Tumor suppressor genes are always found loss of heterozygosity and the presence of homozygous deletions in tumorigenesis. CRMP1 gene is located at 4p16.1 found loss of heterozygosity on chromosome 4 and was proved as a metastasis suppressor in lung cancer. To investigate the epigenetic regulation on CRMP1 promoter region, we have analyzed the methylation status of CRMP1 in 36 primary lung cancers and in various lung cancer cell lines by MS-PCR and Disulfide sequencing methods. The mRNA and protein levels in cells were assayed by real-time PCR and immunohistochemistry. We also transfected a lung cancer cell line with CRMP1 gene to determine whether exogenous expression of CRMP1 would affect in vitro growth and in vivo tumorigenicity. The promoter region of CRMP1 contains two CpG islands prospected to derive two independent CRMP1 isoforms, L-CRMP1 and CRMP1. The disulfide sequence results showed the frequency of DNA methylation in the former CpG island of CRMP1 promoter was increased dependent on increasing invasive ability of lung cancer cells. The methylation specific polymerase chain reaction results presented the promoter of L-CRMP1 was unmethylated in normal lung tissue but highly methylated in its paired lung cancer tissues. Various cancer cell lines showed that L-CRMP1 promoter was hypermethylated. CRMP1 can re-express in cells treated with DNA demethylating agents, 5-aza-2'-deoxycytidine. L-CRMP1 promoter hypermethylation was detected in 100% of lung cancer cell lines, in 67% lung squamous cell carcinomia, or 65% lung adenocarcinoma. Exogenous expression of L-CRMP1 in CL1-0 decreased in vitro colony formation and in vivo tumorigenicity. These data strong implicated that determination of L-CRMP1 methylation status may be a useful tumor marker for lung cancer and CRMP1 family should be a candidate tumor suppressor gene which was silenced by DNA methylaion machinery in tumorigenesis.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA