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Li K, Huang F, Zhang T, Yang F, Duan W, Chen S, Hu T, Huang X. HPV integration status conversion and CIN2 + cancer risk stratification based on HPV integration levels among HPV integration-positive women: a 1-year follow-up study. BMC Cancer 2025; 25:885. [PMID: 40383798 PMCID: PMC12087150 DOI: 10.1186/s12885-025-14138-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2024] [Accepted: 04/11/2025] [Indexed: 05/20/2025] Open
Abstract
BACKGROUND HPV integration is a crucial genetic step in cervical carcinogenesis and the level of HPV integration increases with the grade of precancerous lesion. This study aimed to conduct risk stratification based on HPV integration levels and HPV integration status conversion among HPV integration-positive women after 1 year of follow-up. METHODS This prospective cohort study was conducted in Tongji Hospital between June 2020 and August 2022 and included 1297 consecutive HPV-positive women. The level of integration reads was stratified for risk assessment. RESULTS In total, 194 women were HPV integration-positive and followed for at least 1 year. The immediate risk of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) increased from 36.2% (25/69) among women with 6-20 integration reads to 93.8% (30/32) among women with more than 1000 integration reads (Ptrend < 0.001). The 1-year cumulative risk of CIN2 + increased from 39.1% (27/69) among women with 6-20 integration reads to 96.9% (31/32) among women with more than 1000 integration reads (Ptrend < 0.001). The 1-year cumulative risk of CIN2 + with HPV integration reads more than 40 was 93.8% (90/96), which was significantly higher than that of HPV integration reads less than 40 (38/85, P < 0.001). Among women with HPV integration reads more than 40, 99.0% (95/96) of women progressed with positive outcomes after one year of follow-up (persistent integration at the same site, immediate CIN2+, and 1-year CIN2+). The progression rate of women with persistent integration at the same site was 41.6% (5/12), which was significantly higher than those of HPV integration-negative conversion (0/41, 0%, P < 0.001). CONCLUSION The number of HPV integration reads may help CIN2 + risk stratification and facilitate the clinical management of high-risk patients.
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Affiliation(s)
- Kexin Li
- Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, China
| | - Fanwei Huang
- Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, China
| | - Tao Zhang
- Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, China
| | - Fan Yang
- New Technology Platform, Wuhan KDWS Biological Technology Co., Ltd., Wuhan, Hubei, 430074, China
| | - Weitao Duan
- New Technology Platform, Wuhan KDWS Biological Technology Co., Ltd., Wuhan, Hubei, 430074, China
| | - Shimin Chen
- New Technology Platform, Wuhan KDWS Biological Technology Co., Ltd., Wuhan, Hubei, 430074, China
| | - Ting Hu
- Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, China.
- Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Anv, Wuhan, 430030, China.
| | - Xiaoyuan Huang
- Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, China.
- Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Anv, Wuhan, 430030, China.
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Nakagawa T, Luebeck J, Zhu K, Lange JT, Sasik R, Phillips C, Sadat S, Javadzadeh S, Yang Q, Monther A, Fassardi S, Wang A, Pestonjamasp K, Rosenthal B, Fisch KM, Mischel P, Bafna V, Califano JA. Inhibition of human-HPV hybrid ecDNA enhancers reduces oncogene expression and tumor growth in oropharyngeal cancer. Nat Commun 2025; 16:2964. [PMID: 40140353 PMCID: PMC11947173 DOI: 10.1038/s41467-025-57447-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2024] [Accepted: 02/22/2025] [Indexed: 03/28/2025] Open
Abstract
Extrachromosomal circular DNA (ecDNA) has been found in most types of human cancers, and ecDNA incorporating viral genomes has recently been described, specifically in human papillomavirus (HPV)-mediated oropharyngeal cancer (OPC). However, the molecular mechanisms of human-viral hybrid ecDNA (hybrid ecDNA) for carcinogenesis remains elusive. We characterize the epigenetic status of hybrid ecDNA using HPVOPC cell lines and patient-derived tumor xenografts, identifying HPV oncogenes E6/E7 in hybrid ecDNA are flanked by previously unrecognized somatic DNA enhancers and HPV L1 enhancers, with strong cis-interactions. Targeting of these enhancers by clustered regularly interspaced short palindromic repeats interference or hybrid ecDNA by bromodomain and extra-terminal inhibitor reduces E6/E7 expression, and significantly inhibites in vitro and/or in vivo growth only in ecDNA(+) models. HPV DNA in hybrid ecDNA structures are associated with previously unrecognized somatic and HPV enhancers in hybrid ecDNA that drive HPV ongogene expression and carcinogenesis, and can be targeted with ecDNA disrupting therapeutics.
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Affiliation(s)
- Takuya Nakagawa
- Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA.
- Department of Otorhinolaryngology, Head and Neck Surgery, Chiba University, Graduate School of Medicine, Chiba, Japan.
- Health and Disease Omics Center, Chiba University, Chiba, Japan.
| | - Jens Luebeck
- Department of Computer Science and Engineering, UC San Diego, La Jolla, CA, USA
| | - Kaiyuan Zhu
- Department of Computer Science and Engineering, UC San Diego, La Jolla, CA, USA
| | - Joshua T Lange
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
| | - Roman Sasik
- Center for Computational Biology and Bioinformatics, University of California, San Diego, La Jolla, CA, USA
| | - Chad Phillips
- Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA
| | - Sayed Sadat
- Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA
| | - Sara Javadzadeh
- Department of Computer Science and Engineering, UC San Diego, La Jolla, CA, USA
| | - Qian Yang
- Department of Cellular and Molecular Medicine, Center for Epigenomics, University of California, San Diego, La Jolla, CA, USA
| | - Abdula Monther
- Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA
| | - Santiago Fassardi
- Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA
| | - Allen Wang
- Department of Cellular and Molecular Medicine, Center for Epigenomics, University of California, San Diego, La Jolla, CA, USA
| | - Kersi Pestonjamasp
- Cancer Center Microscopy Core, University of California, San Diego, La Jolla, CA, USA
| | - Brin Rosenthal
- Center for Computational Biology and Bioinformatics, University of California, San Diego, La Jolla, CA, USA
| | - Kathleen M Fisch
- Center for Computational Biology and Bioinformatics, University of California, San Diego, La Jolla, CA, USA
| | - Paul Mischel
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
| | - Vineet Bafna
- Department of Computer Science and Engineering, UC San Diego, La Jolla, CA, USA
| | - Joseph A Califano
- Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA.
- Department of Otolaryngology-Head and Neck Surgery and Gleiberman Head and Neck Cancer Center, University of California, San Diego, La Jolla, CA, USA.
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3
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Prabhakar AT, Morgan IM. A new role for human papillomavirus 16 E2: Mitotic activation of the DNA damage response to promote viral genome segregation. Tumour Virus Res 2024; 18:200291. [PMID: 39245413 PMCID: PMC11416546 DOI: 10.1016/j.tvr.2024.200291] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2024] [Revised: 09/03/2024] [Accepted: 09/05/2024] [Indexed: 09/10/2024] Open
Abstract
Human papillomaviruses (HPV) are causative agents in around 5% of all human cancers. To identify and develop new targeted HPV therapeutics we must enhance our understanding of the viral life cycle and how it interacts with the host. The HPV E2 protein dimerizes and binds to 12bp target sequences in the viral genome and segregates the viral genome during mitosis. In this function, E2 binds to the viral genome and the host chromatin simultaneously, ensuring viral genomes reside in daughter nuclei following cell division. We have demonstrated that a mitotic interaction between E2 and the DNA damage response (DDR) protein TOPBP1 is required for E2 segregation function. In non-infected cells, following DNA damage, TOPBP1 is recruited to the mitotic host genome via interaction with MDC1 and this interaction protects DNA integrity during mitosis. Recently we demonstrated that the E2-TOPBP1 interaction activates the DNA damage response (DDR) during mitosis independently from external stimuli, promoting TOPBP1 interaction with mitotic chromatin and therefore segregation of the viral genome. Therefore, the virus has hijacked an existing host mechanism in order to segregate the viral genome. This intricate E2 function will be described and discussed.
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Affiliation(s)
- Apurva T Prabhakar
- Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, VA, 23298, USA.
| | - Iain M Morgan
- Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, VA, 23298, USA; VCU Massey Cancer Center, Richmond, VA, 23298, USA.
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Lu Z, Haghollahi S, Afzal M. Potential Therapeutic Targets for the Treatment of HPV-Associated Malignancies. Cancers (Basel) 2024; 16:3474. [PMID: 39456568 PMCID: PMC11506301 DOI: 10.3390/cancers16203474] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2024] [Revised: 09/26/2024] [Accepted: 09/27/2024] [Indexed: 10/28/2024] Open
Abstract
This review article aims to summarize broadly recent developments in the treatment of HPV-associated cancers, including cervical cancer and head and neck squamous cell carcinoma. Relatively new treatments targeting the key HPV E6 and E7 oncoproteins, including gene editing with TALENs and CRISPR/Cas9, are discussed. Given the increased immunogenicity of HPV-related diseases, other therapies such as PRR agonists, adoptive cell transfer, and tumor vaccines are reaching the clinical trial phase. Due to the mechanism, immunogenicity, and reversibility of HPV carcinogenesis, HPV-related cancers present unique targets for current and future therapies.
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5
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Nakagawa T, Luebeck J, Zhu K, Lange JT, Sasik R, Phillips C, Sadat S, Javadzadeh S, Yang Q, Wang A, Pestonjamasp K, Rosenthal B, Fisch KM, Mischel P, Bafna V, Califano JA. Inhibition of novel human-HPV hybrid ecDNA enhancers reduces oncogene expression and tumor growth in oropharyngeal cancer. RESEARCH SQUARE 2024:rs.3.rs-4636308. [PMID: 39281879 PMCID: PMC11398563 DOI: 10.21203/rs.3.rs-4636308/v1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 09/18/2024]
Abstract
Extrachromosomal circular DNA (ecDNA) have been found in most types of human cancers, and ecDNA incorporating viral genomes has recently been described, specifically in human papillomavirus (HPV)-mediated oropharyngeal cancer (OPC). However, the molecular mechanisms of human-viral hybrid ecDNA (hybrid ecDNA) for carcinogenesis remains elusive. We characterized the epigenetic status of hybrid ecDNA using HPVOPC cell lines and patient-derived tumor xenografts, identifying HPV oncogenes E6/E7 in hybrid ecDNA were flanked by novel somatic DNA enhancers and HPV L1 enhancers, with strong cis-interaction. Targeting of these enhancers by clustered regularly interspaced short palindromic repeats interference or hybrid ecDNA by bromodomain and extra-terminal inhibitor reduced E6/E7 expression, and significantly inhibited in vitro and/or in vivo growth only in ecDNA(+) models. HPV DNA in hybrid ecDNA structures are associated with novel somatic and HPV enhancers in hybrid ecDNA that drive HPV ongogene expression and carcinogenesis, and can be targeted with ecDNA disrupting therapeutics.
