NFX1-123 and Poly(A) Binding Proteins Synergistically Augment Activation of Telomerase in Human Papillomavirus Type 16 E6-Expressing Cells

ABSTRACT Overcoming senescence signals in somatic cells is critical to cellular immortalization and carcinogenesis. High-risk human papillomavirus (HPV) can immortalize epithelial cells in culture through degradation of the retinoblastoma protein by HPV E7 and activation of hTERT transcription, the catalytic subunit of telomerase, by the heterodimer HPV E6/E6-associated protein (E6AP). Recent work in our laboratory identified a novel repressor of hTERT transcription, NFX1-91, which is targeted for ubiquitin-mediated degradation by HPV type 16 (HPV16) E6/E6AP. In contrast, NFX1-123, a splice variant NFX1, increased expression from an hTERT promoter that was activated by HPV16 E6/E6AP. Here, we show that HPV16 E6 bound both NFX1-91 and NFX1-123 through the common central domain of NFX1 in the absence of E6AP. NFX1-123 positively regulated hTERT expression, as its knockdown decreased hTERT mRNA levels and telomerase activity and its overexpression increased telomerase activity. We identified new protein partners of NFX1-123, including several cytoplasmic poly(A) binding proteins (PABPCs) that interacted with NFX1-123 through its N-terminal PAM2 motif, a protein domain characteristic of other PABPC protein partners. Furthermore, NFX1-123 and PABPCs together had a synergistic stimulatory effect on hTERT-regulated reporter assays. The data suggest that NFX1-123 is integral to hTERT regulation in HPV16 E6-expressing epithelial cells and that the interaction between NFX1-123 and PABPCs is critical to hTERT activity.

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Cytoplasmic Poly(A) Binding Proteins Regulate Telomerase Activity and Cell Growth in Human Papillomavirus Type 16 E6-Expressing Keratinocytes

Journal of Virology ◽

10.1128/jvi.01377-10 ◽

2010 ◽

Vol 84 (24) ◽

pp. 12934-12944 ◽

Cited By ~ 23

Author(s):

Rachel A. Katzenellenbogen ◽

Portia Vliet-Gregg ◽

Mei Xu ◽

Denise A. Galloway

Keyword(s):

Human Papillomavirus ◽

Rna Processing ◽

Posttranscriptional Regulation ◽

Cellular Localization ◽

Binding Proteins ◽

Telomerase Activity ◽

Htert Mrna ◽

Hpv16 E6 ◽

Hpv E6 ◽

E6 And E7

ABSTRACT The high-risk human papillomavirus (HPV) E6 and E7 oncoproteins are critical to the immortalization of keratinocytes. HPV type 16 (HPV16) E6 interacts with endogenous proteins to activate hTERT, the catalytic subunit of telomerase, thus avoiding cellular senescence signals. NFX1-123, the longer splice variant of NFX1, interacts with HPV16 E6, as well as cytoplasmic poly(A) binding proteins 1 and 4 (PABPC1 and PABPC4). HPV16 E6 affects hTERT expression posttranscriptionally through NFX1-123, as NFX1-123 interacts with hTERT mRNA and stabilizes it, leading to greater telomerase activity. The PAM2 motif of NFX1-123, with which it binds PABPCs, is required for the posttranscriptional regulation of hTERT by HPV16 E6 and NFX1-123. There is increasing evidence that RNA and DNA viruses utilize RNA-processing proteins, and specifically PABPCs, in the normal virus life cycle, and there is also evidence that RNA-processing proteins are perturbed in cancers. Here, we show that PABPCs are critical in hTERT regulation by HPV16 E6. Although the amount and cellular localization of PABPCs were largely unchanged in cervical cancer cell lines with or without HPV16 and in human foreskin keratinocytes (HFKs) with or without HPV16 E6, knockdown of PABPCs decreased hTERT mRNA and telomerase activity and overexpression of PABPC4 increased these in HPV16 E6-expressing HFKs. In contrast, knockdown of PABPCs in C33A cells had no effect on hTERT mRNA or telomerase activity. Additionally, overexpression of PABPC4 and hTERT led to greater growth of cultured HPV16 E6-expressing HFKs. This is the first evidence that PABPCs have a targeted role in hTERT regulation leading to a growth advantage in cells expressing HPV16 E6.

