Abstract 482: FKBP51 sustains the constitutive NF-κB activation in melanoma
Authors: Romano Simona , Sun Shao-Cong , D'Angelillo Anna , D'Arrigo Paolo , Bisogni Rita , Romano Maria Fiammetta 
Published: May 2015

Abstract
Constitutive activation of NF-κB occurs in a significant percentage of human cancers, including melanoma. Aberrant NF-κB activation sustains cancer survival, contributes to tumor invasion and accounts for therapy resistance. Several studies support a role for FKBP51 in NF-κB activation, however, mechanisms by which FKBP51 controls NF-κB pathway are still not known. FKBP51 has been previously found to interact with IKKα. We studied FKBP51 interactions with a wide number of -κB proteins by transfecting expression vectors, carrying each tagged-protein. Our results confirmed that FKBP51 strongly interacts with IKKα, in addition, a weaker binding was also found with IKKβ, IKKγ and TAK1. To investigate whether FKBP51 interaction with IKK components was essential for NF-κB activation, we knocked down FKBP51 in two different metastatic melanoma cell lines, namely A375 and SAN. Kinase assays showed a reduced phosphorylation of IκBα in knocked down melanoma cells, in comparison with control melanoma. The reduced phosphorylation of IκBα resulted in its reduced proteasomal degradation, both at the basal level and following TNF-α stimulation. FK506, that inhibits FKBP51 PPIase activity, did not interfere with FKBP51/IKKα interaction in co-IP experiments, but the drug reduced the IκBα phosphorylation levels, suggesting that FKBP51 PPIase activity is required for IKK kinase activity. EMSA confirmed the absence of NF-κB activity in FKBP51 knocked down melanoma, even after TNF-α stimulation. Consistent with this finding, an increased apoptosis was measured in FKBP51 silenced cells, stimulated with TNF-α, or chemotherapeutic agents. TRAF2 (TNF-α receptor associated factor-2) is a prototypical member of the TRAF family, it transduces signals generated by several members of the TNF receptor superfamily. We found FKBP51 modulated TRAF2 at mRNA and protein level. In particular, data obtained by QPCR showed that TRAF2 mRNA decreased by 30% in knocked down melanoma cells, compared with levels in control cells. This finding was in accordance with the notion that TRAF2 is a transcriptional target of NF-κB. In addition, we also found that FKBP51 regulated TRAF ubiquitination via TPR domain. In fact, co-expression of HA-ubiquitin and FKBP51WT prevented TRAF2 ubiquitination. By contrast, co-expression of HA-ubiquitin and FKBP51 mutated in TPR, but not PPIase-domain, determined a TRAF2 degradative ubiquitination. In conclusion, our study provides mechanistic insights into the functions of FKBP51 in regulation of NF-κB activation, namely: 1) FKBP51 isomerization of IKK enables the kinase complex to phosphorylate IκBα, via FK domains; 2) FKBP51 stabilizes TRAF2 and prevents its proteasomal degradation, via TPR domains. Strategies to inhibit NF-κB represent a therapeutic option against cancer, and FKBP51 targeting is a promising strategy.
Citation Format: Simona Romano, Shao-Cong Sun, Anna D'Angelillo, Paolo D'Arrigo, Rita Bisogni, Maria Fiammetta Romano. FKBP51 sustains the constitutive NF-κB activation in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 482. doi:10.1158/1538-7445.AM2014-482