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Affiliation(s)
- Takuya Nakagawa
- Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA
- Department of Otorhinolaryngology, Head and Neck Surgery, Chiba University, Graduate School of Medicine, Chiba, Japan
- Health and Disease Omics Center, Chiba University, Chiba, Japan
| | - Jens Luebeck
- Department of Computer Science and Engineering, UC San Diego, La Jolla, CA, USA
| | - Kaiyuan Zhu
- Department of Computer Science and Engineering, UC San Diego, La Jolla, CA, USA
| | - Joshua T. Lange
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
| | - Roman Sasik
- Center for Computational Biology and Bioinformatics, University of California, San Diego, La Jolla, CA, USA
| | - Chad Phillips
- Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA
| | - Sayed Sadat
- Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA
| | - Sara Javadzadeh
- Department of Computer Science and Engineering, UC San Diego, La Jolla, CA, USA
| | - Qian Yang
- Center for Epigenomics, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA
| | - Allen Wang
- Center for Epigenomics, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA
| | - Kersi Pestonjamasp
- Cancer Center Microscopy Core, University of California, San Diego, La Jolla, CA, USA
| | - Brin Rosenthal
- Center for Computational Biology and Bioinformatics, University of California, San Diego, La Jolla, CA, USA
| | - Kathleen M. Fisch
- Center for Computational Biology and Bioinformatics, University of California, San Diego, La Jolla, CA, USA
| | - Paul Mischel
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
| | - Vineet Bafna
- Department of Computer Science and Engineering, UC San Diego, La Jolla, CA, USA
| | - Joseph A. Califano
- Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA
- Department of Otolaryngology - Head and Neck Surgery, University of California, San Diego, La Jolla, CA, USA
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Janjua D, Thakur K, Aggarwal N, Chaudhary A, Yadav J, Chhokar A, Tripathi T, Joshi U, Senrung A, Bharti AC. Prognostic and therapeutic potential of STAT3: Opportunities and challenges in targeting HPV-mediated cervical carcinogenesis. Crit Rev Oncol Hematol 2024; 197:104346. [PMID: 38608913 DOI: 10.1016/j.critrevonc.2024.104346] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2024] [Revised: 03/28/2024] [Accepted: 04/04/2024] [Indexed: 04/14/2024] Open
Abstract
Cervical cancer (CaCx) ranks as the fourth most prevalent cancer among women globally. Persistent infection of high-risk human papillomaviruses (HR-HPVs) is major etiological factor associated with CaCx. Signal Transducer and Activator of Transcription 3 (STAT3), a prominent member of the STAT family, has emerged as independent oncogenic driver. It is a target of many oncogenic viruses including HPV. How STAT3 influences HPV viral gene expression or gets affected by HPV is an area of active investigation. A better understanding of host-virus interaction will provide a prognostic and therapeutic window for CaCx control and management. In this comprehensive review, we delve into carcinogenic role of STAT3 in development of HPV-induced CaCx. With an emphasis on fascinating interplay between STAT3 and HPV genome, the review explores the diverse array of opportunities and challenges associated with this field to harness the prognostic and therapeutic potential of STAT3 in CaCx.
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Affiliation(s)
- Divya Janjua
- Molecular Oncology Laboratory, Department of Zoology, University of Delhi (North Campus), New Delhi, India
| | - Kulbhushan Thakur
- Molecular Oncology Laboratory, Department of Zoology, University of Delhi (North Campus), New Delhi, India
| | - Nikita Aggarwal
- Molecular Oncology Laboratory, Department of Zoology, University of Delhi (North Campus), New Delhi, India
| | - Apoorva Chaudhary
- Molecular Oncology Laboratory, Department of Zoology, University of Delhi (North Campus), New Delhi, India
| | - Joni Yadav
- Molecular Oncology Laboratory, Department of Zoology, University of Delhi (North Campus), New Delhi, India
| | - Arun Chhokar
- Molecular Oncology Laboratory, Department of Zoology, University of Delhi (North Campus), New Delhi, India; Department of Zoology, Deshbandhu College, University of Delhi, Delhi, India
| | - Tanya Tripathi
- Molecular Oncology Laboratory, Department of Zoology, University of Delhi (North Campus), New Delhi, India
| | - Udit Joshi
- Molecular Oncology Laboratory, Department of Zoology, University of Delhi (North Campus), New Delhi, India
| | - Anna Senrung
- Molecular Oncology Laboratory, Department of Zoology, University of Delhi (North Campus), New Delhi, India; Department of Zoology, Daulat Ram College, University of Delhi, Delhi, India
| | - Alok Chandra Bharti
- Molecular Oncology Laboratory, Department of Zoology, University of Delhi (North Campus), New Delhi, India.
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7
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Kushwah AS, Masood S, Mishra R, Banerjee M. Genetic and epigenetic alterations in DNA repair genes and treatment outcome of chemoradiotherapy in cervical cancer. Crit Rev Oncol Hematol 2024; 194:104240. [PMID: 38122918 DOI: 10.1016/j.critrevonc.2023.104240] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2023] [Revised: 12/06/2023] [Accepted: 12/14/2023] [Indexed: 12/23/2023] Open
Abstract
Cervical cancer (CaCx) is the deadliest malignancy among women which is caused by human papillomavirus (HPV) and anthro-demographical/clinicopathological factors. HPV oncoproteins E6 and E7 target p53 and RB (retinoblastoma) protein degradation, Ataxia telangiectasia mutated (ATM), ATM-RAD3-related (ATR) inactivation and subsequent impairment of non-homologous end joining (NHEJ), homologous recombination, and base excision repair pathways. There is also an accumulation of genetic and epigenetic alterations in Tumor Growth Suppressors (TGS), oncogenes, and DNA repair genes leading to increased genome instability and CaCx development. These alterations might be responsible for differential clinical response to Cisplatin-based chemoradiotherapy (CRT) in patients. This review explores HPV-mediated DNA damage as a risk factor in CaCx development, the mechanistic role of genetic and epigenetic alterations in DNA repair genes and their association with CRT and outcome, It also explores new possibilities for the development of genetic and epigenetic-based biomarkers for diagnostic, prognostic, and molecular therapeutic interventions.
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Affiliation(s)
- Atar Singh Kushwah
- Department of Urology and Oncological Sciences, Icahn School of Medicine at Mount Sinai, 1425 Madison Ave, New York 10029, NY, USA; Molecular & Human Genetics Laboratory, Department of Zoology, University of Lucknow, Lucknow 226007, Uttar Pradesh, India; Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India
| | - Shireen Masood
- Molecular & Human Genetics Laboratory, Department of Zoology, University of Lucknow, Lucknow 226007, Uttar Pradesh, India
| | - Rajnikant Mishra
- Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India
| | - Monisha Banerjee
- Molecular & Human Genetics Laboratory, Department of Zoology, University of Lucknow, Lucknow 226007, Uttar Pradesh, India.
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Borella F, Gallio N, Mangherini L, Cassoni P, Bertero L, Benedetto C, Preti M. Recent advances in treating female genital human papillomavirus related neoplasms with topical imiquimod. J Med Virol 2023; 95:e29238. [PMID: 38009696 DOI: 10.1002/jmv.29238] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2023] [Revised: 10/28/2023] [Accepted: 11/04/2023] [Indexed: 11/29/2023]
Abstract
Human papillomavirus (HPV) encompasses a group of viruses that infect the skin and mucous membranes. In the presence of certain factors, persistent infection with high-risk HPVs can trigger a process of neoplastic transformation. Imiquimod is a topical agent that acts as a Toll-like receptor 7/8 agonist, stimulating the innate and adaptive immune system to exert antitumor and antiviral effects. It has been approved for the treatment of various skin conditions, however, its efficacy and safety in the management of HPV-related-neoplasms of the lower genital tract, such as vulvar, vaginal, and cervical neoplasia, are still under investigation. This review summarizes the current evidence on the use of imiquimod for the treatment of HPV-induced lesions of the female lower genital tract, focusing on its indications, mechanisms of action, outcomes, and predictors of response.
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Affiliation(s)
- Fulvio Borella
- Obstetrics and Gynecology Unit 1, Sant' Anna Hospital, Department of Surgical Sciences, University of Turin, Turin, Italy
| | - Niccolò Gallio
- Obstetrics and Gynecology Unit 2, Sant' Anna Hospital, Department of Surgical Sciences, University of Turin, Turin, Italy
| | - Luca Mangherini
- Pathology Unit, Department of Medical Sciences, University of Turin, Turin, Italy
| | - Paola Cassoni
- Pathology Unit, Department of Medical Sciences, University of Turin, Turin, Italy
| | - Luca Bertero
- Pathology Unit, Department of Medical Sciences, University of Turin, Turin, Italy
| | - Chiara Benedetto
- Obstetrics and Gynecology Unit 1, Sant' Anna Hospital, Department of Surgical Sciences, University of Turin, Turin, Italy
| | - Mario Preti
- Obstetrics and Gynecology Unit 1, Sant' Anna Hospital, Department of Surgical Sciences, University of Turin, Turin, Italy
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Laanemets A, Babok S, Piirsoo A. Characterization and comparative analysis of phosphorylation patterns in HPV18 and HPV11 E1 helicases: Implications for viral genome replication. Virology 2023; 587:109853. [PMID: 37523977 DOI: 10.1016/j.virol.2023.109853] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2023] [Revised: 07/22/2023] [Accepted: 07/24/2023] [Indexed: 08/02/2023]
Abstract
The genome of human papillomaviruses (HPVs) encodes the E1 replication factor, whose biological activities are regulated by cellular protein kinases. Here, the phosphorylation pattern of the E1 helicase of oncogenic mucosotropic HPV18 was investigated both in vitro and in vivo. Four serine residues located in a short peptide within a localization regulatory region were found to be phosphorylated in both experimental settings. We demonstrate that this peptide is targeted in vitro by various protein kinases, including CK2, PKA, and CKD2/cyclin A/B/E complexes. Through point mutagenesis, we show that phosphorylation of this region is essential for E1 subcellular localization, the interaction of E1 with the E2 protein, and replication of the HPV18 genome. Furthermore, we demonstrate the functional conservation of this phosphorylation across the E1 proteins of the low-risk mucosotropic HPV11 and high-risk cutaneotropic HPV5. These findings provide deeper insights into the phosphorylation-mediated regulation of biological activities of the E1 protein.
