Open Access Research article

Thrombospondin-4 is a putative tumour-suppressor gene in colorectal cancer that exhibits age-related methylation

Sonia A Greco1, June Chia1, Kelly J Inglis1, Sarah-Jane Cozzi2, Ingunn Ramsnes1, Ronald L Buttenshaw1, Kevin J Spring1, Glen M Boyle3, Daniel L Worthley1, Barbara A Leggett14 and Vicki LJ Whitehall15*

Author Affiliations

1 Conjoint Gastroenterology Laboratory, Royal Brisbane and Women's Hospital Research Foundation, Clinical Research Centre and Queensland Institute of Medical Research, Brisbane, Australia

2 Immunovirology Laboratory, Queensland Institute of Medical Research, Brisbane, Australia

3 Drug Discovery Group, Queensland Institute of Medical Research, Brisbane, Australia

4 Royal Brisbane and Women's Hospital, Brisbane, Australia

5 Pathology Queensland, Clinical and Statewide Services, Queensland Health, Brisbane, Australia

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BMC Cancer 2010, 10:494  doi:10.1186/1471-2407-10-494

Published: 16 September 2010

Abstract

Background

Thrombospondin-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.

Methods

Fifty-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.

Results

THBS4 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.

Conclusions

THBS4 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.