Background: Cervical carcinogenesis is initiated by infection with high-risk (i.e., carcinogenic) human papillomavirus (HPV) types. The subsequent progression from premalignant cervical intraepithelial neoplasia (CIN) to invasive cancer is driven by both genetic and epigenetic processes. We assessed the role of the gene encoding the adhesion molecule tumor suppressor in lung cancer 1 (TSLC1) in this progression.
Methods: We analyzed TSLC1 gene expression by real-time quantitative reverse transcription-polymerase chain reaction, promoter methylation by sodium bisulfite genomic DNA sequencing, and allelic loss by microsatellite analysis in primary keratinocytes, in four non-tumorigenic HPV-immortalized human keratinocyte cell lines, and in 11 human cervical cancer cell lines that were positive for a high-risk HPV DNA type and in normal cervical epithelial cells. We transfected cervical cancer SiHa cells that did not express TSLC1 mRNA with an expression vector containing the TSLC1 complementary DNA (cDNA) or an empty vector and analyzed transfectants for anchorage-independent growth and tumorigenicity in nude mice. We also examined TSLC1 promoter methylation in premalignant cervical lesions and in cervical carcinomas and smears. All statistical tests were two-sided.
Results: TSLC1 mRNA was strongly reduced, relative to levels in primary keratinocytes, or absent in 10 (91%) of 11 cervical carcinoma cell lines but in none (0%) of the four HPV-immortalized cell lines (difference = 91%, 95% confidence interval [CI] = 74% to 100%; P =.004). The TSLC1 promoter was hypermethylated, relative to normal foreskin and cervical epithelial cells, in nine (82%) of the 11 cervical carcinoma cell lines but in none (0%) of the four HPV-immortalized cell lines (difference = 82%, 95 CI = 59% to 100%; P =.01). Seven (88%, 95% CI = 47% to 100%) of the eight SiHa/TSLC1 transfectants displayed a marked reduction in anchorage-independent growth (i.e., 0-100 colonies per 5000 cells) compared with none of the four (0%, 95% CI = 0% to 60%) SiHa transfectants bearing the empty vector (i.e., SiHa/hygro transfectants; difference = 88%, 95% CI = 65% to 100%; P =.01) or untransfected SiHa cells. All seven mice (100%, 95% CI = 59% to 100%) injected with untransfected SiHa cells or SiHa/hygro transfectants displayed tumors of at least 50 mm(3) by 2-6 weeks after injection compared with none of eight mice (0%, 95% CI = 0% to 37%) injected with the SiHa/TSLC1 transfectants (difference = 100%, 95% CI = 68% to 100%; P<.001). We detected TSLC1 promoter hypermethylation in seven (35%, 95% CI = 15% to 59%) of 20 high-grade CIN lesions (i.e., CIN II and III) and in 30 (58%, 95% CI = 43% to 71%) of 52 cervical squamous cell carcinomas compared with none (0%, 95% CI = 0% to 34%) of nine normal cervical epithelial biopsy samples and none (0%, 95% CI = 0% to 22%) of 12 CIN I lesions (P<.001 for cervical squamous cell cancer versus normal epithelial biopsy samples plus CIN I lesions).
Conclusions: TSLC1 gene silencing via promoter hypermethylation is a frequent event in the progression from high-risk HPV-containing, high-grade CIN lesions to invasive cervical cancer.