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Yin W, Egawa N, Zheng K, Griffin H, Tian P, Aiyenuro A, Bornstein J, Doorbar J. HPV E6 inhibits E6AP to regulate epithelial homeostasis by modulating keratinocyte differentiation commitment and YAP1 activation. PLoS Pathog 2023; 19:e1011464. [PMID: 37379354 DOI: 10.1371/journal.ppat.1011464] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2022] [Accepted: 06/05/2023] [Indexed: 06/30/2023] Open
Abstract
Human papillomaviruses (HPV) cause persistent infections by modulating epithelial homeostasis in cells of the infected basal layer. Using FUCCI and cell-cell competition assays, we have identifed regulatory roles for E6AP and NHERF1, which are the primary HPV11 E6 cellular targets, as well as being targets of the high-risk E6 proteins, in processes governing epithelial homeostasis (i.e. cell density, cell cycle entry, commitment to differentiation and basal layer delamination). Depletion of E6AP, or expression of HPV11 or 16E6 increased keratinocyte cell density and cell cycle activity, and delayed the onset of differentiation; phenotypes which were conspicuously present in HPV11 and 16 infected patient tissue. In line with proposed E6 functions, in HPV11 condyloma tissue, E6AP and NHERF1 were significantly reduced when compared to uninfected epithelium. In experimental systems, loss of HPV11 E6/E6AP binding abolished 11E6's homeostasis regulatory functions, while loss of E6/NHERF1 binding reduced the cell density threshold at which differentiation was triggered. By contrast, a NHERF1-binding mutant of 16E6 was not compromised in its homeostasis functions, while E6AP appeared essential. RNA sequencing revealed similar transcriptional profiles in both 11 and 16E6-expressing cells and E6AP-/- cells, with YAP target genes induced, and keratinocyte differentiation genes being downregulated. HPV11 E6-mediated Yap activation was observed in 2D and 3D (organotypic raft) cell culture systems and HPV-infected lesions, with both NHERF1, which is a regulator of the Hippo and Wnt pathways, and E6AP, playing an important role. As the conserved binding partner of Alpha group HPV E6 proteins, the precise role of E6AP in modulating keratinocyte phenotype and associated signalling pathways has not previously been defined. Our study suggests a model in which the preserved functions of the low and high-risk Alpha E6 proteins modulate epithelial homeostasis via E6AP activity, and lead to alteration of multiple downstream pathways, including those involving NHERF1 and YAP.
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Affiliation(s)
- Wen Yin
- Department of Pathology, University of Cambridge, Cambridge, United Kingdom
| | - Nagayasu Egawa
- Department of Pathology, University of Cambridge, Cambridge, United Kingdom
| | - Ke Zheng
- Department of Pathology, University of Cambridge, Cambridge, United Kingdom
| | - Heather Griffin
- Department of Pathology, University of Cambridge, Cambridge, United Kingdom
| | - Pu Tian
- Department of Pathology, University of Cambridge, Cambridge, United Kingdom
| | - Ademola Aiyenuro
- Department of Pathology, University of Cambridge, Cambridge, United Kingdom
| | - Jacob Bornstein
- Gynecologist & Obstetrician, Colposcopy, Azrieli Faculty of Medicine of Bar-Ilan University, and Galilee Medical Center-Nahariya
| | - John Doorbar
- Department of Pathology, University of Cambridge, Cambridge, United Kingdom
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Prati B, da Silva Abjaude W, Termini L, Morale M, Herbster S, Longatto-Filho A, Nunes RAL, Córdoba Camacho LC, Rabelo-Santos SH, Zeferino LC, Aguayo F, Boccardo E. Three Prime Repair Exonuclease 1 (TREX1) expression correlates with cervical cancer cells growth in vitro and disease progression in vivo. Sci Rep 2019; 9:351. [PMID: 30674977 PMCID: PMC6344518 DOI: 10.1038/s41598-018-37064-x] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2017] [Accepted: 10/17/2018] [Indexed: 12/17/2022] Open
Abstract
Alterations in specific DNA damage repair mechanisms in the presence of human papillomavirus (HPV) infection have been described in different experimental models. However, the global effect of HPV on the expression of genes involved in these pathways has not been analyzed in detail. In the present study, we compared the expression profile of 135 genes involved in DNA damage repair among primary human keratinocytes (PHK), HPV-positive (SiHa and HeLa) and HPV-negative (C33A) cervical cancer derived cell lines. We identified 9 genes which expression pattern distinguishes HPV-positive tumor cell lines from C33A. Moreover, we observed that Three Prime Repair Exonuclease 1 (TREX1) expression is upregulated exclusively in HPV-transformed cell lines and PHK expressing HPV16 E6 and E7 oncogenes. We demonstrated that TREX1 silencing greatly affects tumor cells clonogenic and anchorage independent growth potential. We showed that this effect is associated with p53 upregulation, accumulation of subG1 cells, and requires the expression of E7 from high-risk HPV types. Finally, we observed an increase in TREX1 levels in precancerous lesions, squamous carcinomas and adenocarcinomas clinical samples. Altogether, our results indicate that TREX1 upregulation is important for cervical tumor cells growth and may contribute with tumor establishment and progression.
