Genomic Aberrations in the HTPAP Promoter Affect Tumor Metastasis and Clinical Prognosis of Hepatocellular Carcinoma

We previously reported that the intronic tagSNP +357G/C in the metastasis suppressor HTPAP is associated with metastasis and prognosis of hepatocellular carcinoma (HCC). The aim of this study was to investigate whether SNPs in the HTPAP promoter modulate HTPAP expression and prognosis of HCC. Genomic DNA from 572 microdissected HCCs were genotyped by pyrosequencing and verified by direct sequencing. Haplotype blocks were analyzed. Reporter plasmids were constructed and transfected into HCC cell lines. Transcriptional activities of plasmids were analyzed by dual-luciferase reporter systems. HTPAP expression was measured by real-time quantitative PCR, western blots, and tissue microarrays. Invasion was assessed by Matrigel assays. The prognostic values of HTPAP promoter SNPs in HCC were evaluated by Kaplan-Meier and Cox regression analyses. We identified six SNPs, including -1053A/G and +64G/C, in the HTPAP promoter. The SNPs were in complete linkage disequilibrium, resulting in three promoter haplotypes (promoter I:-1053AA/+64GG, promoter II: -1053AG/+64GC, and promoter III: -1053GG/+64CC). Promoter I manifested the highest luciferase index (p<0.005). However, no significant difference was observed between promoters II and III. We consistently found that HTPAP mRNA and protein levels were significantly higher in promoter I than that of promoter II+III (p<0.001). Invasion was increased in HCC cells transfected with promoters II+III compared to those transfected with promoter I (p<0.05). The HTPAP promoter II+III haplotype was associated with significantly increased metastasis compared to that of promoter I (p = 0.023). The postoperative five-year overall survival of patients with promoters II+III was lower than that of patients with promoter I (p = 0.006). Multivariate analysis showed that the promoter II+III haplotype was an adverse prognostic marker in HCC. The genetic variants at loci –1053 and +64 of the HTPAP promoter affect the expression of HTPAP, which might be a novel determinant and target for HCC prognosis.