hTERT promoter regulation in human oral squamous cell carcinoma cells

OBJECTIVES: Enhanced expression of hTERT (human telomerase gene) occurs during cellular immortalization and is maintained in the vast majority of human oral squamous cell carcinoma (HOSCC) cells. Several cellular factors have been implicated in the control of hTERT mRNA expression by regulating the promoter activity. However, the detailed mode of hTERT promoter activation during oral carcinogenesis is unknown. The current study was undertaken to identify the trans regulatory factors of hTERT promoter using global, high throughput screening of the nuclear proteins that complex with the promoter element in HOSCC-specific manner. METHODS: We employed the promoter magnetic precipitation (PMP) assay followed by mass spectrometry to compare the hTERT promoter binding protein profiles between normal human oral keratinocytes (NHOK) and SCC4 cells, a HOSCC cell line. RESULTS: PMP analysis yielded several novel proteins that complex with the hTERT promoter differentially between NHOK and SCC4 cells. These proteins were identified by mass spectrometry to include MSH2, hnRNP K, hnRNP D, and GRHL2. Association with the hTERT promoter was confirmed in vivo by chromatin immunoprecipitation (ChIP), which revealed stronger association in SCC4 cells compared with NHOK. We serially knocked down the corresponding gene expression by RNA interference in SCC4 cells individually or in various combinations. None of the individual gene knockdown led to reduction of telomerase activity, although the promoter activity was reduced compared with the controls. However, when hnRNP K was knocked down in combination with GRHL2, telomerase activity was completely abolished in cells, and the cells exhibited massive cell death. CONCLUSIONS: This study identified new proteins that form physical complex with the hTERT promoter in vitro and in vivo and demonstrate the necessity of at least hnRNP K and GRHL2 for intact telomerase activity in SCC4 cells. This study was supported by grants from NIDCR/NIH (R01DE18295, R01DE14147, and K22DE15316).