RESEARCH ARTICLES Bone Morphogenic Protein 3 Inactivation Is an Early and Frequent Event in Colorectal Cancer Development Kim Loh, 1 June A. Chia, 1 Sonia Greco, 1 Sarah-Jane Cozzi, 1 Ron L. Buttenshaw, 1 Catherine E. Bond, 1 Lisa A. Simms, 1 Tanya Pike, 1 Joanne P. Young, 2 Jeremy R. Jass, 3 Kevin J. Spring, 1 Barbara A. Leggett, 1 and Vicki L. J. Whitehall 1 * 1 Conjoint Gastroenterology Laboratory,Royal Brisbane and Women’s Hospital Research Foundation Clinical Research Centre and Queensland Institute of Medical Research,Brisbane 4029, Australia 2 Familial Cancer Laboratory,Queensland Institute of Medical Research,Brisbane 4029, Australia 3 Department of Cellular Pathology, St Mark’s Hospital, Harrow HA13UJ,UK Bone morphogenic proteins (BMPs) are members of the TGFB growth factor superfamily with well-described functions in bone formation. Although disrupted BMP signalling in tumor development has more recently been investigated, a role for BMP3 in colorectal cancer (CRC) has remained largely unexplored. The aim of this study was to investigate BMP3 disruption in CRCs in relation to both the traditional and serrated pathways of tumor progression. BMP3 was down-regulated as assessed by real-time PCR in 50 of 56 primary tumors (89%). Bisulfite sequencing of the putative promoter revealed extensive hyper- methylation in the cell line HT29, in which expression could be restored by treatment with a methyltransferase inhibitor. Aber- rant hypermethylation was observed in 33/60 (55%) tumors and was highly correlated with microsatellite instability (P < 0.01), the CpG Island Methylator Phenotype (P < 0.01), BRAF oncogene mutation (P < 0.01), and proximal location (P < 0.001). Methylation was also frequently observed in serrated and traditional adenomatous polyps (22/29, 76%). Re-introduction of BMP3 into cell lines revealed marked growth suppression supporting the functional relevance of this alteration in colorectal tu- mor development. This study provides molecular and functional data supporting the importance of BMP3 silencing as an early and frequent event in colorectal tumors progressing via the serrated and traditional pathways. V V C 2008 Wiley-Liss, Inc. INTRODUCTION Bone morphogenic protein 3 (BMP3) is a mem- ber of the transforming growth factor beta (TGFB) superfamily of cytokines, which includes BMPs, activins, and TGFB isoforms. These growth factors act by binding to type II surface receptors (including BMPRII, ACTRII, and TGFBRII), which then recruit and subsequently phosphoryl- ate type I receptors. This series of events activates a signalling cascade via phosphorylation of recep- tor-mediated SMADs, which ultimately results in the transcriptional regulation of SMAD4 target genes. Receptor specificity of BMP3 has not been defined, but it has been suggested that BMPs may also signal via SMAD-independent pathways to achieve growth suppression (Beck et al., 2006). In vitro studies have suggested a role for BMP3 as a negative growth regulator in bone marrow progeni- tor cells through inhibition of DNA synthesis and proliferation (Amedee et al., 1994). There have been no recent studies to define the function of the BMP3 ligand in tumorigenesis and a potential role in the SMAD signalling pathway has not been demonstrated. BMP family members were initially studied due to their function in development, particularly bone formation, however more recent evidence suggests an important role in tumorigenesis. De-regulated expression in colorectal cancer (CRC) has been reported for family members including BMP4 which is over-expressed (Nosho et al., 2005) and BMP2, BMP3, and BMP5 which are down-regu- lated (Hardwick et al., 2004; Koehler et al., 2004; Koinuma et al., 2005). Aberrant promoter hyper- methylation has been reported as a mechanism of down-regulated expression for some family mem- bers including BMP3B and BMP6 in lung cancer (Kraunz et al., 2005). Perhaps the most compelling *Correspondence to: Vicki L. J. Whitehall, The Queensland Insti- tute of Medical Research, 300 Herston Road, Herston QLD 4029, Australia. E-mail: vicki.whitehall@qimr.edu.au Supported by: The National Health and Medical Research Coun- cil of Australia, Grant number: 290203; Queensland Cancer Fund (145), Queensland Health Pathology and Scientific Services, The Royal Brisbane and Women’s Hospital Research Foundation and The Walter Paulson Tumor Bank. Received 2 October 2007; Accepted 30 January 2008 DOI 10.1002/gcc.20552 Published online 29 February 2008 in Wiley InterScience (www.interscience.wiley.com). V V C 2008 Wiley-Liss, Inc. GENES, CHROMOSOMES & CANCER 47:449–460 (2008)