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Affiliation(s)
- Bruna Prati
- Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo (USP), Av. Prof. Lineu Prestes 1374, 05508-900, São Paulo, SP, Brazil
| | - Walason da Silva Abjaude
- Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo (USP), Av. Prof. Lineu Prestes 1374, 05508-900, São Paulo, SP, Brazil
| | - Lara Termini
- Centro de Investigação Translacional em Oncologia (LIM24), Instituto do Câncer do Estado de São Paulo (ICESP), São Paulo, Brazil
| | - Mirian Morale
- Department of Biochemistry, Institute of Chemistry, USP, São Paulo, Brazil
| | - Suellen Herbster
- Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo (USP), Av. Prof. Lineu Prestes 1374, 05508-900, São Paulo, SP, Brazil
| | - Adhemar Longatto-Filho
- Laboratory of Medical Investigation (LIM 14), Department of Pathology, School of Medicine, USP, Av. Dr. Arnaldo 455, São Paulo, 01246-903, Brazil.,Life and Health Sciences Research Institute, School of Health Sciences, ICVS/3B's - PT Government Associate Laboratory, University of Minho, Braga, Guimarães, Portugal.,Molecular Oncology Research Center, Barretos Cancer Hospital, Pio XII Foundation, Barretos, Rua Antenor Duarte Villela, 1331, Barretos, 14784-400, Brazil
| | - Rafaella Almeida Lima Nunes
- Centro de Investigação Translacional em Oncologia (LIM24), Instituto do Câncer do Estado de São Paulo (ICESP), São Paulo, Brazil
| | - Lizeth Carolina Córdoba Camacho
- Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo (USP), Av. Prof. Lineu Prestes 1374, 05508-900, São Paulo, SP, Brazil.,Laboratório de Oncologia Experimental, Departamento de Radiologia, Faculdade de Medicina, USP, São Paulo, SP, Brazil.,Centro de Investigação Translacional em Oncologia, ICESP, São Paulo, SP, Brazil
| | | | - Luiz Carlos Zeferino
- School of Medical Sciences, State University of Campinas (UNICAMP), Rua Alexander Fleming 101, 13083-881, Campinas, SP, Brazil
| | - Francisco Aguayo
- Basic and Clinical Oncology Department, Faculty of Medicine, University of Chile, Santiago, Chile.,Advanced Center for Chronic Diseases (ACCDiS), Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Enrique Boccardo
- Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo (USP), Av. Prof. Lineu Prestes 1374, 05508-900, São Paulo, SP, Brazil.
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Prati B, Marangoni B, Boccardo E. Human papillomavirus and genome instability: from productive infection to cancer. Clinics (Sao Paulo) 2018; 73:e539s. [PMID: 30208168 PMCID: PMC6113919 DOI: 10.6061/clinics/2018/e539s] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/27/2017] [Accepted: 05/16/2018] [Indexed: 12/29/2022] Open
Abstract
Infection with high oncogenic risk human papillomavirus types is the etiological factor of cervical cancer and a major cause of other epithelial malignancies, including vulvar, vaginal, anal, penile and head and neck carcinomas. These agents affect epithelial homeostasis through the expression of specific proteins that deregulate important cellular signaling pathways to achieve efficient viral replication. Among the major targets of viral proteins are components of the DNA damage detection and repair machinery. The activation of many of these cellular factors is critical to process viral genome replication intermediates and, consequently, to sustain faithful viral progeny production. In addition to the important role of cellular DNA repair machinery in the infective human papillomavirus cycle, alterations in the expression and activity of many of its components are observed in human papillomavirus-related tumors. Several studies from different laboratories have reported the impact of the expression of human papillomavirus oncogenes, mainly E6 and E7, on proteins in almost all the main cellular DNA repair mechanisms. This has direct consequences on cellular transformation since it causes the accumulation of point mutations, insertions and deletions of short nucleotide stretches, as well as numerical and structural chromosomal alterations characteristic of tumor cells. On the other hand, it is clear that human papillomavirus-transformed cells depend on the preservation of a basal cellular DNA repair activity level to maintain tumor cell viability. In this review, we summarize the data concerning the effect of human papillomavirus infection on DNA repair mechanisms. In addition, we discuss the potential of exploiting human papillomavirus-transformed cell dependency on DNA repair pathways as effective antitumoral therapies.
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Affiliation(s)
- Bruna Prati
- Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo, SP, BR
| | - Bruna Marangoni
- Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo, SP, BR
| | - Enrique Boccardo
- Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo, SP, BR
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Ganti K, Broniarczyk J, Manoubi W, Massimi P, Mittal S, Pim D, Szalmas A, Thatte J, Thomas M, Tomaić V, Banks L. The Human Papillomavirus E6 PDZ Binding Motif: From Life Cycle to Malignancy. Viruses 2015; 7:3530-51. [PMID: 26147797 PMCID: PMC4517114 DOI: 10.3390/v7072785] [Citation(s) in RCA: 108] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2015] [Revised: 06/24/2015] [Accepted: 06/25/2015] [Indexed: 01/01/2023] Open
Abstract
Cancer-causing HPV E6 oncoproteins are characterized by the presence of a PDZ binding motif (PBM) at their extreme carboxy terminus. It was long thought that this region of E6 had a sole function to confer interaction with a defined set of cellular substrates. However, more recent studies have shown that the E6 PBM has a complex pattern of regulation, whereby phosphorylation within the PBM can regulate interaction with two classes of cellular proteins: those containing PDZ domains and the members of the 14-3-3 family of proteins. In this review, we explore the roles that the PBM and its ligands play in the virus life cycle, and subsequently how these can inadvertently contribute towards the development of malignancy. We also explore how subtle alterations in cellular signal transduction pathways might result in aberrant E6 phosphorylation, which in turn might contribute towards disease progression.