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NFX1-123 Increases hTERT Expression and Telomerase Activity Posttranscriptionally in Human Papillomavirus Type 16 E6 Keratinocytes

Journal of Virology ◽

10.1128/jvi.02556-08 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6446-6456 ◽

Cited By ~ 41

Author(s):

Rachel A. Katzenellenbogen ◽

Portia Vliet-Gregg ◽

Mei Xu ◽

Denise A. Galloway

Keyword(s):

Human Papillomavirus ◽

Transcriptional Activation ◽

Telomerase Activity ◽

Mrna Levels ◽

Htert Mrna ◽

Cytoplasmic Protein ◽

Nucleic Acid Binding ◽

Htert Expression ◽

Hpv16 E6

ABSTRACT High-risk human papillomavirus (HPV) E6 protein induces telomerase activity through transcriptional activation of hTERT, the catalytic subunit of telomerase. HPV type 16 (HPV16) E6 interacts with two splice variants of NFX1 to increase hTERT expression. NFX1-91 is a transcriptional repressor of hTERT that is polyubiquitinated and targeted for degradation by HPV16 E6 in concert with E6-associated protein. We previously showed that NFX1-123 augments hTERT expression through binding to cytoplasmic poly(A) binding proteins (PABPCs). In this study, we determined that unlike NFX1-91, NFX1-123 is a cytoplasmic protein that colocalized with PABPCs but does not shuttle with PABPCs between the nucleus and cytoplasm. NFX1-123 requires both its PAM2 motif, with which it binds PABPCs, and its R3H domain, which has putative nucleic acid binding capabilities, to increase hTERT mRNA levels and telomerase activity in keratinocytes expressing HPV16 E6. In keratinocytes expressing HPV16 E6 and overexpressing NFX1-123, there was increased protein expression from in vitro-transcribed RNA fused with the 5′ untranslated region (5′ UTR) of hTERT. This posttranscriptional increase in expression required the PAM2 motif and R3H domain of NFX1-123 as well as the coexpression of HPV16 E6. NFX1-123 bound endogenous hTERT mRNA and increased its stability in HPV16 E6-expressing human foreskin keratinocytes, and NFX1-123 increased the stability of in vitro-transcribed RNA fused with the 5′ UTR of hTERT. Together, these studies describe the first evidence of posttranscriptional regulation of hTERT, through the direct interaction of the cytoplasmic protein NFX1-123 with hTERT mRNA, in HPV16 E6-expressing keratinocytes.

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The Global Transcriptional Effects of the Human Papillomavirus E6 Protein in Cervical Carcinoma Cell Lines Are Mediated by the E6AP Ubiquitin Ligase

Journal of Virology ◽

10.1128/jvi.79.6.3737-3747.2005 ◽

2005 ◽

Vol 79 (6) ◽

pp. 3737-3747 ◽

Cited By ~ 68

Author(s):

Melissa L. Kelley ◽

Kerri E. Keiger ◽

Chan Jae Lee ◽

Jon M. Huibregtse

Keyword(s):

Human Papillomavirus ◽

Cell Lines ◽

Ubiquitin Ligase ◽

Telomerase Activity ◽

Transcriptional Program ◽

Hpv E6 ◽

Amplification Protocol ◽

Independent Functions ◽

Interfering Rna ◽

E6 Protein

ABSTRACT The function of the human papillomavirus (HPV) E6 protein that is most clearly linked to carcinogenesis is the targeted degradation of p53, which is dependent on the E6AP ubiquitin ligase. Additional functions have been attributed to E6, including the stimulation of telomerase activity and the targeted degradation of other cellular proteins, but in most cases it is unclear whether these activities are also E6AP dependent. While E6 clearly influences the transcriptional program of HPV-positive cell lines through the inactivation of p53, it has been shown that at least a subset of its p53-independent functions are also reflected in the transcriptional program. For this study, we have determined the extent to which E6AP is involved in mediating the set of E6 functions that impact on the global transcriptional program of HPV-positive cell lines. The transcriptional profiles of ∼31,000 genes were characterized for three cell lines (HeLa, Caski, and SiHa cells) after small interfering RNA (siRNA)-mediated silencing of E6 or E6AP. We found that E6 and E6AP siRNAs elicited nearly identical alterations in the transcriptional profile of each cell line. Some of the expression alterations were apparent secondary effects of p53 stabilization, while the basis of most other changes was not reconcilable with previously proposed E6 functions. While expression changes of the TERT gene (telomerase catalytic subunit) were not revealed by the array, telomerase repeat amplification protocol assays showed that both E6 and E6AP knockouts resulted in a suppression of telomerase activity. Together, these results suggest that E6AP mediates a broad spectrum of E6 functions, including virtually all functions that impact on the transcriptional program of HPV-positive cell lines.

