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 hypermethylation in the cell line HT29, in which expression could be restored by treatment with a methyltransferase inhibitor. Aberrant 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 tumor 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.

BackgroundThrombospondin-4 (THBS4) is a member of the extracellular calcium-binding protein family and is involved in cell adhesion and migration. The aim of this study was to evaluate the potential role of deregulation of THBS4 expression in colorectal carcinogenesis. Of particular interest was the possible silencing of expression by methylation of the CpG island in the gene promoter.MethodsFifty-five sporadic colorectal tumours stratified for the CpG Island Methylator Phenotype (CIMP) were studied. Immunohistochemical staining of THBS4 protein was assessed in normal and tumour specimens. Relative levels of THBS4 transcript expression in matched tumours and normal mucosa were also determined by quantitative RT-PCR. Colony forming ability was examined in 8 cell lines made to overexpress THBS4. Aberrant promoter hypermethylation was investigated as a possible mechanism of gene disruption using MethyLight. Methylation was also assessed in the normal colonic tissue of 99 patients, with samples biopsied from four regions along the length of the colon.ResultsTHBS4 expression was significantly lower in tumour tissue than in matched normal tissue. Immunohistochemical examination demonstrated that THBS4 protein was generally absent from normal epithelial cells and tumours, but was occasionally expressed at low levels in the cytoplasm towards the luminal surface in vesicular structures. Forced THBS4 over-expression caused a 50-60% repression of tumour colony growth in all eight cell lines examined compared to control cell lines. Tumours exhibited significantly higher levels of methylation than matched normal mucosa, and THBS4 methylation correlated with the CpG island methylator phenotype. There was a trend towards decreased gene expression in tumours exhibiting high THBS4 methylation, but the correlation was not significant. THBS4 methylation was detectable in normal mucosal biopsies where it correlated with increasing patient age and negatively with the occurrence of adenomas elsewhere in the colon.ConclusionsTHBS4 shows increased methylation in colorectal cancer, but this is not strongly associated with altered gene expression, either because methylation has not always reached a critical level or because other factors influence THBS4 expression. THBS4 may act as a tumour suppressor gene, demonstrated by its suppression of tumour colony formation in vitro. THBS4 methylation is detectable in normal colonic mucosa and its level may be a biomarker for the occurrence of adenomas and carcinoma.