RESUMO
In most colorectal tumours, APC mutation stabilises beta-catenin and mimics elements of Wnt growth factor signalling, but the high frequency of epigenetic loss of Wnt antagonists indicates an additional role for ligand-mediated Wnt signalling. Here, we have investigated the expression of key components of beta-catenin-independent Wnt response pathways to determine whether their profiles change during the transition from normal mucosa to colorectal adenomas. Transcription of the Wnt/planar cell polarity pathway determinant NKD1 (naked cuticle homologue 1) was induced in adenomas by a median 135-fold and in cancers by 7.4-fold. While some Frizzleds (FZDs) were downregulated in adenomas, the Wnt/Ca(2+) receptors FZD3 and FZD6 were induced by a median factor of 6.5 and 4.6, respectively. Naked cuticle homologue 1, FZD3 and FZD6 expression were coordinated in pre-malignant disease, but this relationship was lost in invasive cancers, where FZD induction was seen less frequently. Naked cuticle homologue 1 expression was associated with nuclear localisation of phospho-c-Jun in adenomas. In cultured cells, NKD1 transcription was induced by lithium chloride but FZD3 expression required Wnt growth factor treatment. These data show that Wnt responses are consistently directed towards both beta-catenin-independent routes in early colorectal tumorigenesis and elements of this are retained in more advanced cancers. These beta-catenin-independent Wnt signalling pathways may provide novel targets for chemoprevention of early colorectal tumours.
Assuntos
Neoplasias Colorretais/etiologia , Transdução de Sinais/fisiologia , Proteínas Wnt/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Adenoma/etiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Receptores Frizzled/biossíntese , Receptores Frizzled/genética , Humanos , Pessoa de Meia-Idade , Proteína Quinase C/fisiologia , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta Catenina/fisiologiaRESUMO
Our previous studies have implicated the Wnt antagonist, sFRP1, as a tumour suppressor gene in advanced colorectal cancer. In this study, we set out to investigate the relationship between sFRP1 expression and large bowel adenomas, a precursor of colorectal cancer. The induction of beta-catenin/TCF mediated transcription is both a frequent early event in colorectal neoplasia, and a key downstream effect of wnt growth factor signalling. Lithium treatment of a small bowel mucosal cell line (FHs 74 int) induced sFRP1 within 8 h, indicating that this gene is positively regulated by beta-catenin, contrasting with the suppression of sFRP1 expression, we saw previously in advanced colorectal cancers. We therefore investigated a series of 12 adenomas and matched large bowel mucosa samples. Real-time RT-PCR analysis showed a reduction in sFRP1 expression in all 12 dysplastic lesions (median 485-fold, IQR 120- to 1500-fold), indicating factors other than beta-catenin influence sFRP1 levels. In a second series of 11 adenomas, we identified methylation of the sFRP1 promotor region in all 11 samples, and this was increased compared with the surrounding normal mucosa in seven cases. Immunohistochemical analysis using a polyclonal antibody supported these findings, with sFRP1 expression reduced in many of the adenoma samples examined. sFRP1 staining in normal mucosa adjacent to the dysplastic tissue was also reduced compared with the normal controls, suggesting that sFRP1 expression may be suppressed in a field of mucosa rather than in individual cells. This study identifies sFRP1 inactivation at the premalignant stage of colorectal cancer development, indicating that these pathways may be useful targets for chemoprevention strategies in this common solid tumour.
