Role of CYB5A in pancreatic cancer prognosis and autophagy modulation.

BACKGROUND Loss of 18q22.3 is a prognostic marker in pancreatic ductal adenocarcinoma (PDAC). This study investigated genes encoded by this cytoband. METHODS We studied mRNA/protein expression in radically resected (n = 130) and metastatic patients (n = 50). The role of CYB5A was tested in 11 PDAC cell lines and five primary cultures through retrovirus-mediated upregulation and small interfering RNA using wound-healing, invasion, annexin-V, electron microscopy, and autophagic assays, as well as autophagy genes and kinases arrays. CYB5A+ orthotopic models (n = 6 mice/group) were monitored by Firefly and Gaussia-luciferase bioluminescence, magnetic resonance imaging, and high-frequency ultrasound. Data were analyzed by t test, Fisher exact-test, log-rank test and Cox proportional hazards models. All statistical tests were two-sided. RESULTS Both resected and metastatic patients with low mRNA or protein expression of CYB5A had statistically significantly shorter survival (eg, median = 16.7 months, 95% confidence interval [CI] = 13.5 to 19.9; vs median = 24.8 months, 95% CI = 12.8 to 36.9; P = .02, two-sided log-rank test; n = 82 radically resected PDACs), and multivariable analyses confirmed prognostic relevance. Moreover, we characterized a novel function to CYB5A, autophagy induction, concomitant with reduced proliferation and migration/invasion of PDAC cells. Network analysis of proautophagic pathways suggested CYB5A interaction with TRAF6, which was confirmed by TRAF6 downregulation after CYB5A reconstitution (-69% in SU.86.86-CYB5A+; P = .005, two-sided t test). CYB5A silencing had opposite effects, restoring TRAF6 expression and wound healing. In vivo studies showed that CYB5A induced autophagy while inhibiting tumor growth/metastasis and increasing survival (median = 57 days, 95% CI = 52 to 61; vs median = 44 days, 95% CI = 21 to 57; P = .03, two-sided log-rank test). CONCLUSIONS These results define CYB5A as a novel prognostic factor for PDAC that exerts its tumor-suppressor function through autophagy induction and TRAF6 modulation.

[1]  A. Kaftory,et al.  Congenital methemoglobinemia with a deficiency of cytochrome b5. , 1986, The New England journal of medicine.

[2]  N. Dubrawsky.  Cancer statistics , 1989, CA: a cancer journal for clinicians.

[3]  M. Talamini,et al.  Six hundred fifty consecutive pancreaticoduodenectomies in the 1990s: pathology, complications, and outcomes. , 1997, Annals of surgery.

[4]  H. Hanafusa,et al.  TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src. , 1999, Molecular cell.

[5]  Yudong D. He,et al.  A Gene-Expression Signature as a Predictor of Survival in Breast Cancer , 2002 .

[6]  Hao Wu,et al.  Distinct molecular mechanism for initiating TRAF6 signalling , 2002, Nature.

[7]  Van,et al.  A gene-expression signature as a predictor of survival in breast cancer. , 2002, The New England journal of medicine.

[8]  Y. Yarden,et al.  Src promotes destruction of c-Cbl: Implications for oncogenic synergy between Src and growth factor receptors , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Christine A Iacobuzio-Donahue,et al.  Exploration of global gene expression patterns in pancreatic adenocarcinoma using cDNA microarrays. , 2003, The American journal of pathology.

[10]  Christian von Mering,et al.  STRING: a database of predicted functional associations between proteins , 2003, Nucleic Acids Res..

[11]  P. Allavena,et al.  Increased Survival, Proliferation, and Migration in Metastatic Human Pancreatic Tumor Cells Expressing Functional CXCR4 , 2004, Cancer Research.

[12]  Murray F. Brennan,et al.  Prognostic Nomogram for Patients Undergoing Resection for Adenocarcinoma of the Pancreas , 2004, Annals of surgery.

[13]  Martin Kuiper,et al.  BiNGO: a Cytoscape plugin to assess overrepresentation of Gene Ontology categories in Biological Networks , 2005, Bioinform..

[14]  R. Siebert,et al.  CD95 and TRAF2 promote invasiveness of pancreatic cancer cells , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[15]  L. Trepanier,et al.  Reductive detoxification of arylhydroxylamine carcinogens by human NADH cytochrome b5 reductase and cytochrome b5. , 2006, Chemical research in toxicology.

[16]  I. Ng,et al.  Identification of Carboxypeptidase of Glutamate Like-B as a Candidate Suppressor in Cell Growth and Metastasis in Human Hepatocellular Carcinoma , 2006, Clinical Cancer Research.

[17]  M. Moran,et al.  Large-scale mapping of human protein–protein interactions by mass spectrometry , 2007, Molecular systems biology.

[18]  Ralph Weissleder,et al.  A secreted luciferase for ex vivo monitoring of in vivo processes , 2008, Nature Methods.

[19]  C. Heldin,et al.  The type I TGF-β receptor engages TRAF6 to activate TAK1 in a receptor kinase-independent manner , 2008, Nature Cell Biology.

[20]  Robert J Gillies,et al.  Gene expression profiling-based identification of cell-surface targets for developing multimeric ligands in pancreatic cancer , 2008, Molecular Cancer Therapeutics.

[21]  Liviu Badea,et al.  Combined gene expression analysis of whole-tissue and microdissected pancreatic ductal adenocarcinoma identifies genes specifically overexpressed in tumor epithelia. , 2008, Hepato-gastroenterology.

[22]  G. Parmigiani,et al.  Core Signaling Pathways in Human Pancreatic Cancers Revealed by Global Genomic Analyses , 2008, Science.

