HDAC4: studying the pro-oncogenic role in human immortalized fibroblasts

In vitro transformation of primary human fibroblasts has been commonly used to understand the specific steps required to generate neoplastic cells following the ordered introduction of cellular and viral oncogenes and/or the down-modulation of tumor suppressor genes. In this thesis I have applied this strategy to explore the pro-oncogenic function of class IIa HDACs. Class IIa histone deacetylases deregulation can contribute to cancer development and progression in different ways. However their real involvement in tumor biology is still debated. To clarify this issue I have investigated the role of HDAC4, a representative member of this class, in human immortalized foreskin fibroblast (BJ/hTERT). I have demonstrated that HDAC4 negatively influences the isolation of clones after retroviral infection. This effect is MEF2-independent and is in part due to the activation of an apoptotic response. Through the generation of BJ/hTERT cells expressing BCL-xL, a Bcl-2 family member characterized by a pro-survival function, it was possible to isolate clones expressing HDAC4 mutated in the 14-3-3 binding sites, suggesting that HDAC4 deregulation can elicit apoptosis. Isolated clones were characterized, and alterations in the cell cycle profile were not observed. However strong repressive forms of HDAC4 were also subject to intense proteolytic degradation. The apoptotic response and the proteasome-mediated degradation were also described using a doxycycline-inducible system. In this case the nuclear resilient mutants of HDAC4 render BJ/hTERT cells more susceptible to apoptosis only when triggered by DNA damage and protein synthesis inhibition, but not by proteasome inhibitors or oxidative stress. In addition all the nuclear resident mutants evidenced a higher rate of proteasomal degradation. Finally, ectopically expressing in BJ/hTERT cells, a form of HDAC4 mutated only in NES sequence (HDAC4-L/A), allowed the isolation of clones characterized by a MEF2-repressed phenotype. This mutation causes the accumulation of the deacetylase in the nuclear compartment, without interfering with 14-3-3 binding. This result suggests a possible implication of these adaptor proteins in the HDAC4 anti-proliferative activity. In parallel murine fibroblast expressing the HDAC4-L/A mutant acquire the ability to growth in an anchorage-independent manner. Overall this thesis sheds some light on the HDAC4 potential of eliciting oncogenic conversion also in human cells managed also by the 14-3-3 binding