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Affiliation(s)
| | - Sofiya Babok
- Institute of Technology, University of Tartu, Tartu, Estonia
| | - Alla Piirsoo
- Institute of Technology, University of Tartu, Tartu, Estonia.
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10
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Zhi W, Wei Y, Lazare C, Meng Y, Wu P, Gao P, Lin S, Peng T, Chu T, Liu B, Ding W, Cao C, Wu P. HPV-CCDC106 integration promotes cervical cancer progression by facilitating the high expression of CCDC106 after HPV E6 splicing. J Med Virol 2023; 95:e28009. [PMID: 35854676 PMCID: PMC9796641 DOI: 10.1002/jmv.28009] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2022] [Revised: 07/11/2022] [Accepted: 07/13/2022] [Indexed: 01/11/2023]
Abstract
Human papillomavirus (HPV) integration and high expression of HPV oncogenes (E6 and E7) are important mechanisms for HPV carcinogenesis in cervical cancer. However, the relationship between HPV integration and HPV E6 spliced transcripts, as well as the underlying mechanisms of HPV integration in carcinogenesis after HPV E6 splicing remains unclear. We analyzed HPV-coiled-coil domain containing 106 (CCDC106) integration samples to characterize the roles of HPV integration, E6 spliceosome I (E6*I), and high CCDC106 expression in cervical carcinogenesis. We found that E6 was alternatively spliced into the E6*I transcript in HPV-CCDC016 integration samples with low p53 expression, in contrast to the role of E6*I in preventing p53 degradation in cervical cancer cells. In addition, CCDC106 was highly expressed after HPV-CCDC106 integration, and interacted with p53, resulting in p53 degradation and cervical cancer cell progression in vitro and in vivo. Importantly, when E6*I was highly expressed in cervical cancer cells, overexpression of CCDC106 independently degraded p53 and promoted cervical cancer cell progression. In this study, we explored the underlying mechanisms of HPV-CCDC106 integration in HPV carcinogenesis after HPV E6 splicing, which should provide insight into host genome dysregulation in cervical carcinogenesis.
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Affiliation(s)
- Wenhua Zhi
- Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina,Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
| | - Ye Wei
- Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina,Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
| | - Cordelle Lazare
- Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina,Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
| | - Yifan Meng
- Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina,Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
| | - Ping Wu
- Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina,Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
| | - Peipei Gao
- Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina,Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
| | - Shitong Lin
- Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina,Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
| | - Ting Peng
- Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina,Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
| | - Tian Chu
- Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina,Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
| | - Binghan Liu
- Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina,Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
| | - Wencheng Ding
- Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina,Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
| | - Canhui Cao
- Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina,Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina,Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Shenzhen HospitalShenzhen Peking University‐The Hong Kong University of Science and Technology Medical CenterGuangdongChina
| | - Peng Wu
- Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina,Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
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11
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Khan A, Das BC, Abiha U, Sisodiya S, Chikara A, Nazir SU, Das AM, Rodrigues AG, Passari AK, Tanwar P, Hussain S, Rashid S, Rashid S. Insights into the role of complement regulatory proteins in HPV mediated cervical carcinogenesis. Semin Cancer Biol 2022; 86:583-589. [PMID: 34087416 DOI: 10.1016/j.semcancer.2021.05.031] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2020] [Revised: 05/28/2021] [Accepted: 05/28/2021] [Indexed: 01/27/2023]
Abstract
The persistent infection of high-risk Human papillomavirus (HR-HPV) induced cervical cancer remains a challenge in women worldwide including India. Recent advances in cancer research have paved the way for advanced cancer treatment modalities including immunotherapy by manipulating the function or number of cytotoxic T cells. It is well established that anaphylatoxins like C3a and C5a of complement system influence tumor growth by evading apoptosis leading to progression of cancer. The role of the complement system, particularly the complement regulatory proteins (CRPs) which are important determinants of immune response play a crucial role in carcinogenesis. In a tumor microenvironment (TME) assisted suppression of immune effector cells may be achieved through CRPs. However, recent advances in pharmacogenomics including drug designing and combination of these approaches have provided a holistic understanding of signaling pathways and their crosstalk, to regulate cellular communications.This review describes the role of complement system; particularly CRPs in HPV induced cervical carcinogenesis which may be used for designing anti- HPV or cervical cancer therapeutics.
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Affiliation(s)
- Asiya Khan
- Centre for Medical Biotechnology, Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India; Laboratory Oncology Unit, Rotary Cancer Center, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India
| | - Bhudev C Das
- Amity Institute of Molecular Medicine & Stem Cell Research (AIMMSCR), Health & Allied Sciences Amity University, Noida, India
| | - Umme Abiha
- Centre for Medical Biotechnology, Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India
| | - Sandeep Sisodiya
- Division of Molecular Oncology & Molecular Diagnostics, ICMR-National Institute of Cancer Prevention and Research, Ministry of Health & Family Welfare, Noida, India; Symbiosis School of Biological Sciences, Symbiosis International (Deemed University), Pune, India
| | - Atul Chikara
- Division of Molecular Oncology & Molecular Diagnostics, ICMR-National Institute of Cancer Prevention and Research, Ministry of Health & Family Welfare, Noida, India; Symbiosis School of Biological Sciences, Symbiosis International (Deemed University), Pune, India
| | - Sheeraz Un Nazir
- Division of Molecular Oncology & Molecular Diagnostics, ICMR-National Institute of Cancer Prevention and Research, Ministry of Health & Family Welfare, Noida, India
| | - Ankan M Das
- Amity Institute of Public Health, Amity University, Noida, India
| | - Alexandre Gomes Rodrigues
- Alpha & Omega Labor, Messe-Alle, 23, 04158, Leipzig, Germany; University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173, Hannover, Germany
| | - Ajit Kumar Passari
- Departmento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), Ciudad de México, Mexico
| | - Pranay Tanwar
- Laboratory Oncology Unit, Rotary Cancer Center, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India
| | - Showket Hussain
- Division of Molecular Oncology & Molecular Diagnostics, ICMR-National Institute of Cancer Prevention and Research, Ministry of Health & Family Welfare, Noida, India.
| | - Sabia Rashid
- Queen Elizabeth Hospital & King's College Hospital, Stadium Road, London, United Kingdom.
| | - Shazia Rashid
- Centre for Medical Biotechnology, Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India.
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12
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Fontan CT, Prabhakar AT, Wang X, Karimi E, Bristol ML, James CD, Morgan IM. Human papillomavirus 16 E2 blocks cellular senescence in response to activation of the DNA damage response. Virology 2022; 575:54-62. [PMID: 36058086 PMCID: PMC10715573 DOI: 10.1016/j.virol.2022.08.007] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2022] [Revised: 08/17/2022] [Accepted: 08/18/2022] [Indexed: 11/21/2022]
Abstract
Following infection by HPV16, the viral proteins E1 and E2 induce viral genome replication in association with host factors. Here we demonstrate that E2 also plays a role in promoting short-term cellular proliferation in the presence of an active DDR. Cisplatin treatment of E2 expressing cells results in short-term proliferation likely due to a block of cellular senescence and apoptosis. However, long-term growth of E2 expressing cells following cisplatin treatment is attenuated due to an accumulation of DNA damage. We discuss a possible role for this E2 function during the viral life cycle. It is also notable that E2 expressing HPV16 positive cancers have a better clinical outcome than non-E2 expressing tumors. While there are a variety of reasons for the better outcome of patients with E2 expressing tumors, this report suggests that E2 regulation of the DNA damage response may be a contributory factor.
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Affiliation(s)
- Christian T Fontan
- Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, Virginia, USA
| | - Apurva T Prabhakar
- Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, Virginia, USA
| | - Xu Wang
- Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, Virginia, USA
| | - Elmira Karimi
- Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, Virginia, USA
| | - Molly L Bristol
- Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, Virginia, USA
| | - Claire D James
- Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, Virginia, USA
| | - Iain M Morgan
- Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, Virginia, USA; VCU Massey Cancer Center, Richmond, Virginia, USA.
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13
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Choi PW, Liu TL, Wong CW, Liu SK, Lum YL, Ming WK. The Dysregulation of MicroRNAs in the Development of Cervical Pre-Cancer—An Update. Int J Mol Sci 2022; 23:ijms23137126. [PMID: 35806128 PMCID: PMC9266862 DOI: 10.3390/ijms23137126] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2022] [Revised: 06/20/2022] [Accepted: 06/24/2022] [Indexed: 02/01/2023] Open
Abstract
Globally in 2020, an estimated ~600,000 women were diagnosed with and 340,000 women died from cervical cancer. Compared to 2012, the number of cases increased by 7.5% and the number of deaths increased by 17%. MiRNAs are involved in multiple processes in the pathogenesis of cervical cancer. Dysregulation of miRNAs in the pre-stage of cervical cancer is the focus of this review. Here we summarize the dysregulated miRNAs in clinical samples from cervical pre-cancer patients and relate them to the early transformation process owing to human papillomavirus (HPV) infection in the cervical cells. When HPV infects the normal cervical cells, the DNA damage response is initiated with the involvement of HPV’s E1 and E2 proteins. Later, cell proliferation and cell death are affected by the E6 and E7 proteins. We find that the expressions of miRNAs in cervical pre-cancerous tissue revealed by different studies seldom agreed with each other. The discrepancy in sample types, samples’ HPV status, expression measurement, and methods for analysis contributed to the non-aligned results across studies. However, several miRNAs (miR-34a, miR-9, miR-21, miR-145, and miR-375) were found to be dysregulated across multiple studies. In addition, there are hints that the DNA damage response and cell growth response induced by HPV during the early transformation of the cervical cells are related to these miRNAs. Currently, no review articles analyse the relationship between the dysregulated miRNAs in cervical pre-cancerous tissue and their possible roles in the early processes involving HPV’s protein encoded by the early genes and DNA damage response during normal cell transformation. Our review provides insight on spotting miRNAs involved in the early pathogenic processes and pointing out their potential as biomarker targets of cervical pre-cancer.
