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Insulin-like growth factor binding protein 7 (IGFBP-7) is considered a tumor suppressor in various cancers, but its role in colorectal cancer (CRC) is still uncertain. The aims of this study were to analyze the IGFBP-7 expression, and explore the mechanism responsible for the inactivation of IGFBP-7 in CRC. mRNA expression was studied by RT-PCR and Northern blot analysis of cultured cells. Methylation status was analyzed by treatment with 5-aza-2′-deoxycytidine followed by sequencing of PCR products of sodium bisulfite-treated genomic DNA. IGFBP-7 protein expression was evaluated by immunohistochemistry (IHC) on tissue microarrays. mRNA expression was lost in six out of eight CRC cell lines as compared to normal colon cells. DNA methylation was found in the region of exon 1 and intron 1 of IGFBP-7. In tumor tissue, 107 out of 279 samples showed a negative expression of IGFBP-7 by IHC, which was significantly associated with poor prognosis. The analysis of 37 paired cancerous and normal mucosa samples confirmed the downregulation in the tumors, but revealed variable basal expression levels of IGFBP-7 in normal mucosal samples. DNA methylation is a mechanism responsible for IGFBP-7 gene silencing providing a target for therapeutic intervention of this tumor suppressor gene. [ABSTRACT FROM AUTHOR]

Frequent allelic loss of the chromosomal region 17p13 in breast cancer has suggested that more tumor suppressor genes, besides p53, are located in this region. By doing suppression subtractive hybridization to detect differentially expressed genes between the breast cancer cell line CAL51 and a nontumorigenic microcell hybrid CAL/17-1, we identified the gene for the gamma-aminobutyric acid type A (GABA(A)) receptor associated protein (GABARAP), located on 17p13.1. GABARAP displayed high expression levels in the microcell hybrid CAL/17-1 but only weak expression in CAL51 and other breast cancer cell lines tested. Furthermore, we observed large vesicles in CAL/17-1 by immunofluorescence staining, whereas no signal could be detected in the tumor cell line. GABARAP mRNA expression and protein expression were significantly down-regulated in invasive ductal and invasive lobular carcinomas compared with normal breast tissue measured by semiquantitative reverse transcription-PCR and immunohistochemistry, respectively. We assessed that neither mutations in the coding region of the gene nor hypermethylation of CpG islands in the promoter region are responsible for loss of gene expression in CAL51; however, 5-aza-2'-deoxycytidine treatment was effective in gene up-regulation, suggesting a methylation-dependent upstream effect. Stable transfection of GABARAP into CAL51 resulted in an increase of gene expression and remarkably influenced the ability of colony formation in soft agar and the growth rate in vitro and, moreover, suppressed the tumorigenicity of the cells in nude mice. In summary, our data suggest that GABARAP acts via a vesicle transport mechanism as a tumor suppressor in breast cancer.