Abstract 1143: Unraveling the role of CYB5A in pancreatic ductal adenocarcinoma (PDAC): correlation with clinical outcome and functional characterization in the modulation of autophagy and oncogenic phenotypes.
Authors: Giovannetti Elisa , Wang Qiuyan , Avan Amir , Funel Niccola , Glavani Elena , Lagerweij Tonny , Chiasserini Davide , Lee Jih-Hsiang , Caretti Viola , Masini Matilde , Boggi Ugo , Wang Yisong , Vasile Enrico , Peters Godefridus J. , Wurdinger Thomas , Giaccone Giuseppe 
Published: August 2014

Abstract
Loss of 18q22.3 was reported to be a prognostic factor in PDAC. This study aimed at evaluating whether the genes of this cytoband were associated with PDAC outcome and provides mechanistic insights on their role in the suppression of oncogenic properties. The expression of FBXO15, C18orf55, CYB5A, CPGL and CPGL-B was evaluated in cells (11 PDAC cell lines, 5 primary cultures, and a normal pancreatic ductal cell line) and in 48 stage IIB PDAC specimens, isolated by laser-microdissection after surgical resection. Low CYB5A expression correlated with 18q22.3 deletion and shorter survival (16.3 vs. 29.5 months, P=0.01). Association with outcome was validated in a tissue-microarray of a second cohort radically resected patients (N=100): patients with CYB5A protein expression levels below median value had significantly shorter OS, and multivariate analysis confirmed CYB5A prognostic relevance (increased risk of death, 2.0, P=0.02). CYB5A expression in primary cultures correlated with expression in their respective tissues, and CYB5A retrovirus-mediated up-regulation of both PDAC-2 primary cells and Su86.86 cells suppressed proliferation and migration/invasion, while enhancing apoptosis and autophagy induction. Marked accumulation of autophagic vacuoles was detected by electron microscopy, while immunofluorescence revealed CYB5A colocalization with LC-3. Activation of pro-autophagic pathways was corroborated by PCR arrays showing a significant up-regulation of several ATG-genes, accompanied by down-regulation of BCL-2 and MAPK14. The phosphorylation status of MAPK14 was also inhibited, together with phospho-EGFR and the pro-invasive kinases phospho-Src/STAT6, as revealed by 144-kinase peptide substrate arrays. Network analysis suggested that this down-regulation was caused by CYB5A interaction with the NF-κB activator TRAF6, whose expression was significantly reduced after reconstitution of CYB5A in a genetic and histopathologic characterized patient-derived orthotopic mouse model. Furthermore, CYB5A upregulation increased survival, while decreasing primary tumor dimension and metastatic spread, as longitudinally monitored with Firefly- and Gaussia-luciferase. We thus identified CYB5A as a novel prognostic factor that modulates autophagy and oncogenic phenotypes, holding a potential as a novel therapeutic approach in the clinical management of PDAC patients.
Citation Format: Elisa Giovannetti, Qiuyan Wang, Amir Avan, Niccola Funel, Elena Glavani, Tonny Lagerweij, Davide Chiasserini, Jih-Hsiang Lee, Viola Caretti, Matilde Masini, Ugo Boggi, Yisong Wang, Enrico Vasile, Godefridus J. Peters, Thomas Wurdinger, Giuseppe Giaccone. Unraveling the role of CYB5A in pancreatic ductal adenocarcinoma (PDAC): correlation with clinical outcome and functional characterization in the modulation of autophagy and oncogenic phenotypes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1143. doi:10.1158/1538-7445.AM2013-1143