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Affiliation(s)
- Ketaki Ganti
- International Center for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, Trieste 34149, Italy.
| | - Justyna Broniarczyk
- International Center for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, Trieste 34149, Italy.
| | - Wiem Manoubi
- International Center for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, Trieste 34149, Italy.
| | - Paola Massimi
- International Center for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, Trieste 34149, Italy.
| | - Suruchi Mittal
- International Center for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, Trieste 34149, Italy.
| | - David Pim
- International Center for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, Trieste 34149, Italy.
| | - Anita Szalmas
- International Center for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, Trieste 34149, Italy.
| | - Jayashree Thatte
- International Center for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, Trieste 34149, Italy.
| | - Miranda Thomas
- International Center for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, Trieste 34149, Italy.
| | - Vjekoslav Tomaić
- International Center for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, Trieste 34149, Italy.
| | - Lawrence Banks
- International Center for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, Trieste 34149, Italy.
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Turunen A, Syrjänen S. Extracellular calcium regulates keratinocyte proliferation and HPV 16 E6 RNA expression in vitro. APMIS 2014; 122:781-9. [PMID: 25295350 PMCID: PMC4282442 DOI: 10.1111/apm.12227] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/03/2022]
Abstract
Human papillomaviruses (HPV) are known to immortalize oral keratinocytes in vitro, but the underlying mechanisms causing the following resistance to differentiation remain unclear. We investigated the effect of extracellular calcium on the proliferation of HPV16-positive keratinocytes and on the mRNA expression of the viral E6-oncogene. HPV16-positive hypopharyngeal carcinoma cells (UD-SCC-2), spontaneously immortalized- (HMK) and HPV16 E6/E7-immortalized human gingival keratinocytes (IHGK) were grown for 3, 6 and 9 days in Keratinocyte Serum-free Medium with calcium concentrations ranging from 0 mM to 6 mM. Calcium concentrations up to 0.09 mM increased cellular proliferation, which decreased at higher concentrations. A shift of calcium concentration from 0 to 4 mM increased E6 expression in UD-SCC-2 cells 2.4-fold by day 9. Simultaneously, E2 expression increased. The most significant upregulation of E6 and E2 expressions was observed at day 9, grown in high-calcium media and the increase in E6 expression coincided with an increase in involucrin expression, likely indicating cell differentiation. Despite this, HPV-positive cells continued to proliferate even at high-calcium media in contrast to HPV-negative cells. Overexpression of E6 mRNA may be an important feature of HPV16-positive cells to resist the natural calcium gradient in differentiating keratinocytes allowing cell proliferation.
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Affiliation(s)
- Aaro Turunen
- Department of Oral Pathology, Institute of Dentistry, University of TurkuTurku, Finland
- Aaro Turunen, Department of Oral Pathology, Institute of Dentistry, Lemminkäisenkatu 2, 20520 Turku, Finland. e-mail:
| | - Stina Syrjänen
- Department of Oral Pathology, Institute of Dentistry, University of TurkuTurku, Finland
- Department of Pathology, Turku University HospitalTurku, Finland
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Manzo-Merino J, Thomas M, Fuentes-Gonzalez AM, Lizano M, Banks L. HPV E6 oncoprotein as a potential therapeutic target in HPV related cancers. Expert Opin Ther Targets 2013; 17:1357-68. [DOI: 10.1517/14728222.2013.832204] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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7
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Vande Pol SB, Klingelhutz AJ. Papillomavirus E6 oncoproteins. Virology 2013; 445:115-37. [PMID: 23711382 DOI: 10.1016/j.virol.2013.04.026] [Citation(s) in RCA: 262] [Impact Index Per Article: 21.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2013] [Revised: 04/22/2013] [Accepted: 04/25/2013] [Indexed: 02/07/2023]
Abstract
Papillomaviruses induce benign and malignant epithelial tumors, and the viral E6 oncoprotein is essential for full transformation. E6 contributes to transformation by associating with cellular proteins, docking on specific acidic LXXLL peptide motifs found on these proteins. This review examines insights from recent studies of human and animal E6 proteins that determine the three-dimensional structure of E6 when bound to acidic LXXLL peptides. The structure of E6 is related to recent advances in the purification and identification of E6 associated protein complexes. These E6 protein-complexes, together with other proteins that bind to E6, alter a broad array of biological outcomes including modulation of cell survival, cellular transcription, host cell differentiation, growth factor dependence, DNA damage responses, and cell cycle progression.
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Affiliation(s)
- Scott B Vande Pol
- Department of Pathology, University of Virginia, Charlottesville, VA 22901, USA.
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8
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Bononi I, Bosi S, Bonaccorsi G, Marci R, Patella A, Ferretti S, Tognon M, Garutti P, Martini F. Establishment of keratinocyte colonies from small-sized cervical intraepithelial neoplasia specimens. J Cell Physiol 2012; 227:3787-95. [PMID: 22392909 DOI: 10.1002/jcp.24088] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The size of human cervical intraepithelial neoplasia (CIN) biopsies is usually very small and standard methods do not allow an adequate number of keratinocytes to be isolated for culturing purposes. In this study, a new approach to establish keratinocyte cultures from small CIN a tissue fragments was developed. Neoplastic specimens and corresponding normal tissues, which were used as controls, were digested with collagenase. Tissue-derived fibroblasts and keratinocytes were co-cultured in calcium and serum medium. Single keratinocyte colonies from primary cultures were expanded using a culture medium optimized in our laboratory. Primary keratinocyte colonies, as well as expanded colonies, were tested for epithelial and cervical markers such as 5, 14, 17, and 19 keratins, and p63 by immunofluorescence. Our results indicate that a variable number of primary keratinocyte colonies could be detected in neoplastic cultures, depending on the grade of cervical lesions from which the colonies originated. Single colonies, when cultured with our new medium, grew at a high rate with uniform size and morphology for some passages. Epithelial and p63 markers were expressed in keratinocyte colonies, as well as in expanded colonies. In conclusion, our study reports a rapid and easy culturing system which enables keratinocyte colonies from minute cervical tumor tissues to be obtained. Moreover, using the new culture medium, keratinocyte colonies can be expanded at a high proliferative rate.
