DISSECTING THE ROLE OF NEDD4 HECT E3 LIGASE AND ITS ISOFORMS IN CANCER

NEDD4 is a E3 ubiquitin ligase belonging to the HECT family and is composed by a N-terminal C2 domain, four WW domains and the catalytic C-terminal HECT domain. Different studies evidenced a correlation between NEDD4 overexpression and tumor progression and in some cases its proto-oncogenic activity has been ascribed to the ubiquitination-mediated degradation of the tumor suppressor PTEN. The proto-oncogenic role of NEDD4 seems to be cellular context dependent and interesting data linked K-RAS activation to NEDD4 overexpression and subsequent PTEN degradation in human colorectal cancer. Since NEDD4 is overexpressed in Non-Small Cell Lung cancer (NSCLC), and high percentage of these tumors show K-RAS mutation, we decided to verify the proto-oncogenic role of NEDD4 in a genetic context characterized by K-RAS mutation in NSCLC. Unfortunately, our attempt to functionally link these events or to validate the phenotype associated to NEDD4 failed. Conducting the experiments, we noticed the presence of few putative NEDD4 isoforms recognized by anti-NEDD4 antibody, and, hypothesizing a possible contribute to the proliferation phenotype, we decided to characterize them. The existence of alternative splice variants of NEDD4 has been reported in online databases, but their relevance is largely unknown. Experiments of siRNA interference followed by western blot together with RT-PCR and sequencing confirmed the identity of NEDD4 isoforms, heterogeneously expressed in cancer cells representative of various tumors. Among the alternative versions of NEDD4, isoform 3 is reported to be one of the mainly expressed across different tissues. Interestingly, this putative isoform, compared to the canonical isoform 1 protein, exhibits a unique domain of 516-amino acid at the N-terminus together with a consistent deletion of the C2 domain. The presence of the N-terminal additional domain, together with the well-known relevance of the C2 domain for the regulation of the catalytic activity of NEDD4, suggests that isoform 3 may promote cellular processes different from the ones exerted by isoform 1. In order to unveil biological and biochemical features of NEDD4 isoform 3, we started analyzing its cellular localization and ubiquitination activity. In addition, co-immunoprecipitation coupled with proteomic experiment led to the identification of exclusive NEDD4 isoform 3 interactors, of which the mitochondrial carrier SLC25A13. To date, significant observations linking NEDD4 E3 ligase to mitochondria are not reported, thus the functional relevance arising from this interaction was explored. In this context, the observed mitochondrial membrane potential dependency upon NEDD4 isoform 3 may provide first evidences of the isoform’s involvement in undisclosed pathways, shedding light on its potential impact on novel cellular functions, different from the ones ascribed to canonical NEDD4.