Genetic and epigenetic alterations of netrin-1 receptors in gastric cancer with chromosomal instability

Abstract

Background: The gene expressions of netrin-1 dependence receptors, DCC and UNC5C, are frequently downregulated in many cancers. We hypothesized that downregulation of DCC and UNC5C has an important growth regulatory function in gastric tumorigenesis.

Results: In the present study, a series of genetic and epigenetic analyses for DCC and UNC5C were performed in a Japanese cohort of 98 sporadic gastric cancers and corresponding normal gastric mucosa specimens. Loss of heterozygosity (LOH) analyses and microsatellite instability (MSI) analysis was applied to determine chromosomal instability (CIN) and MSI phenotypes, respectively. More than 5 % methylation in the DCC and UNC5C promoters were found in 45 % (44/98) and 32 % (31/98) gastric cancers, respectively, and in 9 % (9/105) and 5 % (5/105) normal gastric mucosa, respectively. Overall, 70 % (58 of 83 informative cases) and 51 % (40 of 79 informative cases) of gastric cancers harbored either LOH or aberrant methylation in the DCC and UNC5C genes, respectively. In total, 77 % (51 of 66 informative cases) of gastric cancers showed cumulative defects in these two dependence receptors and were significantly associated with chromosomal instability. Both DCC and UNC5C were inactivated in 97 % of CIN-positive gastric cancers and in 55 % of CIN-negative gastric cancers.

Conclusions: Defect in netrin receptors is a common feature in gastric cancers. DCC alterations are apparent in the early stages, and UNC5C alterations escalate with the progression of the disease, suggesting that the cumulative alterations of netrin-1 receptors was a late event in gastric cancer progression and emphasizing the importance of this growth regulatory pathway in gastric carcinogenesis.

Keywords: Chromosomal instability; DCC; Gastric cancer; Methylation; Netrin-1 receptors; UNC5C.

Fig. 1

DCC promoter methylation and 18q LOH analyses. (a) Schematic representation of the location of the three LOH probes and DCC gene promoter regions in chromosome 18. The red line denotes the DCC gene. Gray and black squares represent the untranslated and the coding exon 1 regions, respectively; arrows on the squares indicate transcriptional starting sites; vertical lines indicate CpG sites; white diamonds represent the restriction sites for HhaI; thick horizontal lines depict the locations of COBRA products; arrows on the thick horizontal lines denote COBRA primers. (b) Representative results of COBRA of DCC. Arrows indicate methylated alleles; M denotes methylation; U denotes unmethylation; Mc denotes the methylated control; SM denotes the size marker. (c) Results of DCC methylation as a continuous variable. In the box plot diagrams, the horizontal line within each box represents the median, the limits of each box represent the interquartile ranges, and the whiskers are the maximum and minimum values. Each green bar represents the mean. NM denotes normal mucosa. T denotes tumor. (d) The frequency of methylation-positivity of cancer and normal tissues according to different thresholds. (e) DCC mRNA expression levels and methylation status in 10 gastric cancer cell lines and a human lung fibroblast cell line. DCC mRNA expression is observed (lower ΔC_T) in GCIY and NHLH cell lines. DW denotes distilled water

Fig. 2

DCC promoter methylation and immunohistochemistry analyses. Immunohistochemistry analysis for DCC (a–c). Nuclei of tumor cells are completely negatively (a), focally negatively (b), and positively (c) stained. (d) Association between epigenetic/genetic alteration and DCC expression

Fig. 3

UNC5C promoter methylation and 4q LOH analyses. (a) Schematic representation of the location of the three LOH probes and UNC5C gene promoter regions in chromosome 4. The red line denotes the UNC5C gene. Gray and black squares represent the untranslated and the coding exon 1 regions, respectively; arrows on the squares indicate transcriptional starting sites; vertical lines indicate CpG sites; white diamonds represent the restriction sites for HhaI; thick horizontal lines depict the location of COBRA products; arrows on the thick horizontal lines denote COBRA primers. (b) Representative results of COBRA of UNC5C. Arrows indicate methylated alleles; M denotes methylation; U denotes unmethylation; Mc denotes the methylated control; SM denotes the size marker. (c) Results of UNC5C methylation as a continuous variable. In the box plot diagrams, the horizontal line within each box represents the median, the limits of each box represent the interquartile ranges, and the whiskers denote the maximum and minimum values. Each green bar represents the mean. NM denotes normal mucosa. T denotes tumor. (d) The frequency of methylation-positivity for cancer and normal tissues according to different thresholds. (e) UNC5C mRNA expression levels and methylation status in 10 gastric cancer cell lines and a human lung fibroblast cell line. UNC5C mRNA expression is observed (lower ΔC_T) in GCIY and NHLH cell lines. DW denotes distilled water

Fig. 4

Association between alteration patterns in netrin-1 receptors and clinicopathological features in gastric cancers. Correlation between alterations in netrin-1 receptors and TNM stage (a), depth of invasion (b), and degree of regional lymph node metastasis (c); association between alteration patterns in netrin-1 receptors and LOH ratio (d), presence of distant metastasis (e), MSI status (f), CIN phenotype (g), and KRAS/BRAF/PIC3CA mutation status (h). *The P value in panel A was calculated between Stage I/II vs. III/IV by Pearson’s chi-square test. ** The P value in panel c was calculated between N0 vs. N1–N3 by Pearson’s chi-square test. In Panel (d), the horizontal line within each box represents the median, the limits of each box represent the interquartile ranges, and the whiskers are the maximum and minimum values in the box plot diagrams. Asterisks and the numbers denote the mean value of the LOH ratio. Pairwise comparisons for each of the subgroups in panel (d) were performed by a non-parametric multiple comparison method using the Steel–Dwass test. ***Two cases could not be evaluated for CIN phenotype