferroptosis and apoptosis, where the predominant form depends on the delivery mode

The co-existence of transcriptional activator and transcriptional repressor MEF2 complexes influences tumor aggressiveness

The contribution of MEF2 TFs to the tumorigenic process is still mysterious. Here we clarify that MEF2 can support both pro-oncogenic or tumor suppressive activities depending on the interaction with co-activators or co-repressors partners. Through these interactions MEF2 supervise histone modifications associated with gene activation/repression, such as H3K4 methylation and H3K27 acetylation. Critical switches for the generation of a MEF2 repressive environment are class IIa HDACs. In leiomyosarcomas (LMS), this two-faced trait of MEF2 is relevant for tumor aggressiveness. Class IIa HDACs are overexpressed in 22% of LMS, where high levels of MEF2, HDAC4 and HDAC9 inversely correlate with overall survival. The knock out of HDAC9 suppresses the transformed phenotype of LMS cells, by restoring the transcriptional proficiency of some MEF2-target loci. HDAC9 coordinates also the demethylation of H3K4me3 at the promoters of MEF2-target genes. Moreover, we show that class IIa HDACs do not bind all the regulative elements bound by MEF2. Hence, in a cell MEF2-target genes actively transcribed and strongly repressed can coexist. However, these repressed MEF2-targets are poised in terms of chromatin signature. Overall our results candidate class IIa HDACs and HDAC9 in particular, as druggable targets for a therapeutic intervention in LMS

Transcriptomic and genomic studies classify NKL54 as a histone deacetylase inhibitor with indirect influence on MEF2-dependent transcription

In leiomyosarcoma class IIa HDACs (histone deacetylases) bind MEF2 and convert these transcription factors into repressors to sustain proliferation. Disruption of this complex with small molecules should antagonize cancer growth. NKL54, a PAOA (pimeloylanilide o-aminoanilide) derivative, binds a hydrophobic groove of MEF2, which is used as a docking site by class IIa HDACs. However, NKL54 could also act as HDAC inhibitor (HDACI). Therefore, it is unclear which activity is predominant. Here, we show that NKL54 and similar derivatives are unable to release MEF2 from binding to class IIa HDACs. Comparative transcriptomic analysis classifies these molecules as HDACIs strongly related to SAHA/vorinostat. Low expressed genes are upregulated by HDACIs, while abundant genes are repressed. This transcriptional resetting correlates with a reorganization of H3K27 acetylation around the transcription start site (TSS). Among the upregulated genes there are several BH3-only family members, thus explaining the induction of apoptosis. Moreover, NKL54 triggers the upregulation of MEF2 and the downregulation of class IIa HDACs. NKL54 also increases the binding of MEF2D to promoters of genes that are upregulated after treatment. In summary, although NKL54 cannot outcompete MEF2 from binding to class IIa HDACs, it supports MEF2-dependent transcription through several actions, including potentiation of chromatin binding

Maria Nallino (1908-1974) and the Birth of Arabic and Islamic studies at Ca’ Foscari

This essay, based on bibliographic and archival material, focuses on the academic figure Maria Nallino, a scholar whose voluminous body of work ranges from classicism to modernity with equal fluency and expertise. Daughter of the famous Orientalist Carlo Alfonso Nallino (1872-1938), whose work she gathered and actively promoted, her arrival at Ca’ Foscari (1962) inaugurated Arabic and Islamic studies in Venice