Synergisticinductionofthe MUC4 mucingenebyinterferon-c andretinoic acid in human pancreatic tumour cells involves a reprogramming of signalling pathways Mahefatiana Andrianifahanana 1 , Anshu Agrawal 1 , Ajay P Singh 1 , Nicolas Moniaux 1 , Isabelle van Seuningen 2 , Jean-Pierre Aubert 2 , Jane Meza 3 and Surinder K Batra* ,1 1 Department of Biochemistry and Molecular Biology, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA; 2 Department of Preventive and Societal Medicine, University of Nebraska Medical Center, Omaha, NE 68198, USA; 3 INSERM, Place de Verdun, 59045 Lille Cedex, France The transmembrane mucin, MUC4, is aberrantly ex- pressed with a high incidence in human pancreatic adenocarcinomas and plays an important role in the pathogenesisofthedisease.Ourrecentstudieshaveshown that interferon-c (IFNc) and retinoic acid (RA) are important regulators of MUC4 in pancreatic tumour cells. Induction of MUC4 by IFNc occurs via a novel pathway involving upregulation of the signal transducer and activator of transcription 1 (STAT-1), whereas its stimulationbyRArequiresmediationbythetransforming growth factor b-2 (TGFb-2). In this study, we have investigated the molecular mechanisms underlying the interaction of IFNc andRAin MUC4 regulation in pan- creatic tumour cells. We demonstrate that these reagents exert a synergistic induction of MUC4. Interestingly, while the upregulation of STAT-1 by IFNc is partially inhibited by RA, IFNc is shown to repress RA-driven TGFb-2 induction, pointing to the involvement of alter- nativemechanism(s)inIFNc–RAsynergism.Moreover,a dose-dependent and cooperative induction of MUC4 promoter activity suggests a regulation at the transcrip- tional level, most likely by STAT-1 and RAR/RXR (RA receptor/retinoic X receptor) or other IFNc/RA- induced secondary intermediate effectors. Our findings provide potential mechanisms that may account for the aberrant expression of MUC4 in pancreatic tumour cells and expose a novel molecular mechanism of gene induction, whereby a reprogramming of signalling path- way through alternative route(s) operates during a synergistic interaction of biological modifiers. Oncogene (2005) 24, 6143–6154. doi:10.1038/sj.onc.1208756; published online 20 June 2005 Keywords: MUC4 mucin; IFNg; retinoic acid; syner- gism; gene regulation; pancreatic cancer Introduction Pancreatic cancer is a highly lethal malignancy char- acterized by an extremely poor prognosis (Warshaw and Fernandez-del Castillo, 1992; Reber, 1998; Jemal et al., 2003). Efforts made to improve the treatment of this neoplastic disorder have been hampered, in large part, by the elusive nature of its initiation and progression. Nonetheless, a wide variety of biochemical and/or genetic aberrations commonly associated with pancrea- tic cancer have been identified, owing to the advent of newer and more efficient molecular biological and genetic techniques (Reber, 1998; Sirivatanauksorn et al., 1998). Among the series of genes whose expression profiles are frequently altered in pancreatic cancer are mucins (MUC), the members of an expanding class of high-molecular-weight glycoproteins (Gendler and Spi- cer, 1995; Moniaux et al., 2001; Hollingsworth and Swanson, 2004). MUC4, a large transmembrane mucin, is aberrantly expressed in the majority (70–80%) of pancreatic tumours and tumour cell lines while remain- ing undetectable in the normal pancreas (Andrianifaha- nana et al., 2001). In recent years, there has been increasing evidence supporting the role of this tumour- associated mucin in the pathogenesis of pancreatic cancer (Andrianifahanana et al., 2001; Swartz et al., 2002; Singh et al., 2004). Comparative expression analyses have revealed a positive correlation between MUC4 levels, the differentiation status of pancreatic tumour cell lines (Andrianifahanana et al., 2001), and tumour grading in accordance with a model of pancreatic tumour progression (Swartz et al., 2002). Moreover, functional studies using antisense- and/or short-interfering RNA (siRNA) oligonucleotide-based knockdown or ectopic expression of MUC4 have provided substantial evidence of its role in the promo- tion of pancreatic tumour cell growth and metastasis in vivo (Singh et al., 2004). Taken together, these observa- tions suggest an intimate link between aberrant MUC4 expression and the pathogenesis of pancreatic cancer. Therefore, an improved understanding of mechanism(s) underlying the regulation of this mucin gene may help to identify key biochemical events and biologically relevant Received 20 September 2004; revised 1 April 2005; accepted 1 April 2005; published online 20 June 2005 *Correspondence: SK Batra; E-mail: sbatra@unmc.edu Oncogene (2005) 24, 6143–6154 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc