Methylation profiling of carcinogenesis-associated genes in sporadic breast cancer

Background

Aberrant methylation of normally unmethylated CpG islands has been associated with transcriptional inactivation of defined tumor suppressor genes (TSG) in human cancer. Abnormal methylation therefore serves as an alternative to the genetic loss of a tumor suppressor gene function by deletion or mutation.

Methods

Methylation profiling was performed by methyl-sensitive PCR with genes involved in cancerogenesis: RB1, p14, p15, p16, CDH1, MGMT, HIC1, N33, LAMC3 and TGFBR1. Methylation profiles of these genes were obtained for 105 breast cancer (BC) specimens. Five specimens (section material) of the normal mammary gland tissue and peripheral blood lymphocytes of 30 healthy subjects were also investigated.

Results

By methylation-sensitive PCR with specific primers we detected no methylation of any investigated genes in control peripheral blood lymphocytes and in five normal breast tissues. High frequencies of promoter methylation were observed for the major TSG involved in controlling the cell cycle through the Cdk-Rb-E2F signaling pathway: RB1, 17%; p16, 56%. Methylation of both genes was revealed in 12% of tumors. p15 was methylated in 2% only. No methylation was observed for the CpG island of p14. The methylation frequency was rather high in the case of the CDH1 promoter (37%), maximal in the case of the HIC1 promoter (79%), and relatively low in the case of MGMT (8%) and N33 (9%). Methylation was shown to be 32.5% for LAMC3. Laminins contain motifs providing cell growth and differentiation via selective activation of signaling pathways dependent on protein kinase C and, probably, involved in metastases spreading. We have detected abnormal methylation of the TGFBR1 CpG island in 35% samples. TGFBR1 takes part in transforming growth factor beta signaling, mainly resulting in inhibition of cell proliferation. None of tumors showed methylation of all genes. No gene was methylated in 11% of tumors, and at least one gene in 89% of tumors.

Conclusion

We have shown that CpG methylation in the promoter regions of TSG is common to sporadic BC. Genes with a higher methylation frequency may be included in the BC methylotype. Identification of the genes with a high methylation frequency is a necessary step in characterizing a particular tumor. Along with other molecular genetic markers, the methylation profile may be employed in early diagnostics and prognostication.