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Affiliation(s)
- Pui-Wah Choi
- Department of Research and Development, WomenX Biotech Limited, Hong Kong Science and Technology Park, Tai Po, Hong Kong; (P.-W.C.); (C.W.W.); (S.K.L.); (Y.-L.L.)
| | - Tin Lun Liu
- International School, Jinan University, Guangzhou 510632, China;
| | - Chun Wai Wong
- Department of Research and Development, WomenX Biotech Limited, Hong Kong Science and Technology Park, Tai Po, Hong Kong; (P.-W.C.); (C.W.W.); (S.K.L.); (Y.-L.L.)
| | - Sze Kei Liu
- Department of Research and Development, WomenX Biotech Limited, Hong Kong Science and Technology Park, Tai Po, Hong Kong; (P.-W.C.); (C.W.W.); (S.K.L.); (Y.-L.L.)
| | - Yick-Liang Lum
- Department of Research and Development, WomenX Biotech Limited, Hong Kong Science and Technology Park, Tai Po, Hong Kong; (P.-W.C.); (C.W.W.); (S.K.L.); (Y.-L.L.)
| | - Wai-Kit Ming
- Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Hong Kong
- Correspondence: ; Tel.: +852-3442-6956
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14
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Abjaude W, Prati B, Munford V, Montenegro A, Lino V, Herbster S, Rabachini T, Termini L, Menck CFM, Boccardo E. ATM Pathway Is Essential for HPV-Positive Human Cervical Cancer-Derived Cell Lines Viability and Proliferation. Pathogens 2022; 11:637. [PMID: 35745491 PMCID: PMC9228918 DOI: 10.3390/pathogens11060637] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2022] [Revised: 05/22/2022] [Accepted: 05/26/2022] [Indexed: 01/01/2023] Open
Abstract
Infection with some mucosal human papillomavirus (HPV) types is the etiological cause of cervical cancer and of a significant fraction of vaginal, vulvar, anal, penile, and head and neck carcinomas. DNA repair machinery is essential for both HPV replication and tumor cells survival suggesting that cellular DNA repair machinery may play a dual role in HPV biology and pathogenesis. Here, we silenced genes involved in DNA Repair pathways to identify genes that are essential for the survival of HPV-transformed cells. We identified that inhibition of the ATM/CHK2/BRCA1 axis selectively affects the proliferation of cervical cancer-derived cell lines, without altering normal primary human keratinocytes (PHK) growth. Silencing or chemical inhibition of ATM/CHK2 reduced the clonogenic and proliferative capacity of cervical cancer-derived cells. Using PHK transduced with HPV16 oncogenes we observed that the effect of ATM/CHK2 silencing depends on the expression of the oncogene E6 and on its ability to induce p53 degradation. Our results show that inhibition of components of the ATM/CHK2 signaling axis reduces p53-deficient cells proliferation potential, suggesting the existence of a synthetic lethal association between CHK2 and p53. Altogether, we present evidence that synthetic lethality using ATM/CHK2 inhibitors can be exploited to treat cervical cancer and other HPV-associated tumors.
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Affiliation(s)
- Walason Abjaude
- Laboratory of Oncovirology, Department of Microbiology, Instituto de Ciências Biomédicas, Universidade de São Paulo, Sao Paulo 05508-900, Brazil; (W.A.); (B.P.); (A.M.); (V.L.); (S.H.)
| | - Bruna Prati
- Laboratory of Oncovirology, Department of Microbiology, Instituto de Ciências Biomédicas, Universidade de São Paulo, Sao Paulo 05508-900, Brazil; (W.A.); (B.P.); (A.M.); (V.L.); (S.H.)
| | - Veridiana Munford
- Laboratory of DNA Repair, Department of Microbiology, Instituto de Ciências Biomédicas, Universidade de São Paulo, Sao Paulo 05508-000, Brazil; (V.M.); (C.F.M.M.)
| | - Aline Montenegro
- Laboratory of Oncovirology, Department of Microbiology, Instituto de Ciências Biomédicas, Universidade de São Paulo, Sao Paulo 05508-900, Brazil; (W.A.); (B.P.); (A.M.); (V.L.); (S.H.)
| | - Vanesca Lino
- Laboratory of Oncovirology, Department of Microbiology, Instituto de Ciências Biomédicas, Universidade de São Paulo, Sao Paulo 05508-900, Brazil; (W.A.); (B.P.); (A.M.); (V.L.); (S.H.)
| | - Suellen Herbster
- Laboratory of Oncovirology, Department of Microbiology, Instituto de Ciências Biomédicas, Universidade de São Paulo, Sao Paulo 05508-900, Brazil; (W.A.); (B.P.); (A.M.); (V.L.); (S.H.)
| | - Tatiana Rabachini
- Institute of Pharmacology, Inselspital, INO-F, University of Bern, CH-3010 Bern, Switzerland;
| | - Lara Termini
- Centro de Investigação Translacional em Oncologia, Instituto do Câncer do Estado de São Paulo, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, Sao Paulo 01246-000, Brazil;
| | - Carlos Frederico Martins Menck
- Laboratory of DNA Repair, Department of Microbiology, Instituto de Ciências Biomédicas, Universidade de São Paulo, Sao Paulo 05508-000, Brazil; (V.M.); (C.F.M.M.)
| | - Enrique Boccardo
- Laboratory of Oncovirology, Department of Microbiology, Instituto de Ciências Biomédicas, Universidade de São Paulo, Sao Paulo 05508-900, Brazil; (W.A.); (B.P.); (A.M.); (V.L.); (S.H.)
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15
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Läsche M, Gallwas J, Gründker C. Like Brothers in Arms: How Hormonal Stimuli and Changes in the Metabolism Signaling Cooperate, Leading HPV Infection to Drive the Onset of Cervical Cancer. Int J Mol Sci 2022; 23:5050. [PMID: 35563441 PMCID: PMC9103757 DOI: 10.3390/ijms23095050] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2022] [Revised: 04/27/2022] [Accepted: 04/28/2022] [Indexed: 02/06/2023] Open
Abstract
Despite all precautionary actions and the possibility of using vaccinations to counteract infections caused by human papillomaviruses (HPVs), HPV-related cancers still account for approximately 5% of all carcinomas. Worldwide, many women are still excluded from adequate health care due to their social position and origin. Therefore, immense efforts in research and therapy are still required to counteract the challenges that this disease entails. The special thing about an HPV infection is that it is not only able to trick the immune system in a sophisticated way, but also, through genetic integration into the host genome, to use all the resources available to the host cells to complete the replication cycle of the virus without activating the alarm mechanisms of immune recognition and elimination. The mechanisms utilized by the virus are the metabolic, immune, and hormonal signaling pathways that it manipulates. Since the virus is dependent on replication enzymes of the host cells, it also intervenes in the cell cycle of the differentiating keratinocytes and shifts their terminal differentiation to the uppermost layers of the squamocolumnar transformation zone (TZ) of the cervix. The individual signaling pathways are closely related and equally important not only for the successful replication of the virus but also for the onset of cervical cancer. We will therefore analyze the effects of HPV infection on metabolic signaling, as well as changes in hormonal and immune signaling in the tumor and its microenvironment to understand how each level of signaling interacts to promote tumorigenesis of cervical cancer.
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Affiliation(s)
| | | | - Carsten Gründker
- Department of Gynecology and Obstetrics, University Medicine Göttingen, 37075 Göttingen, Germany; (M.L.); (J.G.)
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16
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Gao C, Wu P, Yu L, Liu L, Liu H, Tan X, Wang L, Huang X, Wang H. The application of CRISPR/Cas9 system in cervical carcinogenesis. Cancer Gene Ther 2022; 29:466-474. [PMID: 34349239 PMCID: PMC9113934 DOI: 10.1038/s41417-021-00366-w] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2020] [Revised: 05/25/2021] [Accepted: 06/23/2021] [Indexed: 02/02/2023]
Abstract
Integration of high-risk HPV genomes into cellular chromatin has been confirmed to promote cervical carcinogenesis, with HPV16 being the most prevalent high-risk type. Herein, we evaluated the therapeutic effect of the CRISPR/Cas9 system in cervical carcinogenesis, especially for cervical precancerous lesions. In cervical cancer/pre-cancer cell lines, we transfected the HPV16 E7 targeted CRISPR/Cas9, TALEN, ZFN plasmids, respectively. Compared to previous established ZFN and TALEN systems, CRISPR/Cas9 has shown comparable efficiency and specificity in inhibiting cell growth and colony formation and inducing apoptosis in cervical cancer/pre-cancer cell lines, which seemed to be more pronounced in the S12 cell line derived from the low-grade cervical lesion. Furthermore, in xenograft formation assays, CRISPR/Cas9 inhibited tumor formation of the S12 cell line in vivo and affected the corresponding protein expression. In the K14-HPV16 transgenic mice model of HPV-driven spontaneous cervical carcinogenesis, cervical application of CRISPR/Cas9 treatment caused mutations of the E7 gene and restored the expression of RB, E2F1, and CDK2, thereby reversing the cervical carcinogenesis phenotype. In this study, we have demonstrated that CRISPR/Cas9 targeting HPV16 E7 could effectively revert the HPV-related cervical carcinogenesis in vitro, as well as in K14-HPV16 transgenic mice, which has shown great potential in clinical treatment for cervical precancerous lesions.
