HPV16 E7 protein and hTERT proteins defective for telomere maintenance cooperate to immortalize human keratinocytes

Parts used

Key findings

• Telomerase activity and telomere length are decoupled during HPV-driven immortalization. Human foreskin and ectocervical keratinocytes immortalized by HPV-16 E6/E7 show telomerase activity comparable to the cervical cancer lines SiHa, HeLa and C33A, so no further increase in telomerase is needed for progression to malignancy. Yet telomeres continue shortening through immortalization: primary keratinocytes fall from a T/S ratio of 1.0 (about 10 kb) at 2 population doublings to 0.6 at 18 doublings and 0.2 (about 2 kb) by 96 doublings, reaching their shortest by about 74 doublings and then stabilizing at the same ~2 kb length seen in all three cancer lines, including HPV-negative C33A.
• Telomere maintenance is dispensable for hTERT's role in keratinocyte immortalization. The C-terminally epitope-tagged hTERT-HA, which retains telomerase activity but cannot elongate telomeres or immortalize fibroblasts or SV40-transformed epithelial cells on its own, nonetheless immortalizes human foreskin keratinocytes in combination with HPV16 E7, as wild-type hTERT does. Neither hTERT nor E7 alone immortalizes. Telomerase activity is comparable across all hTERT-expressing cells, but telomeres lengthen in hTERT/E7 cells while shortening in hTERT-HA/E7 cells.
• Even catalytically dead hTERT immortalizes keratinocytes with E7, which rules out reverse transcriptase activity as the relevant function. hTERT-D868A, which has no telomerase activity in early-passage keratinocytes, is as efficient at immortalization as wild-type hTERT, and the telomere recruitment-defective hTERT N+T also immortalizes while retaining telomerase activity comparable to E6/E7 cells. Immunofluorescence shows hTERT-D868A at similar expression level and localization to wild-type hTERT, so the effect is not explained by altered abundance or mislocalization.
• Cells immortalized by catalytically dead hTERT-D868A lack telomerase at early passage but display high telomerase activity at late passage, and this is not caused by the introduced protein activating its own promoter: in luciferase reporter assays neither wild-type hTERT nor hTERT-D868A activates the hTERT core promoter, while HPV E6 does. Both proteins remain biologically active in the same assay, since both activate the cyclin D1 promoter. Inactive hTERT therefore mediates transit through crisis, after which continued proliferation correlates with induction of endogenous hTERT, as in E6-immortalized cells.
• hTERT changes host gene expression largely independently of its catalytic function, and its transcriptional footprint overlaps HPV E6's. In primary keratinocytes, 1379 of 6991 E6-induced expression changes (20%) are shared with wild-type hTERT, representing 58% of hTERT's own 2359 changes, and 2077 of those 2359 changes (88%) are reproduced by catalytically inactive hTERT-D868A, which alters 5467 probe sets in total. 1258 changes are common to E6, wild-type hTERT and hTERT-D868A.
• Clustering the 2077 catalysis-independent hTERT expression changes enriches for chromosome organization, chromatin organization and chromatin modification, and identifies Bmi1 — the core subunit of Polycomb Group complex 1 — as a shared target. E6, wild-type hTERT and hTERT-D868A each raise Bmi1 transcript 5- to 7-fold by RT-PCR and increase Bmi1 protein, and two further telomerase-dead hTERT mutants also raise Bmi1 transcript, confirming the induction is reverse-transcriptase independent.
• Bmi1 induction by hTERT is sustained rather than transient: Bmi1 protein is elevated in early-passage hTERT/E7-immortalized keratinocytes at 28 population doublings and remains high after serial passaging to 204 doublings, confirmed by immunohistochemistry showing significantly increased nuclear Bmi1 at both 42 and 220 population doublings relative to control keratinocytes.
• Bmi1 can substitute for hTERT or E6 in immortalization, identifying it as a sufficient downstream effector. Empty-vector keratinocytes fail to reach 25 population doublings, E7 alone or Bmi1 alone extends lifespan by roughly 15 doublings, and immortalization beyond 50 doublings occurs only when Bmi1 and E7 are co-expressed. Bmi1/E7 cells also show shortened telomeres, like E6/E7 cells — reinforcing that immortalization here does not require telomere maintenance.
• Bmi1 elevation tracks cervical disease progression in patient tissue, not just in culture. Across 21 cervical tissues scored by immunohistochemistry, Bmi1 is present modestly in basal and immediate suprabasal nuclei of normal epithelium, CIN1 and CIN2, but increases strikingly through the epithelial layers in CIN3 and invasive carcinoma, where staining is specific to cancerous lesions. Combined intensity and positivity scores for CIN3 and invasive carcinoma are significantly higher than both CIN1 and CIN2 (CIN3 versus CIN1 p = 0.0083, CIN3 versus CIN2 p = 0.0372, invasive versus CIN1 p = 0.0012, invasive versus CIN2 p = 0.0130).

Abstract

Previous studies have shown that wild-type human telomerase reverse transcriptase (hTERT) protein can functionally replace the human papillomavirus type 16 (HPV-16) E6 protein, which cooperates with the viral E7 protein in the immortalization of primary keratinocytes. In the current study, we made the surprising finding that catalytically inactive hTERT (hTERT-D868A), elongation-defective hTERT (hTERT-HA), and telomere recruitment-defective hTERT (hTERT N+T) also cooperate with E7 in mediating bypass of the senescence blockade and effecting cell immortalization. This suggests that hTERT has activities independent of its telomere maintenance functions that mediate transit across this restriction point. Since hTERT has been shown to have a role in gene activation, we performed microarray studies and discovered that E6, hTERT and mutant hTERT proteins altered the expression of highly overlapping sets of cellular genes. Most important, the E6 and hTERT proteins induced mRNA and protein levels of Bmi1, the core subunit of the Polycomb Group (PcG) complex 1. We show further that Bmi1 substitutes for E6 or hTERT in cell immortalization. Finally, tissue array studies demonstrated that expression of Bmi1 increased with the severity of cervical dysplasia, suggesting a potential role in the progression of cervical cancer. Together, these data demonstrate that hTERT has extra-telomeric activities that facilitate cell immortalization and that its induction of Bmi1 is one potential mechanism for mediating this activity.