Cell-Restricted Immortalization by Human Papillomavirus Correlates with Telomerase Activation and Engagement of the hTERT Promoter by Myc

Abstract

The high-risk human papillomaviruses (HPVs) are the causative agents of nearly all cervical cancers and are etiologically linked to additional human cancers, including those of anal, oral, and laryngeal origin. The main transforming genes of the high-risk HPVs are E6 and E7. E6, in addition to its role in p53 degradation, induces hTERT mRNA transcription in genital keratinocytes via interactions with Myc protein, thereby increasing cellular telomerase activity. While the HPV type 16 E6 and E7 genes efficiently immortalize human keratinocytes, they appear to only prolong the life span of human fibroblasts. To examine the molecular basis for this cell-type dependency, we examined the correlation between the ability of E6 to transactivate endogenous and exogenous hTERT promoters and to immortalize genital keratinocytes and fibroblasts. Confirming earlier studies, the E6 and E7 genes were incapable of immortalizing human fibroblasts but did delay senescence. Despite the lack of immortalization, E6 was functional in the fibroblasts, mediating p53 degradation and strongly transactivating an exogenous hTERT promoter. However, E6 failed to transactivate the endogenous hTERT promoter. Coordinately with this failure, we observed that Myc protein was not associated with the endogenous hTERT promoter, most likely due to the extremely low level of Myc expression in these cells and/or to differences in chromatin structure, in contrast with hTERT promoters that we found to be activated by E6 (i.e., the endogenous hTERT promoter in primary keratinoctyes and the exogenous hTERT core promoter in fibroblasts), where Myc is associated with the promoter in either a quiescent or an E6-induced state. These findings are consistent with those of our previous studies on mutagenesis and the knockdown of small interfering RNA, which demonstrated a requirement for Myc in the induction of the hTERT promoter by E6 and suggested that occupancy of the promoter by Myc determines the responsiveness of E6 and the downstream induction of telomerase and cell immortalization.The high-risk papillomaviruses (HPVs; e.g., HPV type 16 and HPV-18) are associated with anogenital carcinomas (65, 66), laryngeal carcinomas, and head and neck carcinomas (15). These HPVs carry two oncogenes, E6 and E7, which are retained and expressed in HPV-positive cervical cancers (1, 2, 50) and are required for maintenance of the tumorigenic phenotype (35, 36). The E6 and E7 proteins were first identified as targeting the p53 and Rb tumor suppressor pathways in host cells (8,9,35,36,47,48), thereby disrupting cell cycle controls.Telomerase is a specialized reverse transcriptase that synthesizes repeat DNA sequences at the ends of chromosomes termed telomeres (17). The absence of telomerase activity in most normal human cells results in the progressive shortening of telomeres with each cell division (19,56,60), eventuating in growth arrest or replicative senescence (6,19). In contrast to most human somatic cells, immortalized and cancer cells contain detectable telomerase ac...

O-linked GlcNAcylation elevated by HPV E6 mediates viral oncogenesis

“…HPV-E6 interacts with c-MYC to reactivate the transcription of human telomerase reverse transcriptase (33)(34)(35)(36). However, the effect of HPV oncoproteins on c-MYC protein levels is inconclusive (33)(34)(35)(36). Due to the pleiotropic activities of c-MYC, its expression and function must be fine-tuned.…”

HPV E6 protein interacts physically and functionally with the cellular telomerase complex

“…Interestingly, overexpression of hTERT or c-Myc (which transactivates the hTERT promoter) can substitute for E6 in the immortalization of primary HFKs, indicating that telomerase activation constitutes a major immortalizing function of E6 (11,12). Our previous studies and those of other laboratories indicate that E6-mediated hTERT transactivation is independent of its ability to degrade p53 or interact with PDZ proteins (11)(12)(13)(14)(15)(16). However, hTERT transactivation by E6 is dependent upon its binding the cellular ubiquitin ligase, E6AP (13,14,(17)(18)(19)(20).…”

“…This would thereby allow us to examine telomerase activity independent of endogenous hTERT transcription. We took advantage of our recent finding that E6 fails to induce the hTERT gene or to induce telomerase in human fibroblasts (15). We used human fibroblasts stably transduced with Flag-tagged hTERT (HFF/hTERT).…”

“…However, hTERT transactivation by E6 is dependent upon its binding the cellular ubiquitin ligase, E6AP (13,14,(17)(18)(19)(20). There appear to be two critical targets for E6/E6AP on the hTERT promoter, Myc (12,15,16) and NFX-1 (13,18,21), which transactivate and transrepress the promoter respectively. The model for E6 regulation of the hTERT promoter is likely to be quite complex and it is anticipated that there might be additional targets for E6 on this promoter.…”

Molecular Markers Implicating Early Malignant Events in Cervical Carcinogenesis

“…HPV E6 has been suggested to induce telomerase activity during the early stages of transformation, giving the cells a growth advantage so that they eventually displace telomerase-deficient cells (27)(28)(29)(30)(31). The E7 protein has also been shown to contribute to telomerase activation and maintenance (32).…”