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Affiliation(s)
- Ilaria Bononi
- Section of Cell Biology and Molecular Genetics, Department of Morphology and Embryology, University of Ferrara, Ferrara, Italy
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9
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p53 acts as a co-repressor to regulate keratin 14 expression during epidermal cell differentiation. PLoS One 2012; 7:e41742. [PMID: 22911849 PMCID: PMC3404013 DOI: 10.1371/journal.pone.0041742] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2012] [Accepted: 06/27/2012] [Indexed: 11/22/2022] Open
Abstract
During epidermal cell differentiation, keratin 14 (K14) expression is down-regulated, p53 expression varies, and the expression of the p53 target genes, p21 and 14-3-3σ, increases. These trends suggest that the relative transcriptional activity of p53 is increased during epidermal cell differentiation. To determine the relationship between K14 and p53, we constructed K14 promoters of various sizes and found that wild-type p53 could repress the promoter activity of all of the K14 promoter constructs in H1299 cells. K14-p160 contains an SP1 binding site mutation that prevents p53 from repressing K14 expression. Using a DNA affinity precipitation assay, we confirmed that p53 forms a complex with SP1 at the SP1 binding site between nucleotides -48 and -43 on the K14 promoter. Thus, our data indicate that p53 acts as a co-repressor to down-regulate K14 expression by binding to SP1. Next, we used a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal cell differentiation model to examine the inhibition of K14 expression caused by increased p53 activity. Human ovarian teratocarcinoma C9 cells were treated with TPA to induce differentiation. Over-expression of the dominant negative p53 mutant ΔTAp53, which inhibits p53 activity, prevented the TPA-induced K14 down-regulation in C9 cells. Furthermore, treatment of normal primary human foreskin keratinocytes (PHFK) with the p53 inhibitor pifithrin-α (PFT-α) showed that the inhibition of p53 activity relieves K14 repression during epidermal cell differentiation. Finally, we found that TPA induces the phosphorylation of p53 at residue 378, which enhances the affinity of p53 to bind to Sp1 and repress K14 expression.
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10
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Cannavo I, Benchetrit M, Loubatier C, Michel G, Lemichez E, Giordanengo V. Characterization of a cluster of oncogenic mutations in E6 of a human papillomavirus 83 variant isolated from a high-grade squamous intraepithelial lesion. J Gen Virol 2011; 92:2428-2436. [PMID: 21752964 DOI: 10.1099/vir.0.032888-0] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
We previously isolated human papillomavirus 83 (HPV83m) from a cervical smear. Sequence analysis of E6 and E7 proteins highlighted five mutations located in the second putative zinc-finger region of E6 (E6m), an important domain for protein-protein or protein-DNA interactions. Here, we show that E6m of HPV83m can trigger human primary cell proliferation and anchorage-independent growth properties, similarly to E6 of HPV16, a high-risk HPV (HR-HPV). Interestingly, we demonstrate that, in contrast to E6 of HPV16, E6m corrupts neither p53 stability nor telomerase activity, but acts as a specific modulator of the transcriptional machinery. By studying E6m reversion mutants, we confirmed the importance of the second zinc-finger domain in triggering the observed upregulation of cell growth and of the transcriptional machinery. Reversion of these mutations in E6m (to yield strain E6r) fully abolished the oncogenic potential of E6m, transforming the phenotype of E6 from a high-risk to a low-risk phenotype. Importantly, our data define the importance of a cluster of mutations in the second zinc finger of E6m in increasing the oncogenic potential of HPV83.