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The High-Risk Human Papillomavirus Type 16 E6 Counters the GAP Function of E6TP1 toward Small Rap G Proteins

Journal of Virology ◽

10.1128/jvi.77.2.1614-1620.2003 ◽

2003 ◽

Vol 77 (2) ◽

pp. 1614-1620 ◽

Cited By ~ 33

Author(s):

Latika Singh ◽

Qingshen Gao ◽

Ajay Kumar ◽

Takaya Gotoh ◽

David E. Wazer ◽

...

Keyword(s):

Human Papillomavirus ◽

High Risk ◽

G Protein ◽

Mutational Analysis ◽

Protein Signaling ◽

Hpv16 E6 ◽

Human Papillomavirus Type 16 ◽

Hpv E6 ◽

Small G Protein ◽

Transforming Activity

ABSTRACT We have recently identified E6TP1 (E6-targeted protein 1) as a novel high-risk human papillomavirus type 16 (HPV16) E6-binding protein. Importantly, mutational analysis of E6 revealed a strong correlation between the transforming activity and its abilities to bind and target E6TP1 for ubiquitin-mediated degradation. As a region within E6TP1 has high homology with GAP domains of known and putative Rap GTPase-activating proteins (GAPs), these results raised the possibility that HPV E6 may alter the Rap small-G-protein signaling pathway. Using two different approaches, we now demonstrate that human E6TP1 exhibits GAP activity for Rap1 and Rap2, confirming recent findings that a closely related rat homologue exhibits Rap-specific GAP activity. Using mutational analysis, we localize the GAP activity to residues 240 to 945 of E6TP1. Significantly, we demonstrate that coexpression of HPV16 E6, by promoting the degradation of E6TP1, enhances the GTP loading of Rap. These results support a role of Rap small-G-protein pathway in E6-mediated oncogenesis.

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NFX1 Plays a Role in Human Papillomavirus Type 16 E6 Activation of NFκB Activity

Journal of Virology ◽

10.1128/jvi.00538-10 ◽

2010 ◽

Vol 84 (21) ◽

pp. 11461-11469 ◽

Cited By ~ 24

Author(s):

Mei Xu ◽

Rachel A. Katzenellenbogen ◽

Carla Grandori ◽

Denise A. Galloway

Keyword(s):

Human Papillomavirus ◽

Human Telomerase Reverse Transcriptase ◽

Hpv16 E6 ◽

Regulatory Pathways ◽

Cellular Processes ◽

Hpv E6 ◽

Host Cell Interactions ◽

Nfκb Pathway ◽

Human Telomerase ◽

Dual Regulator

ABSTRACT High-risk human papillomavirus (HR HPV) requires differentiating epithelial cells to continue to divide in order to replicate the viral DNA. To achieve this, HPV perturbs several regulatory pathways, including cellular apoptosis and senescence signals. HPV E6 has been identified as a regulator of the NFκB signaling pathway, a pathway important in many cellular processes, as well as regulation of virus-host cell interactions. We report here that NFX1-91, an endogenously expressed transcriptional regulator of human telomerase reverse transcriptase (hTERT) that is targeted by HPV type 16 (HPV16) E6/E6-associated protein (E6AP) for degradation, is also critical for regulation of the NFκB pathway by HPV16 E6. Microarray analysis revealed induction of NFκB-responsive genes and reduction of NFκB inhibitors with knockdown of NFX1-91. Knockdown of NFX1-91 induced downregulation of p105, an NFκB inhibitor in both primary human foreskin keratinocytes (HFKs) and HCT116 cells. Chromatin immunoprecipitation assays further confirmed that NFX1-91 bound to the p105 promoter and upregulated its expression. Similarly, in HPV16 E6-positive cells, reduction of p105 expression was observed, paralleling knockdown of NFX1-91 expression. Overall, our data suggest a mechanism for HPV16 E6 activation of the NFκB pathway through NFX1-91. Also, it provides evidence that NFX1-91 can function as a dual regulator, not only a transcriptional repressor, but also a transcriptional activator, when bound to DNA.

