Human Telomerase Reverse Transcriptase Activated by E6 Oncoprotein Is Required for Human Papillomavirus-16/18-Infected Lung Tumorigenesis

Purpose: Our recent report indicates that human papillomavirus (HPV)-16/18 E6 oncoprotein is expressed in lung tumors and is related to p53 inactivation. We further explored whether human telomerase reverse transcriptase (hTERT) transcription is up-regulated by E6 and contributes to lung tumor development. Experimental Design: Immunohistochemistry detected HPV-16 E6 oncoprotein in 135 lung tumors, and hTERT mRNA was evaluated by real-time reverse transcription-PCR and in situ hybridization, respectively. A small RNA interference (RNAi), Western blotting, and chromatin immunoprecipitation analysis were used to clarify whether hTERT transcription was regulated by c-Myc and Sp1. The telomerase activity and oncogenic potential of TL-1 with or without E6- or hTERT-RNAi was determined by real-time quantitative telomeric repeat amplification protocol analysis and soft-agar assay, respectively. Results: hTERT mRNA levels in EIS-positive tumors, which were prevalent in females, nonsmokers, and adenocarcinomas, were significantly higher than in E6-negative tumors. In addition, hTERT mRNA levels in early tumors (stage 1) were greater than levels in advanced tumors (stages 11 and 111). Chromatin immunoprecipitation assay showed that Sp1 cooperated with c-Myc to activate hTERT transcription in TL-1 cells, which was similar to the SiHa cells. The telomerase activity of the TL-1 cells decreased concomitantly with the transfection of various doses of E6- or hTERT-RNAi. A soft-agar assay showed that the oncogenic potential of TL-1 cells was significantly reduced after being transfected with E6-RNAi. Moreover, a colony of TL-1 cells could not form after transfection with hTERT-RNAi. Conclusion: Transcriptional activation of hTERT by E6 oncoprotein is required for HPV-16/18-infected lung tumorigenesis.