Interplay between promoter methylation and chromosomal loss in gene silencing at 3p11-p14 in cervical cancer

Abstract

Loss of 3p11-p14 is a frequent event in epithelial cancer and a candidate prognostic biomarker in cervical cancer. In addition to loss, promoter methylation can participate in gene silencing and promote tumor aggressiveness. We have performed a complete mapping of promoter methylation at 3p11-p14 in two independent cohorts of cervical cancer patients (n = 149, n = 121), using Illumina 450K methylation arrays. The aim was to investigate whether hyperm-ethylation was frequent and could contribute to gene silencing and disease aggressiveness either alone or combined with loss. By comparing the methylation level of individual CpG sites with corresponding data of normal cervical tissue, 26 out of 41 genes were found to be hypermethylated in both cohorts. The frequency of patients with hypermethylation of these genes was found to be higher at tumor stages of 3 and 4 than in stage 1 tumors. Seventeen of the 26 genes were transcriptionally downregulated in cancer compared to normal tissue, whereof 6 genes showed a significant correlation between methylation and expression. Integrated analysis of methylation, gene dosage, and expression of the 26 hypermethylated genes identified 3 regulation patterns encompassing 8 hypermethylated genes; a methylation driven pattern (C3orf14, GPR27, ZNF717), a gene dosage driven pattern (THOC7, PSMD6), and a combined methylation and gene dosage driven pattern (FHIT, ADAMTS9, LRIG1). In survival analysis, patients with both hypermethylation and loss of LRIG1 had a worse outcome compared to those harboring only hypermethylation or none of the events. C3orf14 emerged as a novel methylation regulated suppressor gene, for which knockdown was found to promote invasive growth in human papilloma virus (HPV)-transformed keratinocytes. In conclusion, hypermethylation at 3p11-p14 is common in cervical cancer and may exert a selection pressure during carcinogenesis alone or combined with loss. Information on both events could lead to improved prognostic markers.

Keywords: 3p; cervical cancer; chromosomal loss; gene expression; integrative genomic profiling; promoter methylation; tumor suppressor genes.

Figure 1.

Hypermethylated CpG sites. Methylation status of 150 CpG sites on 26 genes for 149 cervical cancer patients in cohort 1. Patients are shown in columns and CpG sites found to be hypermethylated in tumors compared to normal tissue in at least 10% of the patients are ordered by chromosomal location in rows with gene symbols indicated. Hypermethylated (M) and not hypermethylated (NM) sites are indicated with red and blue color, respectively. Frequency of patients with hypermethylation and chromosomal loss is shown for each site by the black and green curves, respectively.

Figure 2.

Validation of hypermethylation frequency in cohort 2. Frequency of 121 cohort 2 patients with hypermethylation versus the corresponding frequency for 149 cohort 1 patients. In total, 150 CpG sites found to be hypermethylated in cohort 1 are shown, and each dot represents the hypermethylation frequency of an individual site. Pearson's correlation coefficient and P-value are indicated. Line of unity is included.

Figure 3.

Identification and validation of silenced genes. (A) Difference in expression of 26 hypermethylated genes between tumors and normal cervical samples. The mean fold change based on 3 external datasets (GSE6791, GSE7803, GSE9750) is shown for each gene. Seventeen genes which were significantly downregulated at an adjusted P ≤ 0.10 in at least one of the data sets are indicated in red and their symbol is listed. (B) Correlation coefficients (rho) from Spearman's rank correlation analysis of methylation (β-value) against expression for 26 hypermethylated genes in 147 cervical tumors (cohort 1). Only negative correlations are shown, and in cases of several methylation and expression probes for the same gene, the most significant probes are presented. The horizontal line indicates the cut-off significance level, corresponding to rho = −0.20 (adj P ≤ 0.10). Eight significant genes are indicated in red and their symbol is listed. (C) Schematic illustration of the CpG sites for 6 significant genes in (B), which were validated in cohort 2. Significant CpG sites are indicated in green (adj P ≤ 0.10) and not significant sites in blue for 147 patients in cohort 1 and 121 patients in cohort 2. Sites in white were filtered during preprocessing due to their location closer than 10 bp from a SNP or low variation across the patients (hatched white; IQR < 0.08). Gray sites were hypermethylated in <10% of the patients. TSS: transcription start site.

Figure 4.

Individual and combined effect of loss and hypermethylation on gene expression. Box plots of gene expression in 4 groups of tumors with different combination of gene dosage and methylation status, demonstrating individual effect of methylation (A), individual effect of gene dosage (B), and combined effect of methylation and gene dosage (C) on gene expression. In total, 73 tumors from cohort 1 were included, for which gene expression, gene dosage, and methylation data were available. NL: no loss; L: loss; NM: not hypermethylated; M: hypermethylated. The median expression value of each group is indicated by the horizontal lines, and the edges of the boxes represent the first and third quartiles. P-values from Welch's t-test are indicated. All indicated differences had an adjusted P ≤ 0.10.

Figure 5.

Individual and combined effect of LRIG1 loss and hypermethylation on clinical outcome. Kaplan-Meier curves of progression free survival for cervical cancer patients (cohort 1) with and without loss of LRIG1 (A), with and without hypermethylation of LRIG1 (B), the 2 combined (C), and high and low LRIG1 expression (D). P-values in log-rank test and number of patients are indicated. NL: no loss; L: loss; NM: not hypermethylated; M: hypermethylated.

Figure 6.

Methylation characteristics and suppressor function of C3orf14. (A) Frequency of cervical cancer patients with hypermethylation of individual CpG sites ordered by location. (B) Methylation (β-value) against number of hypermethylated CpG sites. (C) Density plots (kernel density estimation with band width 0.02, black line; histogram, gray bars) of C3orf14 methylation in normal cervical tissue from GSE46306 (left) and tumors in cohort 1 (right). The blue lines indicate cut-off β-value for scoring hypermethylation. (D) C3orf14 expression against number of hypermethylated CpG sites. (E) C3orf14 expression against methylation (β-value). Invasion (F) and cell viability (G) of control and C3orf14 siRNA treated FK16A cells at early (p40/42) and late (p304) passages. The columns and bars show the mean and standard deviation of triplicates for one representative experiment for each passage. The difference between siRNA treated and control cells was significant for invasion (p42: P = 0.010, p304: P = 0.006, t-test), but not for cell viability. In B, C, and E, methylation data of the CpG site correlating most strongly with gene expression were used. In B, D, and E, correlation coefficient (rho) and P-value in Spearman's rank correlation analysis are indicated. In A-E, data from cohort 1 are presented; similar results were found for cohort 2.