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Affiliation(s)
- Chun Gao
- grid.412793.a0000 0004 1799 5032Cancer Biology Research Center (Key laboratory of the ministry of education), Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei China ,grid.412793.a0000 0004 1799 5032Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei China
| | - Ping Wu
- grid.412793.a0000 0004 1799 5032Cancer Biology Research Center (Key laboratory of the ministry of education), Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei China ,grid.412793.a0000 0004 1799 5032Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei China
| | - Lan Yu
- grid.488530.20000 0004 1803 6191Department of Gynecologic Oncology, Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, China
| | - Liting Liu
- grid.412793.a0000 0004 1799 5032Cancer Biology Research Center (Key laboratory of the ministry of education), Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei China
| | - Hong Liu
- grid.412793.a0000 0004 1799 5032Cancer Biology Research Center (Key laboratory of the ministry of education), Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei China
| | - Xiangyu Tan
- grid.412793.a0000 0004 1799 5032Cancer Biology Research Center (Key laboratory of the ministry of education), Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei China
| | - Liming Wang
- grid.412793.a0000 0004 1799 5032Cancer Biology Research Center (Key laboratory of the ministry of education), Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei China ,grid.412793.a0000 0004 1799 5032Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei China
| | - Xiaoyuan Huang
- grid.412793.a0000 0004 1799 5032Cancer Biology Research Center (Key laboratory of the ministry of education), Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei China ,grid.412793.a0000 0004 1799 5032Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei China
| | - Hui Wang
- grid.412793.a0000 0004 1799 5032Cancer Biology Research Center (Key laboratory of the ministry of education), Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei China ,grid.412793.a0000 0004 1799 5032Department of Gynecologic Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei China ,grid.431048.a0000 0004 1757 7762Department of Gynecologic Oncology, Women’s Hospital, School of Medicine, Zhejiang University, Zhejiang, China
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17
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Warburton A, Markowitz TE, Katz JP, Pipas JM, McBride AA. Recurrent integration of human papillomavirus genomes at transcriptional regulatory hubs. NPJ Genom Med 2021; 6:101. [PMID: 34848725 PMCID: PMC8632991 DOI: 10.1038/s41525-021-00264-y] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2021] [Accepted: 10/28/2021] [Indexed: 01/15/2023] Open
Abstract
Oncogenic human papillomavirus (HPV) genomes are often integrated into host chromosomes in HPV-associated cancers. HPV genomes are integrated either as a single copy or as tandem repeats of viral DNA interspersed with, or without, host DNA. Integration occurs frequently in common fragile sites susceptible to tandem repeat formation and the flanking or interspersed host DNA often contains transcriptional enhancer elements. When co-amplified with the viral genome, these enhancers can form super-enhancer-like elements that drive high viral oncogene expression. Here we compiled highly curated datasets of HPV integration sites in cervical (CESC) and head and neck squamous cell carcinoma (HNSCC) cancers, and assessed the number of breakpoints, viral transcriptional activity, and host genome copy number at each insertion site. Tumors frequently contained multiple distinct HPV integration sites but often only one “driver” site that expressed viral RNA. As common fragile sites and active enhancer elements are cell-type-specific, we mapped these regions in cervical cell lines using FANCD2 and Brd4/H3K27ac ChIP-seq, respectively. Large enhancer clusters, or super-enhancers, were also defined using the Brd4/H3K27ac ChIP-seq dataset. HPV integration breakpoints were enriched at both FANCD2-associated fragile sites and enhancer-rich regions, and frequently showed adjacent focal DNA amplification in CESC samples. We identified recurrent integration “hotspots” that were enriched for super-enhancers, some of which function as regulatory hubs for cell-identity genes. We propose that during persistent infection, extrachromosomal HPV minichromosomes associate with these transcriptional epicenters and accidental integration could promote viral oncogene expression and carcinogenesis.
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Affiliation(s)
- Alix Warburton
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 33 North Drive, MSC3209, Bethesda, MD, 20892, USA
| | - Tovah E Markowitz
- NIAID Collaborative Bioinformatics Resource (NCBR), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.,Advanced Biomedical Computational Science, Frederick National Laboratory for Cancer Research, Frederick, MD, USA
| | - Joshua P Katz
- Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - James M Pipas
- Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Alison A McBride
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 33 North Drive, MSC3209, Bethesda, MD, 20892, USA.
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18
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Liblekas L, Piirsoo A, Laanemets A, Tombak EM, Laaneväli A, Ustav E, Ustav M, Piirsoo M. Analysis of the Replication Mechanisms of the Human Papillomavirus Genomes. Front Microbiol 2021; 12:738125. [PMID: 34733254 PMCID: PMC8558456 DOI: 10.3389/fmicb.2021.738125] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2021] [Accepted: 09/22/2021] [Indexed: 11/27/2022] Open
Abstract
The life-cycle of human papillomaviruses (HPVs) includes three distinct phases of the viral genome replication. First, the viral genome is amplified in the infected cells, and this amplification is often accompanied by the oligomerization of the viral genomes. Second stage includes the replication of viral genomes in concert with the host cell genome. The viral genome is further amplified during the third stage of the viral-life cycle, which takes place only in the differentiated keratinocytes. We have previously shown that the HPV18 genomes utilize at least two distinct replication mechanisms during the initial amplification. One of these mechanisms is a well-described bidirectional replication via theta type of replication intermediates. The nature of another replication mechanism utilized by HPV18 involves most likely recombination-dependent replication. In this paper, we show that the usage of different replication mechanisms is a property shared also by other HPV types, namely HPV11 and HPV5. We further show that the emergence of the recombination dependent replication coincides with the oligomerization of the viral genomes and is dependent on the replicative DNA polymerases. We also show that the oligomeric genomes of HPV18 replicate almost exclusively using recombination dependent mechanism, whereas monomeric HPV31 genomes replicate bi-directionally during the maintenance phase of the viral life-cycle.
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Affiliation(s)
- Lisett Liblekas
- Institute of Technology, University of Tartu, Tartu, Estonia
| | - Alla Piirsoo
- Institute of Technology, University of Tartu, Tartu, Estonia
| | | | | | | | - Ene Ustav
- Institute of Technology, University of Tartu, Tartu, Estonia
| | - Mart Ustav
- Institute of Technology, University of Tartu, Tartu, Estonia
| | - Marko Piirsoo
- Institute of Technology, University of Tartu, Tartu, Estonia
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19
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Warburton A, Della Fera AN, McBride AA. Dangerous Liaisons: Long-Term Replication with an Extrachromosomal HPV Genome. Viruses 2021; 13:1846. [PMID: 34578427 PMCID: PMC8472234 DOI: 10.3390/v13091846] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2021] [Revised: 09/09/2021] [Accepted: 09/14/2021] [Indexed: 01/17/2023] Open
Abstract
Papillomaviruses cause persistent, and usually self-limiting, infections in the mucosal and cutaneous surfaces of the host epithelium. However, in some cases, infection with an oncogenic HPV can lead to cancer. The viral genome is a small, double-stranded circular DNA molecule that is assembled into nucleosomes at all stages of infection. The viral minichromosome replicates at a low copy number in the nucleus of persistently infected cells using the cellular replication machinery. When the infected cells differentiate, the virus hijacks the host DNA damage and repair pathways to replicate viral DNA to a high copy number to generate progeny virions. This strategy is highly effective and requires a close association between viral and host chromatin, as well as cellular processes associated with DNA replication, repair, and transcription. However, this association can lead to accidental integration of the viral genome into host DNA, and under certain circumstances integration can promote oncogenesis. Here we describe the fate of viral DNA at each stage of the viral life cycle and how this might facilitate accidental integration and subsequent carcinogenesis.
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Affiliation(s)
| | | | - Alison A. McBride
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA; (A.W.); (A.N.D.F.)
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Das D, Bristol ML, Pichierri P, Morgan IM. Using a Human Papillomavirus Model to Study DNA Replication and Repair of Wild Type and Damaged DNA Templates in Mammalian Cells. Int J Mol Sci 2020; 21:E7564. [PMID: 33066318 PMCID: PMC7589113 DOI: 10.3390/ijms21207564] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2020] [Revised: 10/06/2020] [Accepted: 10/12/2020] [Indexed: 12/13/2022] Open
Abstract
Human papillomaviruses have 8kbp DNA episomal genomes that replicate autonomously from host DNA. During initial infection, the virus increases its copy number to 20-50 copies per cell, causing torsional stress on the replicating DNA. This activates the DNA damage response (DDR) and HPV replicates its genome, at least in part, using homologous recombination. An active DDR is on throughout the HPV life cycle. Two viral proteins are required for replication of the viral genome; E2 binds to 12bp palindromic sequences around the A/T rich origin of replication and recruits the viral helicase E1 via a protein-protein interaction. E1 forms a di-hexameric complex that replicates the viral genome in association with host factors. Transient replication assays following transfection with E1-E2 expression plasmids, along with an origin containing plasmid, allow monitoring of E1-E2 replication activity. Incorporating a bacterial lacZ gene into the origin plasmid allows for the determination of replication fidelity. Here we describe how we exploited this system to investigate replication and repair in mammalian cells, including using damaged DNA templates. We propose that this system has the potential to enhance the understanding of cellular components involved in DNA replication and repair.
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Affiliation(s)
- Dipon Das
- Department of Oral and Craniofacial Molecular Biology, VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Richmond, VA 23298, USA; (D.D.); (M.L.B.)
| | - Molly L. Bristol
- Department of Oral and Craniofacial Molecular Biology, VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Richmond, VA 23298, USA; (D.D.); (M.L.B.)
| | - Pietro Pichierri
- Department of Environment and Health, Istituto Superiore di Sanita’, 00161 Rome, Italy;
| | - Iain M. Morgan
- Department of Oral and Craniofacial Molecular Biology, VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Richmond, VA 23298, USA; (D.D.); (M.L.B.)
