Chapter 12 CYB 5 A role in pancreatic cancer prognosis and autophagy modulation

Background. Loss of 18q22.3 is a prognostic marker in pancreatic ductal adenocarcinoma (PDAC). The present study investigated genes encoded by this cytoband. Methods. We studied mRNA/protein expression in radically-resected (N=148) and metastatic patients (N=50). The role of CYB5A was tested in 11 PDAC cell lines and 5 primary cultures, through retrovirus-mediated up-regulation and siRNA, using woundhealing, 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, MRI and high-frequencyultrasound. Data were analyzed by t-test, Fisher's exact-test, Log-rank-test and Cox’s 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 (e.g., 16.7, 95% confidence interval [CI]=13.5-19.9 vs. 24.8 months, 95%CI=12.8-36.9, two-sided log-rank P=0.02, in 82 radically-resected PDACs), and multivariate analyses confirmed its 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 down-regulation after CYB5A reconstitution (-69% in SU.86.86-CYB5A+, twosided t-test P=0.005). 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 (57, 95%CI=52-61, vs. 44 days, 95%CI=21-57, two-sided log-rank P=0.03). Conclusions. These results define CYB5A as a novel prognostic factor for PDAC, exerting its tumor-suppressor function via autophagy-induction and TRAF6 modulation. CYB5A in pancreatic cancer 233 | P a g e Introduction Pancreatic ductal adenocarcinoma (PDAC) carries one of the worst prognoses of any major malignancy and exhibits profound chemoresistance [1-3]. The inefficacy of currently available therapeutic strategies has been attributed to the dense desmoplastic reaction, which reduces drug penetration, and to the high rate of genetic alterations affecting multiple pathways [4-5]. Genetic analyses uncovered mechanisms controlling pancreatic carcinogenesis [6], and studies to identify aberrancies associated with outcome are warranted. We previously investigated genomic imbalances using array-comparative genomic hybridization (aCGH) in a cohort of 44 radically-resected patients, the largest PDAC series ever investigated by aCGH [7]. In this series the median overall survivals (OS) for patients with and without loss of the cytoband 18q22.3 were 7.6 and 21.4 months, respectively (P=0.019, two-sided log-rank test). The cytoband 18q22.3 contains five known genes (FBXO15, c18orf55, CYB5A, c18orf51, and CPGL). In agreement with previous findings [8], CPGL reduced proliferation and inhibited migration in SU.86.86 cells carrying FLAG-tagged-CPGL. However, knockdown of CPGL in the PANC-1 cells did not affect proliferation, cell cycle distribution and wound-healing [7]. The aim of the present study was to evaluate whether the mRNAs and/or proteins coded by the genes in the 18q22.3 cytoband were associated with outcome in two cohorts of radically-resected patients and one cohort of metastatic PDAC patients. Further, we aimed at characterizing key factors affecting proliferative and invasive capacity, as well as autophagy induction, which may provide mechanistic insights on PDAC aggressive behaviour and contribute to the rational development of new prognostic and therapeutic approaches.

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

[2]  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.

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

[4]  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.

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

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

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

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

[9]  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.

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

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

[12]  Robert Clarke,et al.  Guidelines for the use and interpretation of assays for monitoring autophagy , 2012 .

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

[14]  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.

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

[16]  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.

[17]  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.

[18]  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.

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

[20]  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.

[21]  V. Caretti,et al.  Monitoring of Tumor Growth and Post‐Irradiation Recurrence in a Diffuse Intrinsic Pontine Glioma Mouse Model , 2011, Brain pathology.

[22]  Qiuyan Wang,et al.  A ubiquitin ligase-associated chaperone holdase maintains polypeptides in soluble states for proteasome degradation. , 2011, Molecular cell.

[23]  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.

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

[25]  J. Aten,et al.  In silico analysis of kinase expression identifies WEE1 as a gatekeeper against mitotic catastrophe in glioblastoma. , 2010, Cancer cell.

[26]  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.

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

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

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

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

[31]  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.

[32]  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.

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

[34]  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.

[35]  Franco Mosca,et al.  Laser microdissection and primary cell cultures improve pharmacogenetic analysis in pancreatic adenocarcinoma , 2008, Laboratory Investigation.

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

[37]  P. Vineis,et al.  Exposure to the tobacco smoke constituent 4-aminobiphenyl induces chromosomal instability in human cancer cells. , 2007, Cancer research.

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

[39]  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.

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

[41]  H. Friess,et al.  Detection of 2-amino-1-methyl-6-phenylimidazo [4,5-b]-pyridine (PhIP)–DNA adducts in human pancreatic tissues , 2006, Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals.

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

[43]  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.

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

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

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

[47]  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.

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

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

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

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

[52]  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. , 2013, Current pharmaceutical design.

[53]  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.].

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

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

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