[23]  David Allard,et al.  Inhibition of Hedgehog Signaling Enhances Delivery of Chemotherapy in a Mouse Model of Pancreatic Cancer , 2009, Science.

[24]  Sridhar Ramaswamy,et al.  Synthetic Lethal Interaction between Oncogenic KRAS Dependency and STK33 Suppression in Human Cancer Cells , 2009, Cell.

[25]  Krishna R. Kalari,et al.  FKBP51 affects cancer cell response to chemotherapy by negatively regulating Akt. , 2009, Cancer cell.

[26]  J. Iovanna,et al.  Gemcitabine Induces the VMP1-Mediated Autophagy Pathway to Promote Apoptotic Death in Human Pancreatic Cancer Cells , 2010, Pancreatology.

[27]  Ugo Boggi,et al.  MicroRNA-21 in pancreatic cancer: correlation with clinical outcome and pharmacologic aspects underlying its role in the modulation of gemcitabine activity. , 2010, Cancer research.

[28]  M. Nowak,et al.  Distant Metastasis Occurs Late during the Genetic Evolution of Pancreatic Cancer , 2010, Nature.

[29]  P. Meltzer,et al.  Loss of 18q22.3 Involving the Carboxypeptidase of Glutamate-like Gene Is Associated with Poor Prognosis in Resected Pancreatic Cancer , 2011, Clinical Cancer Research.

[30]  D. Melisi,et al.  Modulation of pancreatic cancer chemoresistance by inhibition of TAK1. , 2011, Journal of the National Cancer Institute.

[31]  E. Giovannetti,et al.  Correlation of basal EGFR expression with pancreatic cancer grading but not with clinical outcome after gemcitabine-based treatment. , 2011, Annals of oncology : official journal of the European Society for Medical Oncology.

[32]  S. Lam,et al.  TRAF6 is an amplified oncogene bridging the RAS and NF-κB pathways in human lung cancer. , 2011, The Journal of clinical investigation.

[33]  H. Zentgraf,et al.  Autophagy and cell death signaling following dietary sulforaphane act independently of each other and require oxidative stress in pancreatic cancer. , 2011, International journal of oncology.

[34]  E. Giovannetti,et al.  Defining a prognostic molecular profile for ductal adenocarcinoma of the pancreas highlights known key signaling pathways , 2012, Expert review of anticancer therapy.

[35]  Molecular Mechanisms and Modulation of Key Pathways Underlying the Synergistic Interaction of Sorafenib with Erlotinib in Non-Small-Cell-Lung Cancer (NSCLC) Cells , 2012 .

[36]  K. Brown,et al.  Array-Comparative Genomic Hybridization Reveals Loss of SOCS6 Is Associated with Poor Prognosis in Primary Lung Squamous Cell Carcinoma , 2012, PloS one.

[37]  Manuel Hidalgo,et al.  Translational Therapeutic Opportunities in Ductal Adenocarcinoma of the Pancreas , 2012, Clinical Cancer Research.

[38]  M. Vaccaro,et al.  Chemotherapy and autophagy-mediated cell death in pancreatic cancer cells. , 2012, Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.].

[39]  Paul,et al.  Loss of 18 q 22 . 3 involving the carboxypeptidase of glutamate-like gene is associated with poor prognosis in resected pancreatic cancer , 2012 .

[40]  Haiyuan Yu,et al.  Detecting overlapping protein complexes in protein-protein interaction networks , 2012, Nature Methods.

[41]  Junjie Zhang,et al.  Autophagy is induced through the ROS-TP53-DRAM1 pathway in response to mitochondrial protein synthesis inhibition , 2012, Autophagy.

[42]  M. Penichet,et al.  TRAF6 activation in multiple myeloma: a potential therapeutic target. , 2012, Clinical lymphoma, myeloma & leukemia.

[43]  L. Qin,et al.  A Novel Survival-Based Tissue Microarray of Pancreatic Cancer Validates MUC1 and Mesothelin as Biomarkers , 2012, PloS one.

[44]  M. Lotze,et al.  AGER/RAGE-mediated autophagy promotes pancreatic tumorigenesis and bioenergetics through the IL6-pSTAT3 pathway , 2012, Autophagy.

[45]  Lincoln D. Stein,et al.  Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes , 2012, Nature.

[46]  Ruiying Zhao,et al.  KrasG12D-induced IKK2/β/NF-κB activation by IL-1α and p62 feedforward loops is required for development of pancreatic ductal adenocarcinoma. , 2012, Cancer cell.

[47]  A. Jemal,et al.  Cancer statistics, 2012 , 2012, CA: a cancer journal for clinicians.

[48]  C. Sigman,et al.  Cancer biomarkers: selecting the right drug for the right patient , 2012, Nature Reviews Drug Discovery.

[49]  Sharmeela Kaushal,et al.  KRas induces a Src/PEAK1/ErbB2 kinase amplification loop that drives metastatic growth and therapy resistance in pancreatic cancer. , 2012, Cancer research.

[50]  M. Bugliani,et al.  Palmitate Activates Autophagy in INS-1E β-Cells and in Isolated Rat and Human Pancreatic Islets , 2012, PloS one.

[51]  E. Giovannetti,et al.  Crizotinib inhibits metabolic inactivation of gemcitabine in c-Met-driven pancreatic carcinoma. , 2013, Cancer research.

[52]  Y. Tajima,et al.  The HSP70 and Autophagy Inhibitor Pifithrin-μ Enhances the Antitumor Effects of TRAIL on Human Pancreatic Cancer , 2013, Molecular Cancer Therapeutics.

[53]  E. Giovannetti,et al.  Molecular mechanisms and modulation of key pathways underlying the synergistic interaction of sorafenib with erlotinib in non-small-cell-lung cancer (NSCLC) cells. , 2012, Current pharmaceutical design.