- VCU Massey Cancer Center, Richmond, VA 23298, USA
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21
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James CD, Das D, Morgan EL, Otoa R, Macdonald A, Morgan IM. Werner Syndrome Protein (WRN) Regulates Cell Proliferation and the Human Papillomavirus 16 Life Cycle during Epithelial Differentiation. mSphere 2020; 5:e00858-20. [PMID: 32938703 PMCID: PMC7494838 DOI: 10.1128/msphere.00858-20] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2020] [Accepted: 09/01/2020] [Indexed: 12/13/2022] Open
Abstract
Human papillomaviruses recruit a host of DNA damage response factors to their viral genome to facilitate homologous recombination replication in association with the viral replication factors E1 and E2. We previously demonstrated that SIRT1 deacetylation of WRN promotes recruitment of WRN to E1-E2 replicating DNA and that WRN regulates both the levels and fidelity of E1-E2 replication. The deacetylation of WRN by SIRT1 results in an active protein able to complex with replicating DNA, but a protein that is less stable. Here, we demonstrate an inverse correlation between SIRT1 and WRN in CIN cervical lesions compared to normal control tissue, supporting our model of SIRT1 deacetylation destabilizing WRN protein. We CRISPR/Cas9 edited N/Tert-1 and N/Tert-1+HPV16 cells to knock out WRN protein expression and subjected the cells to organotypic raft cultures. In N/Tert-1 cells without WRN expression, there was enhanced basal cell proliferation, DNA damage, and thickening of the differentiated epithelium. In N/Tert-1+HPV16 cells, there was enhanced basal cell proliferation, increased DNA damage throughout the epithelium, and increased viral DNA replication. Overall, the results demonstrate that the expression of WRN is required to control the proliferation of N/Tert-1 cells and controls the HPV16 life cycle in these cells. This complements our previous data demonstrating that WRN controls the levels and fidelity of HPV16 E1-E2 DNA replication. The results describe a new role for WRN, a tumor suppressor, in controlling keratinocyte differentiation and the HPV16 life cycle.IMPORTANCE HPV16 is the major human viral carcinogen, responsible for around 3 to 4% of all cancers worldwide. Our understanding of how the viral replication machinery interacts with host factors to control/activate the DNA damage response to promote the viral life cycle remains incomplete. Recently, we demonstrated a SIRT1-WRN axis that controls HPV16 replication, and here we demonstrate that this axis persists in clinical cervical lesions induced by HPV16. Here, we describe the effects of WRN depletion on cellular differentiation with or without HPV16; WRN depletion results in enhanced proliferation and DNA damage irrespective of HPV16 status. Also, WRN is a restriction factor for the viral life cycle since replication is disrupted in the absence of WRN. Future studies will focus on enhancing our understanding of how WRN regulates viral replication. Our goal is to ultimately identify cellular factors essential for HPV16 replication that can be targeted for therapeutic gain.
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Affiliation(s)
- Claire D James
- Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, Virginia, USA
| | - Dipon Das
- Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, Virginia, USA
| | - Ethan L Morgan
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK
| | - Raymonde Otoa
- Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, Virginia, USA
| | - Andrew Macdonald
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK
| | - Iain M Morgan
- Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, Virginia, USA
- VCU Massey Cancer Center, Richmond, Virginia, USA
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James CD, Das D, Bristol ML, Morgan IM. Activating the DNA Damage Response and Suppressing Innate Immunity: Human Papillomaviruses Walk the Line. Pathogens 2020; 9:E467. [PMID: 32545729 PMCID: PMC7350329 DOI: 10.3390/pathogens9060467] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Revised: 06/08/2020] [Accepted: 06/10/2020] [Indexed: 12/25/2022] Open
Abstract
Activation of the DNA damage response (DDR) by external agents can result in DNA fragments entering the cytoplasm and activating innate immune signaling pathways, including the stimulator of interferon genes (STING) pathway. The consequences of this activation can result in alterations in the cell cycle including the induction of cellular senescence, as well as boost the adaptive immune response following interferon production. Human papillomaviruses (HPV) are the causative agents in a host of human cancers including cervical and oropharyngeal; HPV are responsible for around 5% of all cancers. During infection, HPV replication activates the DDR in order to promote the viral life cycle. A striking feature of HPV-infected cells is their ability to continue to proliferate in the presence of an active DDR. Simultaneously, HPV suppress the innate immune response using a number of different mechanisms. The activation of the DDR and suppression of the innate immune response are essential for the progression of the viral life cycle. Here, we describe the mechanisms HPV use to turn on the DDR, while simultaneously suppressing the innate immune response. Pushing HPV from this fine line and tipping the balance towards activation of the innate immune response would be therapeutically beneficial.
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Affiliation(s)
- Claire D. James
- Philips Institute for Oral Health Research, School of Dentistry, Virginia Commonwealth University (VCU), Richmond, VA 23298, USA; (C.D.J.); (D.D.); (M.L.B.)
| | - Dipon Das
- Philips Institute for Oral Health Research, School of Dentistry, Virginia Commonwealth University (VCU), Richmond, VA 23298, USA; (C.D.J.); (D.D.); (M.L.B.)
| | - Molly L. Bristol
- Philips Institute for Oral Health Research, School of Dentistry, Virginia Commonwealth University (VCU), Richmond, VA 23298, USA; (C.D.J.); (D.D.); (M.L.B.)
| | - Iain M. Morgan
- Philips Institute for Oral Health Research, School of Dentistry, Virginia Commonwealth University (VCU), Richmond, VA 23298, USA; (C.D.J.); (D.D.); (M.L.B.)
- VCU Massey Cancer Center, Richmond, VA 23298, USA
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James CD, Morgan IM, Bristol ML. The Relationship between Estrogen-Related Signaling and Human Papillomavirus Positive Cancers. Pathogens 2020; 9:E403. [PMID: 32455952 PMCID: PMC7281727 DOI: 10.3390/pathogens9050403] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Revised: 05/19/2020] [Accepted: 05/20/2020] [Indexed: 12/15/2022] Open
Abstract
High risk-human papillomaviruses (HPVs) are known carcinogens. Numerous reports have linked the steroid hormone estrogen, and the expression of estrogen receptors (ERs), to HPV-related cancers, although the exact nature of the interactions remains to be fully elucidated. Here we will focus on estrogen signaling and describe both pro and potentially anti-cancer effects of this hormone in HPV-positive cancers. This review will summarize: (1) cell culture-related evidence, (2) animal model evidence, and (3) clinical evidence demonstrating an interaction between estrogen and HPV-positive cancers. This comprehensive review provides insights into the potential relationship between estrogen and HPV. We suggest that estrogen may provide a potential therapeutic for HPV-related cancers, however additional studies are necessary.
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Affiliation(s)
- Claire D. James
- School of Dentistry, Philips Institute for Oral Health Research, Virginia Commonwealth University (VCU), Richmond, VA 23298, USA;
| | - Iain M. Morgan
- School of Dentistry, Philips Institute for Oral Health Research, Virginia Commonwealth University (VCU), Richmond, VA 23298, USA;
- VCU Massey Cancer Center, Virginia Commonwealth University (VCU), Richmond, VA 23298, USA
| | - Molly L. Bristol
- School of Dentistry, Philips Institute for Oral Health Research, Virginia Commonwealth University (VCU), Richmond, VA 23298, USA;
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Ferreira AR, Ramalho AC, Marques M, Ribeiro D. The Interplay between Antiviral Signalling and Carcinogenesis in Human Papillomavirus Infections. Cancers (Basel) 2020; 12:cancers12030646. [PMID: 32164347 PMCID: PMC7139948 DOI: 10.3390/cancers12030646] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2020] [Revised: 03/05/2020] [Accepted: 03/06/2020] [Indexed: 12/20/2022] Open
Abstract
Human papillomaviruses (HPV) are the causative agents of the most common sexually transmitted infection worldwide. While infection is generally asymptomatic and can be cleared by the host immune system, when persistence occurs, HPV can become a risk factor for malignant transformation. Progression to cancer is actually an unintended consequence of the complex HPV life cycle. Different antiviral defence mechanisms recognize HPV early in infection, leading to the activation of the innate immune response. However, the virus has evolved several specific strategies to efficiently evade the antiviral immune signalling. Here, we review and discuss the interplay between HPV and the host cell innate immunity. We further highlight the evasion strategies developed by different HPV to escape this cellular response and focus on the correlation with HPV-induced persistence and tumorigenesis.
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Affiliation(s)
| | | | | | - Daniela Ribeiro
- Correspondence: ; Tel.: +351-234-247 014; Fax: +351-234-372-587
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James CD, Fontan CT, Otoa R, Das D, Prabhakar AT, Wang X, Bristol ML, Morgan IM. Human Papillomavirus 16 E6 and E7 Synergistically Repress Innate Immune Gene Transcription. mSphere 2020; 5:e00828-19. [PMID: 31915229 PMCID: PMC6952203 DOI: 10.1128/msphere.00828-19] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2019] [Accepted: 12/12/2019] [Indexed: 11/22/2022] Open
Abstract
Human papillomaviruses (HPV) are causative agents in 5% of all cancers, including the majority of anogenital and oropharyngeal cancers. Downregulation of innate immune genes (IIGs) by HPV to promote the viral life cycle is well documented; E6 and E7 are known repressors of these genes. More recently, we demonstrated that E2 could also repress IIGs. These studies have been carried out in cells overexpressing the viral proteins, and to further investigate the role of individual viral proteins in this repression, we introduced stop codons into E6 and/or E7 in the entire HPV16 genome and generated N/Tert-1 cells stably maintaining the HPV16 genomes. We demonstrate that E6 or E7 individually is not sufficient to repress IIG expression in the context of the entire HPV16 genome; both are required for a synergistic repression. The DNA damage response (DDR) is activated by HPV16 irrespective of E6 and E7 expression, presumably due to viral replication; E1 is a known activator of the DDR. In addition, replication stress was apparent in HPV16-positive cells lacking E6 and E7, manifested by attenuated cellular growth and activation of replication stress genes. These studies led us to the following model. Viral replication per se can activate the DDR following infection, and this activation is a known inducer of IIG expression, which may induce cellular senescence. To combat this, E6 and E7 synergistically combine to manipulate the DDR and actively repress innate immune gene expression promoting cellular growth; neither protein by itself is able to do this.IMPORTANCE The role of human papillomavirus 16 (HPV16) in human cancers is well established; however, to date there are no antiviral therapeutics that are available for combatting these cancers. To identify such targets, we must enhance the understanding of the viral life cycle. Innate immune genes (IIGs) are repressed by HPV16, and we have reported that this repression persists through to cancer. Reversal of this repression would boost the immune response to HPV16-positive tumors, an area that is becoming more important given the advances in immunotherapy. This report demonstrates that E6 and E7 synergistically repress IIG expression in the context of the entire HPV16 genome. Removal of either protein activates the expression of IIGs by HPV16. Therefore, gaining a precise understanding of how the viral oncogenes repress IIG expression represents an opportunity to reverse this repression and boost the immune response to HPV16 infections for therapeutic gain.