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Affiliation(s)
- Isabelle Cannavo
- INSERM, U895, Centre Méditerranéen de Médecine Moléculaire, C3M, Toxines Microbiennes dans la relation hote pathogènes, Nice, France
- Université de Nice-Sophia-Antipolis, UFR Médecine, Nice, France
- Laboratoire de Virologie, Centre Hospitalier Universitaire de Nice, Hôpital Archet 2, Nice, France
| | - Maxime Benchetrit
- Université de Nice-Sophia-Antipolis, UFR Médecine, Nice, France
- Laboratoire d'Anatomo-Pathologie, Centre Hospitalier Universitaire de Nice, Hôpital Archet 2, Nice, France
| | - Céline Loubatier
- INSERM, U895, Centre Méditerranéen de Médecine Moléculaire, C3M, Toxines Microbiennes dans la relation hote pathogènes, Nice, France
- Université de Nice-Sophia-Antipolis, UFR Médecine, Nice, France
| | - Gregory Michel
- INSERM, U895, Centre Méditerranéen de Médecine Moléculaire, C3M, Toxines Microbiennes dans la relation hote pathogènes, Nice, France
| | - Emmanuel Lemichez
- INSERM, U895, Centre Méditerranéen de Médecine Moléculaire, C3M, Toxines Microbiennes dans la relation hote pathogènes, Nice, France
| | - Valérie Giordanengo
- INSERM, U895, Centre Méditerranéen de Médecine Moléculaire, C3M, Toxines Microbiennes dans la relation hote pathogènes, Nice, France
- Université de Nice-Sophia-Antipolis, UFR Médecine, Nice, France
- Laboratoire de Virologie, Centre Hospitalier Universitaire de Nice, Hôpital Archet 2, Nice, France
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Jabbar S, Strati K, Shin MK, Pitot HC, Lambert PF. Human papillomavirus type 16 E6 and E7 oncoproteins act synergistically to cause head and neck cancer in mice. Virology 2010; 407:60-7. [PMID: 20797753 DOI: 10.1016/j.virol.2010.08.003] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2010] [Revised: 05/10/2010] [Accepted: 08/02/2010] [Indexed: 12/11/2022]
Abstract
High-risk human papillomaviruses (HPVs) contribute to cervical and other anogenital cancers, and they are also linked etiologically to a subset of head and neck squamous cell carcinomas (HNSCC). We previously established a model for HPV-associated HNSCC in which we treated transgenic mice expressing the papillomaviral oncoproteins with the chemical carcinogen 4-nitroquinoline-1-oxide (4-NQO). We found that the HPV-16 E7 oncoprotein was highly potent in causing HNSCC, and its dominance masked any potential oncogenic contribution of E6, a second papillomaviral oncoprotein commonly expressed in human cancers. In the current study, we shortened the duration of treatment with 4-NQO to reduce the incidence of cancers and discovered a striking synergy between E6 and E7 in causing HNSCC. Comparing the oncogenic properties of wild-type versus mutant E6 genes in this model for HNSCC uncovered a role for some but not other cellular targets of E6 previously shown to contribute to cervical cancer.
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Affiliation(s)
- Sean Jabbar
- McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, 1400 University Avenue, Madison, WI 53706, USA
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12
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Sun L, Song Y, Tong T, Wu L, Zhang W, Zhan Q. Down-modulation of Notch1 expression in cervical cancer is associated with HPV-induced carcinogenesis. ACTA ACUST UNITED AC 2010. [DOI: 10.1007/s11805-009-0401-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
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13
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HPV16 E6 augments Wnt signaling in an E6AP-dependent manner. Virology 2009; 396:47-58. [PMID: 19896689 DOI: 10.1016/j.virol.2009.10.011] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2009] [Revised: 08/05/2009] [Accepted: 10/06/2009] [Indexed: 01/29/2023]
Abstract
In this study we investigated the effect of HPV16 E6 on the Wnt/beta-catenin oncogenic signaling pathway. Luciferase reporter assays indicated that ectopically expressed E6 significantly augmented the Wnt/beta-catenin/TCF-dependent signaling response in a dose-dependent manner. This activity was independent of the ability of E6 to target p53 for degradation or bind to the PDZ-containing E6 targets. Epistasis experiments suggested that the stimulatory effect is independent of GSK3beta or APC. Coexpression, half-life determination, cell fractionation and immunofluorescence analyses indicated that E6 did not alter the expression levels, stability or cellular distribution of beta-catenin. Further experiments using E6 mutants defective for E6AP binding and E6AP knockdown cells indicated the absolute requirement of the ubiquitin ligase E6AP for enhancement of the Wnt signal by E6. Thus, this study suggests a role for the E6/E6AP complex in augmentation of the Wnt signaling pathway which may contribute to HPV induced carcinogenesis.
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14
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Honoré B. The rapidly expanding CREC protein family: members, localization, function, and role in disease. Bioessays 2009; 31:262-77. [PMID: 19260022 DOI: 10.1002/bies.200800186] [Citation(s) in RCA: 108] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Although many aspects of the physiological and pathophysiological mechanisms remain unknown, recent advances in our knowledge suggest that the CREC proteins are promising disease biomarkers or targets for therapeutic intervention in a variety of diseases. The CREC family of low affinity, Ca2+-binding, multiple EF-hand proteins are encoded by five genes, RCN1, RCN2, RCN3, SDF4, and CALU, resulting in reticulocalbin, ER Ca2+-binding protein of 55 kDa (ERC-55), reticulocalbin-3, Ca2+-binding protein of 45 kDa (Cab45), and calumenin. Alternative splicing increases the number of gene products. The proteins are localized in the cytosol, in various parts of the secretory pathway, secreted to the extracellular space or localized on the cell surface. The emerging functions appear to be highly diverse. The proteins interact with several different ligands. Rather well-described functions are attached to calumenin with the inhibition of several proteins in the endoplasmic or sarcoplasmic reticulum membrane, the vitamin K(1) 2,3-epoxide reductase, the gamma-carboxylase, the ryanodine receptor, and the Ca2+-transporting ATPase. Other functions concern participation in the secretory process, chaperone activity, signal transduction as well as participation in a large variety of disease processes.
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Affiliation(s)
- Bent Honoré
- Department of Medical Biochemistry, Aarhus University, Aarhus C, Denmark.
