Osteopontin provides early proliferative drive and may be dependent upon aberran c-myc signalling in murine intestinal tumours Cristina Martinez a , Michael Churchman b , Tom Freeman c , Mohammad Ilyas d, ⁎ a Digestive Diseases Research Unit, Institut Fundacio Recerca, Hospital General Vall D'hebron, 08035 Barcelona, Spain b Genome Variation Laboratory Service, Cancer Res. UK,Level 6,Clinical Sciences Building, St James's University Hospital, Leeds, LS9 7TF, UK c Chancellors Building, University of Edinburgh, Edinburgh, EH16 4SB, UK d Division of Pathology, School of Molecular Medical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH,UK a b s t r a c t a r t i c l e i n f o Article history: Received 8 September 2009 and in revised form 9 December 2009 Available online 4 January 2010 Keywords: Osteopontin Wnt signalling c-myc Pea3 Intestine Tumourigenesis Osteopontin is thought to play an important role in tumour metastasis. In a previous expression profiling study of murine intestinal adenomas, we found that Opn was up-regulated. We also found β-catenin binding motifs in the Opn promoter implying that, contrary to current beliefs, induction of Opn may occur during early tumourigenesis. We studied 59 murine intestinaladenomas for Opn expression and every tumour showed up-regulation compared to normalmucosa confirming early deregulation in these tumours. To determine whether Opn makes a functional contribution to tumourigenesis, Opn was knocked down in the murine colorectal cancer cell line CMT93.Inhibition of Opn expression resulted in decreased cell numbers. To determine the mechanism of Opn induction in these tumours, the Opn promoter was cloned and each of the putative β-catenin binding motifs was mutated. No major change in Opn promoter activity was observed thereby excluding Opn as a direct β-catenin target gene. However,mutation of one of two putative c-myc binding sites in the Opn promoter led to near complete loss of promoter activity whilst mutation of one of four PEA3 binding sites led to a 50% reduction in promoter activity. We conclude that Opn deregulation is an early event in intestinal tumourigenesis which may promote tumour development by altering either proliferation or apoptosis to increase tumour cell numbers.Opn expression in the intestine is dependent on c-myc binding sites in the promoter. Since c-myc is a known β- catenin target gene, deregulation of Opn may be a secondary effect of aberrant Wnt signalling. © 2010 Elsevier Inc. All rights reserved. Introduction Osteopontin (also known as Eta-1, secreted phosphoprotein (Spp1)) is a cytokine which is secreted by a variety of cell types.It has a wide range of postulated physiological roles including cellular immunity,macrophage homing, wound healing (through neovascu- larisation and inhibition of apoptosis),angiogenesisand calcium homeostasis (reviewed by Weber, 2001). It is a small (32.5 kDa), variably phosphorylated, acidic glycoprotein which contains a central thrombin cleavage site. The C-terminus ofthe intact molecule is a ligand for the CD44 receptor whilst the N-terminus, after thrombin cleavage,functions as a ligand for integrin α v β 3 . Osteopontin secretion has been described in both epithelial and mesenchymal cancers. It is generally considered to play a role in promoting tumour metastasis through induction of cell migration, induction and activation ofmetalloproteinases, angiogenesis and bone resorption through activation of osteoclasts (Weber, 2001; Oates et al., 1997). Inhibition of osteopontin has been shown to inhibit the metastatic phenotype in cell lines ( Behrend et al., 1994) and, concordant with a postulated role as a “metastasis gene,” a number of studies have shown an association between levels of osteopontin expression and progression/prognosis (Agrawal et al., 2002; Kim et al., 2002; Omalley et al.,1996; Cappia et al., 2008). We have recently completed a study comparing the transcripto- somes of intestinal adenomas and corresponding normalmucosa obtained from Multiple Intestinal Neoplasia (Min) mice (Martinez et al.,2005).The Min mouse carries a germline Apc mutation (Su et al., 1992) and is generally regarded as a model for Familial Adenomatous Polyposis (FAP).Our data identified Opn as a gene up-regulated in tumours from both the small and the large intestine. In-silico examination of the murine Opn promoter using the SIGSCAN program (Prestridge,1991) found six putative binding motifs for LEF/TCF proteins (characterised by the consensus sequence (A/T)(A/T)CAAAG (Roose and Clevers, 1999)) within 2 kb of sequence upstream of the transcription initiation site. These proteins form a complex with β- catenin to activate transcription ofgenes which are targets ofthe canonical Wnt signalling pathway (Ilyas, 2005). Aberrant activation of Wnt signalling is generally accepted as the first step in the deve- Experimental and Molecular Pathology 88 (2010) 272–277 ⁎ Corresponding author. E-mail address: mohammad.ilyas@nottingham.ac.uk (M. Ilyas). 0014-4800/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2009.12.008 Contents lists available at ScienceDirect Experimental and Molecular Pathology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x m p