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Affiliation(s)
- Claire D James
- Philips Institute for Oral Health Research, School of Dentistry, Virginia Commonwealth University, Richmond, Virginia, USA
| | - Christian T Fontan
- Philips Institute for Oral Health Research, School of Dentistry, Virginia Commonwealth University, Richmond, Virginia, USA
| | - Raymonde Otoa
- Philips Institute for Oral Health Research, School of Dentistry, Virginia Commonwealth University, Richmond, Virginia, USA
| | - Dipon Das
- Philips Institute for Oral Health Research, School of Dentistry, Virginia Commonwealth University, Richmond, Virginia, USA
| | - Apurva T Prabhakar
- Philips Institute for Oral Health Research, School of Dentistry, Virginia Commonwealth University, Richmond, Virginia, USA
| | - Xu Wang
- Philips Institute for Oral Health Research, School of Dentistry, Virginia Commonwealth University, Richmond, Virginia, USA
| | - Molly L Bristol
- Philips Institute for Oral Health Research, School of Dentistry, Virginia Commonwealth University, Richmond, Virginia, USA
| | - Iain M Morgan
- Philips Institute for Oral Health Research, School of Dentistry, Virginia Commonwealth University, Richmond, Virginia, USA
- Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia, USA
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E2 protein is the major determinant of specificity at the human papillomavirus origin of replication. PLoS One 2019; 14:e0224334. [PMID: 31644607 PMCID: PMC6808437 DOI: 10.1371/journal.pone.0224334] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2019] [Accepted: 10/11/2019] [Indexed: 01/21/2023] Open
Abstract
The replication of human papillomavirus (HPV) genomes requires E1 and E2 proteins as the viral trans-factors and the replication origin, located in the URR, as a cis-element. The minimal requirements for an HPV replication origin vary among different virus types but always include one or more binding sites for the E2 protein. The requirements for an E1 binding site seem to vary among different HPV genera, with alpha-HPV11 and -18 minimal origins able to replicate without E1 binding site in contrast to beta-HPV8. In the present article, we analysed the sequence requirements for the beta-HPV5 minimal origin of replication. We show that the HPV5 URR is able to replicate in U2OS cells without the sequence proposed as an E1 binding site, albeit at lower levels than wt URR, given that three E2 binding sites are intact and both viral replication proteins are present. The lack of an absolute requirement of the E1 binding site for the origin of replication of HPV5 led us to analyse whether the viral E1 and E2 proteins from other HPV types are competent to support replication from this origin. Surprisingly, the E1 and E2 proteins from beta-HPV types support replication from the origin in contrast to proteins from alpha-HPV types 11, -16, or -18. Furthermore, the replication proteins E1 and E2 of these alpha-HPV types are unable to support the replication of HPV5 URR, even if the E1 binding site is intact. In light of these results, we performed a detailed analysis of the ability of different combinations of E1 and E2 proteins from various alpha- and beta-HPV types to support the replication of URR sequences from the respective HPV types in the U2OS cell line.
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27
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Das D, Bristol ML, Smith NW, James CD, Wang X, Pichierri P, Morgan IM. Werner Helicase Control of Human Papillomavirus 16 E1-E2 DNA Replication Is Regulated by SIRT1 Deacetylation. mBio 2019; 10:e00263-19. [PMID: 30890607 PMCID: PMC6426601 DOI: 10.1128/mbio.00263-19] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2019] [Accepted: 02/04/2019] [Indexed: 01/03/2023] Open
Abstract
Human papillomaviruses (HPV) are double-stranded DNA viruses causative in a host of human diseases, including several cancers. Following infection, two viral proteins, E1 and E2, activate viral replication in association with cellular factors and stimulate the DNA damage response (DDR) during the replication process. E1-E2 uses homologous recombination (HR) to facilitate DNA replication, but an understanding of host factors involved in this process remains incomplete. Previously, we demonstrated that the class III deacetylase SIRT1, which can regulate HR, is recruited to E1-E2-replicating DNA and regulates the level of replication. Here, we demonstrate that SIRT1 promotes the fidelity of E1-E2 replication and that the absence of SIRT1 results in reduced recruitment of the DNA repair protein Werner helicase (WRN) to E1-E2-replicating DNA. CRISPR/Cas9 editing demonstrates that WRN, like SIRT1, regulates the quantity and fidelity of E1-E2 replication. This is the first report of WRN regulation of E1-E2 DNA replication, or a role for WRN in the HPV life cycle. In the absence of SIRT1 there is an increased acetylation and stability of WRN, but a reduced ability to interact with E1-E2-replicating DNA. We present a model in which E1-E2 replication turns on the DDR, stimulating SIRT1 deacetylation of WRN. This deacetylation promotes WRN interaction with E1-E2-replicating DNA to control the quantity and fidelity of replication. As well as offering a crucial insight into HPV replication control, this system offers a unique model for investigating the link between SIRT1 and WRN in controlling replication in mammalian cells.IMPORTANCE HPV16 is the major viral human carcinogen responsible for between 3 and 4% of all cancers worldwide. Following infection, this virus activates the DNA damage response (DDR) to promote its life cycle and recruits DDR proteins to its replicating DNA in order to facilitate homologous recombination during replication. This promotes the production of viable viral progeny. Our understanding of how HPV16 replication interacts with the DDR remains incomplete. Here, we demonstrate that the cellular deacetylase SIRT1, which is a part of the E1-E2 replication complex, regulates recruitment of the DNA repair protein WRN to the replicating DNA. We demonstrate that WRN regulates the level and fidelity of E1-E2 replication. Overall, the results suggest a mechanism by which SIRT1 deacetylation of WRN promotes its interaction with E1-E2-replicating DNA to control the levels and fidelity of that replication.
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Affiliation(s)
- Dipon Das
- Department of Oral and Craniofacial Molecular Biology, VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Richmond, Virginia, USA
| | - Molly L Bristol
- Department of Oral and Craniofacial Molecular Biology, VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Richmond, Virginia, USA
| | - Nathan W Smith
- Department of Oral and Craniofacial Molecular Biology, VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Richmond, Virginia, USA
| | - Claire D James
- Department of Oral and Craniofacial Molecular Biology, VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Richmond, Virginia, USA
| | - Xu Wang
- Department of Oral and Craniofacial Molecular Biology, VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Richmond, Virginia, USA
| | - Pietro Pichierri
- Department of Environment and Health, Istituto Superiore di Sanità, Rome, Italy
| | - Iain M Morgan
- Department of Oral and Craniofacial Molecular Biology, VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Richmond, Virginia, USA
- VCU Massey Cancer Center, Richmond, Virginia, USA
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Luff J, Mader M, Rowland P, Britton M, Fass J, Yuan H. Viral genome integration of canine papillomavirus 16. PAPILLOMAVIRUS RESEARCH 2019; 7:88-96. [PMID: 30771493 PMCID: PMC6402295 DOI: 10.1016/j.pvr.2019.02.002] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/31/2018] [Revised: 01/30/2019] [Accepted: 02/12/2019] [Indexed: 01/03/2023]
Abstract
Papillomaviruses infect humans and animals, most often causing benign proliferations on skin or mucosal surfaces. Rarely, these infections persist and progress to cancer. In humans, this transformation most often occurs with high-risk papillomaviruses, where viral integration is a critical event in carcinogenesis. The first aim of this study was to sequence the viral genome of canine papillomavirus (CPV) 16 from a pigmented viral plaque that progressed to metastatic squamous cell carcinoma in a dog. The second aim was to characterize multiple viral genomic deletions and translocations as well as host integration sites. The full viral genome was identified using a combination of PCR and high throughput sequencing. CPV16 is most closely related to chipapillomaviruses CPV4, CPV9, and CPV12 and we propose CPV16 be classified as a chipapillomavirus. Assembly of the full viral genome enabled identification of deletion of portions of the E1 and E2/E4 genes and two viral translocations within the squamous cell carcinoma. Genome walking was performed which identified four sites of viral integration into the host genome. This is the first description of integration of a canine papillomavirus into the host genome, raising the possibility that CPV16 may be a potential canine high-risk papillomavirus type.
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Affiliation(s)
- Jennifer Luff
- Department of Population Health and Pathobiology, North Carolina State University, Raleigh, North Carolina, USA.
| | - Michelle Mader
- Department of Population Health and Pathobiology, North Carolina State University, Raleigh, North Carolina, USA
| | | | - Monica Britton
- UC Davis Genome Center-Bioinformatics Core, University of California, Davis, CA, USA
| | - Joseph Fass
- UC Davis Genome Center-Bioinformatics Core, University of California, Davis, CA, USA
| | - Hang Yuan
- Department of Pathology, Georgetown University Medical Center, Washington, DC, USA
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Krump NA, Liu W, You J. Mechanisms of persistence by small DNA tumor viruses. Curr Opin Virol 2018; 32:71-79. [PMID: 30278284 DOI: 10.1016/j.coviro.2018.09.002] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2018] [Revised: 09/07/2018] [Accepted: 09/14/2018] [Indexed: 12/13/2022]
Abstract
Virus infection contributes to nearly 15% of human cancers worldwide. Many of the oncogenic viruses tend to cause cancer in immunosuppressed individuals, but maintain asymptomatic, persistent infection for decades in the general population. In this review, we discuss the tactics employed by two small DNA tumor viruses, Human papillomavirus (HPV) and Merkel cell polyomavirus (MCPyV), to establish persistent infection. We will also highlight recent key findings as well as outstanding questions regarding the mechanisms by which HPV and MCPyV evade host immune control to promote their survival. Since persistent infection enables virus-induced tumorigenesis, identifying the mechanisms by which small DNA tumor viruses achieve latent infection may inform new approaches for preventing and treating their respective human cancers.