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15
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Yugawa T, Kiyono T. Molecular mechanisms of cervical carcinogenesis by high-risk human papillomaviruses: novel functions of E6 and E7 oncoproteins. Rev Med Virol 2009; 19:97-113. [PMID: 19156753 DOI: 10.1002/rmv.605] [Citation(s) in RCA: 155] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Over the last two decades, since the initial discovery of human papillomavirus (HPV) type 16 and 18 DNAs in cervical cancers by Dr. Harald zur Hausen (winner of the Nobel Prize in Physiology or Medicine, 2008), the HPVs have been well characterised as causative agents for cervical cancer. Viral DNA from a specific group of HPVs can be detected in at least 90% of all cervical cancers and two viral genes, E6 and E7, are invariably expressed in HPV-positive cervical cancer cells. Their gene products are known to inactivate the major tumour suppressors, p53 and retinoblastoma protein (pRB), respectively. In addition, one function of E6 is to activate telomerase, and E6 and E7 cooperate to effectively immortalise human primary epithelial cells. Though expression of E6 and E7 is itself not sufficient for cancer development, it seems to be either directly or indirectly involved in every stage of multi-step carcinogenesis. Epidemiological and biological studies suggest the potential efficacy of prophylactic vaccines to prevent genital HPV infection as an anti-cancer strategy. However, given the widespread nature of HPV infection and unresolved issues about the duration and type specificity of the currently available HPV vaccines, it is crucial that molecular details of the natural history of HPV infection as well as the biological activities of the viral oncoproteins be elucidated in order to provide the basis for development of new therapeutic strategies against HPV-associated malignancies. This review highlights novel functions of E6 and E7 as well as the molecular mechanisms of HPV-induced carcinogenesis.
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Affiliation(s)
- Takashi Yugawa
- Virology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
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16
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Epshtein A, Jackman A, Gonen P, Sherman L. HPV16 E6 oncoprotein increases cell adhesion in human keratinocytes. Arch Virol 2008; 154:55-63. [PMID: 19066713 DOI: 10.1007/s00705-008-0273-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2008] [Accepted: 11/06/2008] [Indexed: 11/28/2022]
Abstract
Expression of the E6 oncoprotein of human papillomavirus (HPV) 16 in primary human keratinocytes (PHKs) was previously shown to significantly reduce apoptosis. This could be due to increased cell adhesion. Adhesion ability was tested by seeding cells on tissue culture dishes coated with different concentrations of poly(HEME) and determination of the proportion of attached cells. Assays were carried out with PHKs, immortalized human keratinocytes (HaCaT) and human 293T cells. The E6 gene was transduced via retroviral infection or DNA transfection. Results of these assays showed that expression of E6 increased the proportion of cells that attached to poly(HEME). Several HPV16 E6 mutants were also tested in the above assay in 293T cells. These assays showed that the p53 targeting region of E6 is dispensable for this activity. Assays of inhibition of tyrosine kinases by bombesin showed that E6 probably utilizes other pathways to increase cell adhesion.
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Affiliation(s)
- Alexander Epshtein
- Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
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17
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Narisawa-Saito M, Yoshimatsu Y, Ohno SI, Yugawa T, Egawa N, Fujita M, Hirohashi S, Kiyono T. An In vitro Multistep Carcinogenesis Model for Human Cervical Cancer. Cancer Res 2008; 68:5699-705. [DOI: 10.1158/0008-5472.can-07-6862] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
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18
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Mammas IN, Sourvinos G, Giannoudis A, Spandidos DA. Human papilloma virus (HPV) and host cellular interactions. Pathol Oncol Res 2008; 14:345-54. [PMID: 18493868 DOI: 10.1007/s12253-008-9056-6] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/05/2008] [Accepted: 04/17/2008] [Indexed: 12/17/2022]
Abstract
Viral-induced carcinogenesis has been attributed to the ability of viral oncoproteins to target and interact with the host cellular proteins. It is generally accepted that Human papilloma virus (HPV) E6 and E7 function as the dominant oncoproteins of 'high-risk' HPVs by altering the function of critical cellular proteins. Initially it was shown that HPV E6 enhances the degradation of p53, while HPV E7 inactivates the function of the retinoblastoma tumor suppressor protein Rb. However, recent studies during the last decade have identified a number of additional host cellular targets of both HPV E6 and E7 that may also play an important role in malignant cellular transformation. In this review we present the interactions of HPV E6 and E7 with the host cellular target proteins. We also present the role of DNA integration in the malignant transformation of the epithelial cell.
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Affiliation(s)
- Ioannis N Mammas
- Department of Virology, School of Medicine, University of Crete, Heraklion, 71100, Crete, Greece
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19
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HPV E7 contributes to the telomerase activity of immortalized and tumorigenic cells and augments E6-induced hTERT promoter function. Virology 2008; 375:611-23. [PMID: 18367227 DOI: 10.1016/j.virol.2008.02.025] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2008] [Revised: 02/01/2008] [Accepted: 02/14/2008] [Indexed: 01/31/2023]
Abstract
The E6 and E7 proteins of high-risk HPVs are both required for the immortalization of primary human keratinocytes and the maintenance of the malignant phenotype of HPV-positive cancer cell lines. Our previous studies have shown that E6 protein binds Myc protein and that both E6 and Myc associate with and cooperatively activate the hTERT promoter, thereby increasing cellular telomerase activity. In this study, we evaluated the role of E7 in the maintenance and activation of telomerase in immortalized and tumorigenic cells. siRNA knockdown of either E6 or E7 (or both) in HPV-immortalized cells or an HPV-positive cancer cell line reduced hTERT transcription and telomerase activity. Since telomerase was inhibited by E7 siRNA in cells that independently expressed the E6 and E7 genes, our results reveal an independent role for E7 in the maintenance of telomerase activity. However, E7 alone was insufficient to increase endogenous hTERT mRNA or telomerase activity, although it significantly augmented E6-induced hTERT transcription and telomerase activity. To further explore this apparent E7-induced promoter augmentation, we analyzed an exogenous hTERT core promoter in transduced keratinocytes. E7 alone induced the wt hTERT promoter and augmented E6-induced hTERT promoter activity. Mutation of the E2F site in the hTERT promoter abrogated the ability of E7 to induce the hTERT promoter or to enhance the ability of E6 to induce the promoter. Correspondingly, keratinocytes expressing E6 and a mutant E7 (defective for binding pRb pocket proteins) showed lower telomerase activity than cells expressing wt E6 and wt E7. Thus, HPV E7 plays a role in the maintenance of telomerase activity in stable cell lines and augments acute, E6-induced hTERT promoter activity.
