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.