Regular articleBiomarkers, genomics, proteomics, and gene regulationSynergistic Silencing by Promoter Methylation and Reduced AP-2α Transactivation of the Proapoptotic HRK Gene Confers Apoptosis Resistance and Enhanced Tumor Growth

The Harakiri (HRK) gene encodes an important proapoptotic mitochondrial protein of the Bcl-2 family. HRK is expressed in normal tissues but is decreased in many cancers such as melanoma, the mechanisms of which have not been fully elucidated. Here, we demonstrate that HRK is silenced by hypermethylation of a major proximal CpG island in the HRK promoter. Furthermore, we show that HRK is a novel target gene regulated by the transcription factor AP-2α, which interacts with an AP-2α binding site in the HRK promoter. Hypermethylation of the major proximal CpG island (which contains the AP-2α binding site within the most densely methylated −218- to −194-bp region) inhibited AP-2α binding and transcriptional activity. Artificial overexpression of AP-2α in melanoma cells up-regulated HRK transcription, which was further restored by treatment with DNA methyltransferase inhibitor 5-azacytidine. Artificial overexpression of HRK by recombinant adenovirus induced caspase-dependent apoptosis, inhibited melanoma cell growth in vitro, and markedly reduced in vivo melanoma growth in a nude mouse xenograft model. RNA interference by siHRK or siAP-2α reversed the above effects. We conclude that the synergistic effects of HRK promoter hypermethylation and loss of AP-2α transactivation lead to HRK gene silencing and confer resistance to apoptosis and enhanced tumor growth. These novel molecular lesions may provide the basis for new therapeutic approaches to treating AP-2α- and HRK-deficient cancers.