Abstract 2787: Modulation of autophagy and oncogenic phenotypes through CYB5A-TRAF6 signaling influence prognosis of resected and metastatic pancreatic cancer
Authors: Giovannetti Elisa , Wang Qiuyan , Funel Niccola , Avan Amir , Lagerweij Tonny , Caretti Viola , Boggi Ugo , Wang Yisong , Caponi Sara , Velde Arjan van der , Vasile Enrico , Verheul Henk M. , Wurdinger Thomas , Giaccone Giuseppe 
Published: May 2015

Abstract
High-resolution array comparative genomic hybridization in a cohort of 44 radically resected pancreatic cancer patients revealed a correlation between shorter survival and loss of the 18q22.3cytoband. The present study investigated the genes encoded by this cytoband and provided mechanistic insights on their role in the suppression of oncogenic properties. We studied mRNA and protein expression of FBXO15, C18orf55, CYB5A, CPGL and CPGL-B in frozen laser-microdissected tissues from radically resected (N=48) and biopsies from metastatic patients (N=50). Low CYB5A expression correlated with 18q22.3 deletion and shorter survival, both in resected and metastatic patients (i.e. 6.5 vs. 12.7 months, P=0.004). Association with outcome was validated in a tissue-microarray of a second cohort of radically resected patients (N=100): patients with low expression levels of CYB5A had significantly shorter OS, and multivariate analysis confirmed CYB5A prognostic relevance (increased risk of death=2.0, P=0.02). The role of CYB5A was evaluated in 11 pancreatic cancer cell lines, 5 primary cultures, and a normal pancreatic ductal cell line through retrovirus-mediated up-regulation and siRNA. With these analyses we characterized a novel function of CYB5A, autophagy induction, concomitant with reduced proliferation and migration/invasion of pancreatic cancer cells. Marked accumulation of autophagic vacuoles was detected by electron microscopy, while immunofluorescence revealed CYB5A modulation of LC-3. Activation of pro-autophagic pathways was corroborated by gene and kinase arrays showing a significant up-regulation of several ATG-genes, accompanied by down-regulation of BCL-2 and MAPK14. Additionally, inhibition of multiple components of EGFR, Akt and Src signalling favored cancer cell death while preventing potentially deleterious cross-talk between key oncogenic players. Network analysis suggested CYB5A interaction with TRAF6, which was confirmed by TRAF6 down-regulation after CYB5A reconstitution (-69% in SU.86.86-CYB5A+, P=0.005). CYB5A silencing had opposite effects, restoring TRAF6 expression and wound-healing. In vivo studies were performed in genetically and histopathologically characterized patient-derived CYB5A+ orthotopic models (N=6 mice/group), monitored by Firefly and Gaussia-luciferase bioluminescence, MRI and high-frequency-ultrasound. CYB5A induced autophagy, as demonstrated by immunohistochemical analyses of p62 down-regulation, and ATG7/ATG16L2 overexpression, while inhibiting tumor growth/metastasis and increasing survival (57 vs. 44 days, P=0.03). These results define CYB5A as a novel prognostic factor, exerting its tumor-suppressor function via autophagy-induction and TRAF6 modulation, which holds a potential as a novel therapeutic approach in the clinical management of pancreatic cancer.
Citation Format: Elisa Giovannetti, Qiuyan Wang, Niccola Funel, Amir Avan, Tonny Lagerweij, Viola Caretti, Ugo Boggi, Yisong Wang, Sara Caponi, Arjan van der Velde, Enrico Vasile, Henk M. Verheul, Thomas Wurdinger, Giuseppe Giaccone. Modulation of autophagy and oncogenic phenotypes through CYB5A-TRAF6 signaling influence prognosis of resected and metastatic pancreatic cancer. [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 2787. doi:10.1158/1538-7445.AM2014-2787