Identification of novel tumor markers in prostate, colon and breast cancer by unbiased methylation profiling

Abstract

DNA hypermethylation is a common epigenetic abnormality in cancer and may serve as a useful marker to clone cancer-related genes as well as a marker of clinical disease activity. To identify CpG islands methylated in prostate cancer, we used methylated CpG island amplification (MCA) coupled with representational difference analysis (RDA) on prostate cancer cell lines. We isolated 34 clones that corresponded to promoter CpG islands, including 5 reported targets of hypermethylation in cancer. We confirmed the data for 17 CpG islands by COBRA and/or pyrosequencing. All 17 genes were methylated in at least 2 cell lines of a 21-cancer cell line panel containing prostate cancer, colon cancer, leukemia, and breast cancer. Based on methylation in primary tumors compared to normal adjacent tissues, NKX2-5, CLSTN1, SPOCK2, SLC16A12, DPYS and NSE1 are candidate biomarkers for prostate cancer (methylation range 50%-85%). The combination of NSE1 or SPOCK2 hypermethylation showed a sensitivity of 80% and specificity of 95% in differentiating cancer from normal. Similarly NKX2-5, SPOCK2, SLC16A12, DPYS and GALR2 are candidate biomarkers for colon cancer (methylation range 60%-95%) and GALR2 hypermethylation showed a sensitivity of 85% and specificity of 95%. Finally, SLC16A12, GALR2, TOX, SPOCK2, EGFR5 and DPYS are candidate biomarkers for breast cancer (methylation range 33%-79%) with the combination of EGFR5 or TOX hypermethylation showing a sensitivity of 92% and specificity of 92%. Expression analysis for eight genes that had the most hypermethylation confirmed the methylation associated silencing and reactivation with 5-aza-2'-deoxycytidine treatment. Our data identify new targets of transcriptional silencing in cancer, and provide new biomarkers that could be useful in screening for prostate cancer and other cancers.

Figure 1. CpG island map and DNA methylation status of identified clones.

CpG maps of the seventeen genes analyzed in this study. For each gene, short vertical bars indicate CpG sites. Exon1 is indicated by black rectangles at the top, whereas location of the MCA-RDA clones is indicated by red rectangles at the bottom. Arrows point to known or presumed transcription start sites. Blue bars at the bottom indicate areas that analyzed by COBRA or pyrosequencing.

Figure 2. Representative COBRA and pyrosequencing for GALR2 and TFAP2C.

For GALR2 and TFAP2C methylation analyses, we used COBRA (left panel) and pyrosequencing (right panel). The methylation density of pyrosequencing is presented in the top of each tracing as the averaged methylation of the CpG sites analyzed. Both methods analyzed almost the same sites in the promoter region as presented in Figure 1. A. In GALR2 methylation analysis, normal adjacent sample showed 9% methylation by COBRA and 6% by pyrosequencing but tumor sample showed 45% by COBRA and 42% by pyrosequencing. B. In TFAP2C methylation analysis, normal sample showed 1% methylation by COBRA and 3% methylation by pyrosequencing but tumor sample showed 61% by COBRA and 64% by pyrosequencing.

Figure 3. Heat map of DNA methylation profiling of identified clones by MCA-RDA.

The bottom red-scale bar refers to the degree of methylation as measured by COBRA and/or pyrosequencing. NL represents normal leukocyte. A. All of the 17 genes investigated were methylated in at least 2 cell lines of a 21-cell line panel investigated. B. Methylation map for 8 genes studied in twenty primary prostate cancers (T) and paired normal (N) prostate samples. C. Methylation map for 9 genes studied in twenty primary colon cancers (T) and paired normal (N) colon samples. D. Methylation map of 7 genes in twenty-four breast cancers (T) and paired normal (N) samples. Pat.; patient samples.

Figure 4. Scatter plots of methylation analysis in primary prostate cancer (A), colorectal cancer (B) and breast cancer (C).

N and T represent tumor and adjacent normal. *Significantly different from normal adjacent samples, P<0.05.

Figure 5. Silencing in cancer and reactivation of hypermethylation.

Expression of target genes and GAPDH (control) was measured by RT-PCR in various cell lines. The sizes of the PCR products are listed in Table 1. Most cell lines chosen for analysis had high methylation level (>50%), and all had very low levels of expression at baseline, which was markedly increased by treatment with the methylation inhibitor, 5-azadC with or without the HDAC inhibitor TSA. Ctl.; no treatment, D; 5-AzadC alone, T; TSA alone, D+T; 5-AzadC and TSA in combination.