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Human Papillomavirus Antibodies and Future Risk of Anogenital Cancer: A Nested Case-Control Study in the European Prospective Investigation Into Cancer and Nutrition Study

Journal of Clinical Oncology ◽

10.1200/jco.2014.57.8435 ◽

2015 ◽

Vol 33 (8) ◽

pp. 877-884 ◽

Cited By ~ 37

Author(s):

Aimée R. Kreimer ◽

Paul Brennan ◽

Krystle A. Lang Kuhs ◽

Tim Waterboer ◽

Gary Clifford ◽

...

Keyword(s):

Human Papillomavirus ◽

Anal Cancer ◽

Cancer Diagnosis ◽

Blood Samples ◽

Hpv16 E6 ◽

Hpv E6 ◽

Nutrition Study ◽

Nested Case Control ◽

Control Study ◽

Prospective Investigation

Purpose Human papillomavirus (HPV) type 16 (HPV16) causes cancer at several anatomic sites. In the European Prospective Investigation Into Cancer and Nutrition study, HPV16 E6 seropositivity was present more than 10 years before oropharyngeal cancer diagnosis and was nearly absent in controls. The current study sought to evaluate the extent to which HPV16 E6 antibodies are present before diagnosis of anogenital cancers within the same cohort. Methods Four hundred incident anogenital cancers (273 cervical, 24 anal, 67 vulvar, 12 vaginal, and 24 penile cancers) with prediagnostic blood samples (collected on average 3 and 8 years before diagnosis for cervix and noncervix cancers, respectively) and 718 matched controls were included. Plasma was analyzed for antibodies against HPV16 E6 and multiple other HPV proteins and genotypes and evaluated in relation to risk using unconditional logistic regression. Results HPV16 E6 seropositivity was present in 29.2% of individuals (seven of 24 individuals) who later developed anal cancer compared with 0.6% of controls (four of 718 controls) who remained cancer free (odds ratio [OR], 75.9; 95% CI, 17.9 to 321). HPV16 E6 seropositivity was less common for cancers of the cervix (3.3%), vagina (8.3%), vulva (1.5%), and penis (8.3%). No associations were seen for non–type 16 HPV E6 antibodies, apart from anti-HPV58 E6 and anal cancer (OR, 6.8; 95% CI, 1.4 to 33.1). HPV16 E6 seropositivity tended to increase in blood samples drawn closer in time to cancer diagnosis. Conclusion HPV16 E6 seropositivity is relatively common before diagnosis of anal cancer but rare for other HPV-related anogenital cancers.

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Binding of Human Papillomavirus Type 16 E6 to E6AP Is Not Required for Activation of hTERT

Journal of Virology ◽

10.1128/jvi.01776-07 ◽

2007 ◽

Vol 82 (1) ◽

pp. 71-76 ◽

Cited By ~ 30

Author(s):

Pedja Sekaric ◽

Jonathan J. Cherry ◽

Elliot J. Androphy

Keyword(s):

Human Papillomavirus ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Htert Promoter ◽

Hpv16 E6 ◽

Human Papillomavirus Type 16 ◽

Human Mammary Epithelial ◽

E6 Protein ◽

The Relationship

ABSTRACT The human papillomavirus (HPV) type 16 (HPV16) E6 protein stimulates transcription of the catalytic subunit of telomerase, hTERT, in epithelial cells. It has been reported that binding to the ubiquitin ligase E6AP is required for this E6 activity, with E6 directing E6AP to the hTERT promoter. We previously reported two E6AP binding-defective HPV16 E6 mutations that induced immortalization of human mammary epithelial cells. Because activation of hTERT is proposed to be necessary for epithelial cell immortalization, we sought to further characterize the relationship between E6/E6AP association and telomerase induction. We demonstrate that while these E6 mutants do not bind E6AP, they retain the capability to stimulate the expression of hTERT. Chromatin immunoprecipitation assays confirmed the presence of Myc, wild-type E6, and the E6AP binding-defective E6 mutants, but not E6AP itself, at the endogenous hTERT promoter. Interestingly, an immortalization-defective E6 mutant localized to the hTERT promoter but failed to increase transcription. We conclude that binding to E6AP is not necessary for E6 localization to or activation of the hTERT promoter and that another activity of E6 is involved in hTERT activation.

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Identification of High-Risk Human Papillomavirus DNA, p16 and E6/E7 Oncoproteins in Laryngeal and Hypopharyngeal Squamous Cell Carcinomas

Viruses ◽

10.3390/v13061008 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1008

Author(s):

Andrejs Lifsics ◽

Valerija Groma ◽

Maksims Cistjakovs ◽

Sandra Skuja ◽

Renars Deksnis ◽

...