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20
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Yugawa T, Handa K, Narisawa-Saito M, Ohno SI, Fujita M, Kiyono T. Regulation of Notch1 gene expression by p53 in epithelial cells. Mol Cell Biol 2007; 27:3732-42. [PMID: 17353266 PMCID: PMC1899981 DOI: 10.1128/mcb.02119-06] [Citation(s) in RCA: 157] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
The E6 protein of cervical cancer-associated human papillomaviruses (HPVs) is known to suppress keratinocyte differentiation through unidentified mechanisms. Notch1 is a determinant of keratinocyte differentiation and functions as a tumor suppressor in mammalian epidermis. Here, we report that the Notch1 gene is a novel target of p53 and can be down-regulated by E6 through p53 degradation in normal human epithelial cells. Thus, inactivation of p53 by E6 or short-hairpin RNA (shRNA) resulted in reduced Notch1 expression at the transcription level, and a p53-responsive element could be identified in the Notch1 promoter. The expression of E6, p53 shRNA, or Notch1 shRNA suppressed both spontaneous keratinocyte differentiation in culture and its induction upon DNA damage. Furthermore, the induction of Notch1 and differentiation makers as well as thickening of the epidermal layer upon UV irradiation was observed in wild-type but not in p53-deficient mouse skin. Together, our findings not only demonstrate a novel link between p53 and Notch1 in keratinocyte differentiation upon genotoxic stress but also suggest a novel tumor suppressor mechanism of p53 in the development of squamous cell carcinomas, including HPV-induced tumors.
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Affiliation(s)
- Takashi Yugawa
- Virology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
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21
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Lichtig H, Algrisi M, Botzer LE, Abadi T, Verbitzky Y, Jackman A, Tommasino M, Zehbe I, Sherman L. HPV16 E6 natural variants exhibit different activities in functional assays relevant to the carcinogenic potential of E6. Virology 2006; 350:216-27. [PMID: 16519914 DOI: 10.1016/j.virol.2006.01.038] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2005] [Revised: 01/09/2006] [Accepted: 01/26/2006] [Indexed: 01/16/2023]
Abstract
Genetic studies have revealed natural amino acid variations within the human papillomavirus (HPV) type 16 E6 oncoprotein. To address the functional significance of E6 polymorphisms, 10 HPV16 E6 variants isolated from cervical lesions of Swedish women were evaluated for their activities in different in vitro and in vivo assays relevant to the carcinogenic potential of E6. Small differences between E6 prototype and variants, and among variants, were observed in transient expression assays that assessed p53 degradation, Bax degradation, and inhibition of p53 transactivation. More variable levels of activities were exhibited by the E6 proteins in assays that evaluated binding to the E6-binding protein (E6BP) or the human discs large protein (hDlg). The E6 prototype expressed moderate to high activity in the above assays. The L83V polymorphism, previously associated with risk for cancer progression in some populations, expressed similar levels of activity as that of the E6 prototype in most functional assays. On the other hand, L83V displayed more efficient degradation of Bax and binding to E6BP, but lower binding to hDlg. Results of this study indicate that naturally occurring amino acid variations in HPV16 E6 can alter activities of the protein important for its carcinogenic potential.
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Affiliation(s)
- Hava Lichtig
- Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel, and Thunder Bay Regional Health Sciences Centre, Regional Cancer Care Program, Ontario, Canada
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22
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Black AF, Bouez C, Perrier E, Schlotmann K, Chapuis F, Damour O. Optimization and Characterization of an Engineered Human Skin Equivalent. ACTA ACUST UNITED AC 2005; 11:723-33. [PMID: 15998214 DOI: 10.1089/ten.2005.11.723] [Citation(s) in RCA: 99] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Skin equivalents (SEs) have been designed to meet both basic and applied research needs. The successful application of tissue-engineered SEs requires that the reconstituted tissues be endowed with the correct organization and function. A large body of experimental evidence now supports the notion that the inducing effects of mesenchymal tissue on epithelial cell morphogenesis are mediated, at least in part, by extracellular matrix components in addition to cell-cell interactions. A coculture model including both fibroblasts and keratinocytes was used to study the effects of progressive serum reduction on epidermal differentiation, quality of dermal and dermal-epidermal junctions, and expression of extracellular matrix proteins. The cells were successively added to a dermal substrate composed of collagen, glycosaminoglycans, and chitosan. The main aim of this study was to optimize this model for pharmacotoxicological trials. Control skin equivalents were cultured with medium containing 10% serum throughout the production process. Serum content was reduced to 1 and 0% at the air-liquid interface and compared with control skin equivalents. First, we demonstrated that serum deprivation at the air-liquid interface improves keratinocyte terminal differentiation. Second, we showed that, in the absence of serum, the specific characteristics of the SE are maintained, including epidermal and dermal ultrastructure, the expression of major dermal extracellular matrix components (human collagen types I, III, and V, fibronectin, elastin, and fibrillin 1), and the dermal-epidermal junction (laminin, human type IV collagen, alpha6 integrin). Furthermore, our results indicate that coculture models using keratinocytes and fibroblasts have both morphological and functional properties required for biologically useful tissues.
