Identification of Novel Tumor Markers in Prostate, Colon and Breast Cancer by Unbiased Methylation Profiling

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.

[1]  Hiroyuki Takahashi,et al.  Altered methylation of multiple genes in carcinogenesis of the prostate , 2003, International journal of cancer.

[2]  D. Williams,et al.  Prostate specific antigen and androgen receptor induction and characterization of an immortalized adult human prostatic epithelial cell line. , 1996, Carcinogenesis.

[3]  W. El-Deiry,et al.  AP2 inhibits cancer cell growth and activates p21WAF1/CIP1 expression , 1997, Nature Genetics.

[4]  Masatoshi Watanabe,et al.  Aberrant methylation of the vascular endothelial growth factor receptor‐1 gene in prostate cancer , 2003, Cancer science.

[5]  P. Jones,et al.  Involvement of DNA methylation in human carcinogenesis. , 1998, Biological chemistry.

[6]  Jeanne Kowalski,et al.  Hypermethylation of CpG Islands in Primary and Metastatic Human Prostate Cancer , 2004, Cancer Research.

[7]  P. Laird.  Early detection: The power and the promise of DNA methylation markers , 2003, Nature Reviews Cancer.

[8]  C. Reznikoff,et al.  Characterization of human uroepithelial cells immortalized in vitro by simian virus 40. , 1987, Cancer research.

[9]  P. Laird,et al.  COBRA: a sensitive and quantitative DNA methylation assay. , 1997, Nucleic acids research.

[10]  S. Baylin,et al.  Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification. , 1999, Cancer research.

[11]  K A Baggerly,et al.  Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites. , 2003, BioTechniques.

[12]  P. Laird,et al.  Sensitive Detection of DNA Methylation , 2003, Annals of the New York Academy of Sciences.

[13]  N. Dubrawsky.  Cancer statistics , 1989, CA: a cancer journal for clinicians.

[14]  J. Herman,et al.  Alterations in DNA methylation: a fundamental aspect of neoplasia. , 1998, Advances in cancer research.

[15]  S. Baylin,et al.  Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon , 1994, Nature Genetics.

[16]  A. Jemal,et al.  Cancer Statistics, 2005 , 2005, CA: a cancer journal for clinicians.

[17]  C Eng,et al.  Excessive CpG island hypermethylation in cancer cell lines versus primary human malignancies. , 2001, Human molecular genetics.

[18]  J. Herman,et al.  Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer , 1999, Nature Genetics.

[19]  G. Pfeifer,et al.  Frequent hypermethylation of the RASSF1A gene in prostate cancer , 2002, Oncogene.

[20]  J. Herman,et al.  E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas. , 1995, Cancer research.

[21]  Gary L Rosner,et al.  Tumour vasculature: On the verge of collapse , 2005, Nature Reviews Cancer.

[22]  Pearlly S Yan,et al.  High-throughput methylation profiling by MCA coupled to CpG island microarray. , 2007, Genome research.

[23]  R. Kuick,et al.  Combined restriction landmark genomic scanning and virtual genome scans identify a novel human homeobox gene, ALX3, that is hypermethylated in neuroblastoma , 2002, Genes, chromosomes & cancer.

[24]  Hyeon Joo Lee,et al.  Aberrant CpG island hypermethylation of multiple genes in prostate cancer and prostatic intraepithelial neoplasia , 2004, The Journal of pathology.

[25]  M. Tainsky,et al.  Loss of AP‐2 results in downregulation of c‐KIT and enhancement of melanoma tumorigenicity and metastasis , 1998, The EMBO journal.

[26]  Dustin E. Schones,et al.  High-Resolution Profiling of Histone Methylations in the Human Genome , 2007, Cell.

[27]  J. Issa,et al.  Age-Related DNA Methylation Changes in Normal Human Prostate Tissues , 2007, Clinical Cancer Research.

[28]  M. Toyota,et al.  DNA Methylation Down-regulates CDX1 Gene Expression in Colorectal Cancer Cell Lines* , 2002, The Journal of Biological Chemistry.

[29]  Masatoshi Watanabe,et al.  The Role of Epigenetic Modifications in Retinoic Acid Receptor β2 Gene Expression in Human Prostate Cancers , 2001, Laboratory Investigation.

[30]  S. Clark,et al.  High sensitivity mapping of methylated cytosines. , 1994, Nucleic acids research.

[31]  Wei Chen,et al.  Comparing the DNA Hypermethylome with Gene Mutations in Human Colorectal Cancer , 2007, PLoS genetics.

[32]  R. Singal,et al.  Cytosine methylation represses glutathione S-transferase P1 (GSTP1) gene expression in human prostate cancer cells. , 2001, Cancer research.

[33]  J. Herman,et al.  Gene silencing in cancer in association with promoter hypermethylation. , 2003, The New England journal of medicine.