Keyword(s):

Human Papillomavirus ◽

High Risk ◽

Squamous Cell ◽

Surrogate Marker ◽

Hpv Infection ◽

Hpv16 E6 ◽

Hpv Dna ◽

Molecular Virology ◽

Hpv E6 ◽

E6 And E7

Human papillomavirus (HPV) was proven to play a significant role in cancer development in the oropharynx. However, its role in the development of laryngeal (LSCC) and hypopharyngeal squamous cell carcinoma (HPSCC) remains to be clarified. High-risk HPV (HR-HPV) viral proteins E6 and E7 are considered to be pertinent to HPV-related carcinogenesis. Hence, our aim was to estimate LSCC and HPSCC for HR-HPV DNA, p16, and E6/E7 oncoprotein status by using molecular virology and immunohistochemistry methods. The prevalence of HPV16 infection was 22/41 (53.7%) and 20/31 (64.5%) for LSCC and HPSCC, accordingly. The majority of HPV16+ tumor samples were stage III or IV. In most samples, the presence of either HPV16 E6 or HPV16 E7 viral protein in dysplastic or tumor cells was confirmed using immunohistochemistry. Our results suggest a high prevalence of HPV16 as a primary HR-HPV type in LSCC and HPSCC. The lack of HPV E6/E7 oncoproteins in some tumor samples may suggest either the absence of viral integration or the presence of other mechanisms of tumorigenesis. The utilization of p16 IHC as a surrogate marker of HR-HPV infection is impractical in LSCC and HPSCC.

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Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor

Journal of Virology ◽

10.1128/jvi.02413-16 ◽

2017 ◽

Vol 91 (6) ◽

Cited By ~ 29

Author(s):

Seiichiro Mori ◽

Takamasa Takeuchi ◽

Yoshiyuki Ishii ◽

Takashi Yugawa ◽

Tohru Kiyono ◽

...

Keyword(s):

Transcription Factor ◽

Human Papillomavirus ◽

Molecular Mechanisms ◽

Cytidine Deaminase ◽

Ectopic Expression ◽

Human Keratinocytes ◽

Hek293 Cells ◽

Mrna Levels ◽

Hpv16 E6 ◽

Human Cancers

ABSTRACT The cytidine deaminase APOBEC3B (A3B) underlies the genetic heterogeneity of several human cancers, including cervical cancer, which is caused by human papillomavirus (HPV) infection. We previously identified a region within the A3B promoter that is activated by the viral protein HPV16 E6 in human keratinocytes. Here, we discovered three sites recognized by the TEAD family of transcription factors within this region of the A3B promoter. Reporter assays in HEK293 cells showed that exogenously expressed TEAD4 induced A3B promoter activation through binding to these sites. Normal immortalized human keratinocytes expressing E6 (NIKS-E6) displayed increased levels of TEAD1/4 protein compared to parental NIKS. A series of E6 mutants revealed that E6-mediated degradation of p53 was important for increasing TEAD4 levels. Knockdown of TEADs in NIKS-E6 significantly reduced A3B mRNA levels, whereas ectopic expression of TEAD4 in NIKS increased A3B mRNA levels. Finally, chromatin immunoprecipitation assays demonstrated increased levels of TEAD4 binding to the A3B promoter in NIKS-E6 compared to NIKS. Collectively, these results indicate that E6 induces upregulation of A3B through increased levels of TEADs, highlighting the importance of the TEAD-A3B axis in carcinogenesis. IMPORTANCE The expression of APOBEC3B (A3B), a cellular DNA cytidine deaminase, is upregulated in various human cancers and leaves characteristic, signature mutations in cancer genomes, suggesting that it plays a prominent role in carcinogenesis. Viral oncoproteins encoded by human papillomavirus (HPV) and polyomavirus have been reported to induce A3B expression, implying the involvement of A3B upregulation in virus-associated carcinogenesis. However, the molecular mechanisms causing A3B upregulation remain unclear. Here, we demonstrate that exogenous expression of the cellular transcription factor TEAD activates the A3B promoter. Further, the HPV oncoprotein E6 increases the levels of endogenous TEAD1/4 protein, thereby leading to A3B upregulation. Since increased levels of TEAD4 are frequently observed in many cancers, an understanding of the direct link between TEAD and A3B upregulation is of broad oncological interest.

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