Low expression of the folate-associated genes RFC-1 and FPGS correlates with lack of a DCC splice variant in mucosa adjacent to carcinomas in colorectal cancer patients

Y Wettergren PhD^a, E Odin BSc^a, S Nilsson PhD^b, R Willén MD PhD^c, G Carlsson MD PhD^a, L Larsson MD^a, B Gustavsson MD PhD^a

^aDepartment of General Surgery, ^bDepartment of Clinical Genetics; Sahlgrenska University Hospital/Östra, Göteborg University, Göteborg, Sweden, ^cDepartment of Pathology and Cytology, Uppsala University Hospital, Uppsala, Sweden

Folate deficiency leads to DNA strand breaks, mutations, and aberrant methylation and is a risk factor for colorectal cancer. In a recent study we analyzed the expression of two folate-associated genes, reduced folate carrier (RFC-1) and folylpolyglutamate synthase (FPGS), in normal-appearing mucosa obtained 10 cm apart from primary colon carcinomas. Low gene expression correlated with low folate levels and influenced patients’ survival negatively. The tumour suppressor gene deleted in colorectal carcinoma (DCC) functions as a dependence receptor inducing apoptosis when unoccupied by the ligand netrin-1. The biological function of the DCC gene product is not fully elucidated but it is known to guide neural crest cells in the spinal cord as well as in the colonic mucosa. Inactivation of DCC may be caused by aberrant DNA methylation, or by mutations resulting in loss of heterozygosity (LOH) i.e. by alterations associated with folate deficiency. Folate supplementation has been shown to prevent LOH of DCC in colonic mucosa of colorectal adenoma patients. AIM: The aim of the present study was to investigate if DCC was expressed differently in mucosa according to the gene expression levels of RFC-1 and FPGS. METHODS: Normal-appearing mucosa samples (n=53) were taken 10 cm apart from carcinomas. cDNA was prepared by reverse-transcribed PCR. DCC gene expression was detected by automated capillary gel electrophoresis and fragment analysis. Quantification of RFC-1 and FPGS gene expression was performed using real-time PCR. RESULTS: Two DCC exon 17-derived PCR products, 341 bp (DCC341) and 281 bp (DCC281) long, were detected in the mucosa. DCC281 was present in 98% (53/54) and DCC341 in 55% (29/53) of the samples. The relative gene expression levels of RFC-1 and FPGS in mucosa lacking DCC341 were significantly lower than in mucosa expressing DCC341. Multivariate analysis of pairwise gene expression values showed that the RFC-1 and FPGS levels were significantly correlated in mucosa expressing DCC341 (R = 0.49, p = 0.01, n = 27) whereas no significant RFC-1 and FPGS correlation was found in mucosa that lacked DCC341. CONCLUSIONS: The results imply that the folate status, reflected by the expression levels of RFC-1 and FPGS, in mucosa adjacent to carcinomas may influence the expression of DCC splice variants. Lack of DCC341 seems to be common in mucosa with low folate and may be indicative of a pre-malignant state.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in poster session 797 (Manifestations of cancer).