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Affiliation(s)
- Annie F Black
- Banque de Tissus et Cellules HCL, Laboratoire des Substituts Cutanés CNRS UPR-412, Hôpital Edouard Herriot, Lyon, France
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23
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Magal SS, Jackman A, Ish-Shalom S, Botzer LE, Gonen P, Schlegel R, Sherman L. Downregulation of Bax mRNA expression and protein stability by the E6 protein of human papillomavirus 16. J Gen Virol 2005; 86:611-621. [PMID: 15722521 DOI: 10.1099/vir.0.80453-0] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
Previous studies have shown that human papillomavirus (HPV) 16 E6 inhibits apoptosis induced during terminal differentiation of primary human keratinocytes (PHKs) triggered by serum and calcium. E6 inhibition of apoptosis was accompanied with prolonged expression of Bcl-2 and reduced elevation of Bax levels. In the present study, the effect of E6 on Bax mRNA expression and protein stability was investigated. These studies indicate that stable E6 expression in differentiating keratinocytes reduced the steady-state levels of Bax mRNA and shortened the half-life of Bax protein. These results were confirmed in transiently transfected 293T cells where E6 degraded Bax in a dose-dependent manner. Bax degradation was also exhibited in Saos-2 cells that lack p53, indicating its p53 independence. E6 did not form complexes with Bax and did not induce Bax degradation in vitro under experimental conditions where p53 was degraded. Finally, E6 aa 120–132 were shown to be necessary for Bax destabilization and, more importantly, for abrogating the ability of Bax to induce cellular apoptosis, highlighting the functional consequences of the E6-induced alterations in Bax expression.
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Affiliation(s)
- Sharon Shnitman Magal
- Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel
| | - Anna Jackman
- Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel
| | - Shahar Ish-Shalom
- Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel
| | - Liat Edri Botzer
- Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel
| | - Pinhas Gonen
- Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel
| | - Richard Schlegel
- Department of Pathology, Georgetown University Medical School, Washington, DC 2007, USA
| | - Levana Sherman
- Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel
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24
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Abstract
Infection with human papillomaviruses is strongly associated with the development of multiple cancers including esophageal squamous cell carcinoma. The HPV E6 gene is essential for the oncogenic potential of HPV. The regulation of apoptosis by oncogene has been related to carcinogenesis closely; therefore, the modulation of E6 on cellular apoptosis has become a hot research topic recently. Inactivation of the pro-apoptotic tumor suppressor p53 by E6 is an important mechanism by which E6 promotes cell growth; it is expected that inactivation of p53 by E6 should lead to a reduction in cellular apoptosis, numerous studies showed that E6 could in fact sensitize cells to apoptosis. The molecular basis for apoptosis modulation by E6 is poorly understood. In this article, we will present an overview of observations and current understanding of molecular basis for E6-induced apoptosis.
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Affiliation(s)
- Ting-Ting Li
- Institute of Gastroenterology, 15 West Changle Road, Xijing Hospital Xi'an 710032, Shaanxi Province, China
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25
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Tao M, Kruhlak M, Xia S, Androphy E, Zheng ZM. Signals that dictate nuclear localization of human papillomavirus type 16 oncoprotein E6 in living cells. J Virol 2004; 77:13232-47. [PMID: 14645580 PMCID: PMC296047 DOI: 10.1128/jvi.77.24.13232-13247.2003] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Human papillomavirus (HPV) type 16 E6 (16E6) is an oncogenic, multifunctional nuclear protein that induces p53 degradation and perturbs normal cell cycle control, leading to immortalization and transformation of infected keratinocytes and epithelial cells. Although it is unclear how 16E6 disrupts the epigenetic profile of host genes, its presence in the nucleus is a key feature. The present report describes intrinsic properties of 16E6 that influence its nuclear import in living cells. When the coding region of full-length 16E6 was inserted in frame into the C terminus of green fluorescent protein (GFP), it effectively prevented the 16E6 pre-mRNA from being spliced and led to the expression of a GFP-E6 fusion which localized predominantly to the nucleus. Further studies identified three novel nuclear localization signals (NLSs) in 16E6 that drive the protein to accumulate in the nucleus. We found that all three NLS sequences are rich in positively charged basic residues and that point mutations in these key residues could abolish the retention of 16E6 in the nucleus as well as the p53 degradation and cell immortalization activities of the protein. When inserted into corresponding regions of low-risk HPV type 6 E6, the three NLS sequences described for 16E6 functioned actively in converting the normally cytoplasmic HPV type 6 E6 into a nuclear protein. The separate NLS sequences, however, appear to play different roles in nuclear import and retention of HPV E6. The discovery of three unique NLS sequences in 16E6 provides new insights into the nuclear association of 16E6 which may reveal other novel activities of this important oncogenic protein.
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Affiliation(s)
- Mingfang Tao
- HIV and AIDS Malignancy Branch. Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA
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