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Affiliation(s)
- Nathan A Krump
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Wei Liu
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Jianxin You
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
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30
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Li S, Liu Z, Yan J, Sun S, Hou X, Liu D, Zhang K, Li JT. Integration sites of human papillomavirus 18 in esophageal cancer samples. Oncol Lett 2018; 15:7438-7442. [PMID: 29731894 DOI: 10.3892/ol.2018.8191] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2016] [Accepted: 07/27/2017] [Indexed: 11/06/2022] Open
Abstract
To investigate the association between human papillomavirus (HPV) infection and esophageal cancer, genomic DNA was isolated from 189 samples obtained from patients with esophageal carcinoma, and HPV DNA was identified using the polymerase chain reaction (PCR) with the following specific primers: My09/11 for HPV L1 and HPV18 E6 for HPV18. The HPV18 gene products were sequenced to identify the HPV genotype and the HPV18 integration site was verified using PCR amplification of papillomavirus oncogene transcripts. HPV18 oncogene transcript products were ligated into a pMD-18T plasmid vector and sequenced to confirm the physical location of HPV18 integration. Of the 189 samples, 168 were positive for HPV, of which 33 were positive for HPV18. The sequencing analysis identified two HPV18 E6-positive samples containing one mutation and two samples containing two mutations in the viral DNA. In total ~600 bp of the HPV18 oncogene transcript was detected in three esophageal cancer samples. Sequence analysis revealed that, in two patients, the HPV18 infection was integrated into human chromosome 5, whereas in the remaining sample the virus was integrated into human chromosome 2. The high prevalence of HPV18 infection suggested that HPV18 infection is a pathogenic factor in esophageal carcinoma progression. The integration of HPV18 DNA into the host cell genome suggests that persistent HPV infection has a role in esophageal epithelial cell malignant transformation and carcinogenesis.
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Affiliation(s)
- Shuying Li
- School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, Hebei 063000, P.R. China
| | - Zhanjun Liu
- School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, Hebei 063000, P.R. China
| | - Jianghong Yan
- School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, Hebei 063000, P.R. China
| | - Shangbo Sun
- School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, Hebei 063000, P.R. China
| | - Xiaoli Hou
- School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, Hebei 063000, P.R. China
| | - Dianqing Liu
- School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, Hebei 063000, P.R. China
| | - Ke Zhang
- School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, Hebei 063000, P.R. China
| | - Jin-Tao Li
- College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, P.R. China
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31
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Cruz-Gregorio A, Manzo-Merino J, Lizano M. Cellular redox, cancer and human papillomavirus. Virus Res 2018; 246:35-45. [DOI: 10.1016/j.virusres.2018.01.003] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2017] [Revised: 01/10/2018] [Accepted: 01/10/2018] [Indexed: 12/28/2022]
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Sankovski E, Abroi A, Ustav M, Ustav M. Nuclear myosin 1 associates with papillomavirus E2 regulatory protein and influences viral replication. Virology 2018; 514:142-155. [PMID: 29179037 DOI: 10.1016/j.virol.2017.11.013] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2017] [Revised: 11/09/2017] [Accepted: 11/16/2017] [Indexed: 11/25/2022]
Abstract
Nuclear myosin 1c (NM1) associates with RNA polymerases and is a partner in the chromatin remodeling complex B-WICH. This complex, which also contains WSTF and SNF2h proteins, is involved in transcriptional regulation. We report herein that papillomavirus protein E2 binds to NM1 and co-precipitates with the WSTF and SNF2h proteins. Our data suggest that E2 associates with the cellular B-WICH complex through binding to NM1. E2 and NM1 associate via their N-terminal domains and this interaction is ATP dependent. The cellular multifunctional protein Brd4 and beta-actin are also present in the NM1-E2 complex. NM1 downregulation by siRNA increases the replication of the BPV1 and HPV5 genomes but does not affect HPV18 genome replication. These results suggest that the B-WICH complex may play a role in the papillomavirus life cycle through NM1 and E2 protein interaction.
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Affiliation(s)
- Eve Sankovski
- University of Tartu, Institute of Technology, Nooruse 1, 50411 Tartu, Estonia
| | - Aare Abroi
- Estonian Biocentre, Riia 23, 51010 Tartu, Estonia
| | - Mart Ustav
- University of Tartu, Institute of Technology, Nooruse 1, 50411 Tartu, Estonia; Icosagen Cell Factory OÜ, Eerika tee 1, Õssu küla, Ülenurme vald, 61713 Tartumaa, Estonia
| | - Mart Ustav
- University of Tartu, Institute of Technology, Nooruse 1, 50411 Tartu, Estonia; Icosagen Cell Factory OÜ, Eerika tee 1, Õssu küla, Ülenurme vald, 61713 Tartumaa, Estonia; Estonian Academy of Sciences, Kohtu 6, 10130 Tallinn, Estonia.
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Warburton A, Redmond CJ, Dooley KE, Fu H, Gillison ML, Akagi K, Symer DE, Aladjem MI, McBride AA. HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression. PLoS Genet 2018; 14:e1007179. [PMID: 29364907 PMCID: PMC5798845 DOI: 10.1371/journal.pgen.1007179] [Citation(s) in RCA: 72] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2017] [Revised: 02/05/2018] [Accepted: 01/01/2018] [Indexed: 12/12/2022] Open
Abstract
Integration of human papillomavirus (HPV) genomes into cellular chromatin is common in HPV-associated cancers. Integration is random, and each site is unique depending on how and where the virus integrates. We recently showed that tandemly integrated HPV16 could result in the formation of a super-enhancer-like element that drives transcription of the viral oncogenes. Here, we characterize the chromatin landscape and genomic architecture of this integration locus to elucidate the mechanisms that promoted de novo super-enhancer formation. Using next-generation sequencing and molecular combing/fiber-FISH, we show that ~26 copies of HPV16 are integrated into an intergenic region of chromosome 2p23.2, interspersed with 25 kb of amplified, flanking cellular DNA. This interspersed, co-amplified viral-host pattern is frequent in HPV-associated cancers and here we designate it as Type III integration. An abundant viral-cellular fusion transcript encoding the viral E6/E7 oncogenes is expressed from the integration locus and the chromatin encompassing both the viral enhancer and a region in the adjacent amplified cellular sequences is strongly enriched in the super-enhancer markers H3K27ac and Brd4. Notably, the peak in the amplified cellular sequence corresponds to an epithelial-cell-type specific enhancer. Thus, HPV16 integration generated a super-enhancer-like element composed of tandem interspersed copies of the viral upstream regulatory region and a cellular enhancer, to drive high levels of oncogene expression.
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Affiliation(s)
- Alix Warburton
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Catherine J. Redmond
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Katharine E. Dooley
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Haiqing Fu
- Developmental Therapeutics Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Maura L. Gillison
- Department of Internal Medicine, The Ohio State University, Columbus, Ohio, United States of America
| | - Keiko Akagi
- Department of Cancer Biology and Genetics, The Ohio State University Comprehensive Cancer Center, Columbus, Ohio, United States of America
| | - David E. Symer
- Department of Cancer Biology and Genetics, The Ohio State University Comprehensive Cancer Center, Columbus, Ohio, United States of America
- Human Cancer Genetics Program, Department of Cancer Biology and Genetics, The Ohio State University Comprehensive Cancer Center, Columbus, Ohio, United States of America
- Department of Biomedical Informatics (adjunct), The Ohio State University Comprehensive Cancer Center, Columbus, Ohio, United States of America
| | - Mirit I. Aladjem
- Developmental Therapeutics Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Alison A. McBride
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
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Zhang K, Liu Z, Li J, Li J, Yan J, Su Y, Li S, Li J. Analysis of human papilloma virus type 52 integration status in exfoliated cervical cells. Exp Ther Med 2017; 14:5817-5824. [PMID: 29285126 PMCID: PMC5740806 DOI: 10.3892/etm.2017.5279] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2016] [Accepted: 08/22/2017] [Indexed: 11/25/2022] Open
Abstract
To explore the significance of human papilloma virus type 52 (HPV52) infection and its integration in cells within cervical lesions, the expression levels of HPV52 were detected using polymerase chain reaction (PCR). The copy numbers of HPV52 E2, HPV52 E6 and the reference gene β-actin were determined by quantitative PCR to analyze the association between HPV52 integration and cervical lesions. HPV52 integration was analyzed by the amplification of papillomavirus oncogene transcripts. A total of 13 samples from 468 cases were positive for HPV52. Among the samples, 1 case with an E2/E6 ratio >1 was purely episomal, 3 cases with an E2/E6 ratio of 0 were purely integrated, and 9 cases with an E2/E6 ratio of between 0 and 1 were a mixture of integrated and episomal. With the progression of cervical disease, the prevalence of the episomal type decreased gradually, and the prevalence of the integrated (episomal and integrated) forms increased. The pure integration of HPV52 occurred in chromosomes 2, 5 and 8. These results indicate that HPV52 integration into the host genome may be a key factor in cervical lesions. Thus, patients at high risk for cervical lesions may potentially be identified by screening for HPV52 infection and integration.
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Affiliation(s)
- Ke Zhang
- Hebei Key Laboratory for Chronic Diseases/Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Diseases, School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, Hebei 063000, P.R. China
| | - Zhanjun Liu
- Hebei Key Laboratory for Chronic Diseases/Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Diseases, School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, Hebei 063000, P.R. China
| | - Ji Li
- Hebei Key Laboratory for Chronic Diseases/Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Diseases, School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, Hebei 063000, P.R. China
| | - Juan Li
- Hebei Key Laboratory for Chronic Diseases/Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Diseases, School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, Hebei 063000, P.R. China
| | - Jianghong Yan
- Hebei Key Laboratory for Chronic Diseases/Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Diseases, School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, Hebei 063000, P.R. China
| | - Yunchuan Su
- Hebei Key Laboratory for Chronic Diseases/Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Diseases, School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, Hebei 063000, P.R. China
| | - Shuying Li
- Hebei Key Laboratory for Chronic Diseases/Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Diseases, School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, Hebei 063000, P.R. China
| | - Jintao Li
- College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, P.R. China
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Why Human Papillomaviruses Activate the DNA Damage Response (DDR) and How Cellular and Viral Replication Persists in the Presence of DDR Signaling. Viruses 2017; 9:v9100268. [PMID: 28934154 PMCID: PMC5691620 DOI: 10.3390/v9100268] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2017] [Revised: 09/12/2017] [Accepted: 09/15/2017] [Indexed: 12/15/2022] Open
Abstract
Human papillomaviruses (HPV) require the activation of the DNA damage response (DDR) in order to undergo a successful life cycle. This activation presents a challenge for the virus and the infected cell: how does viral and host replication proceed in the presence of a DDR that ordinarily arrests replication; and how do HPV16 infected cells retain the ability to proliferate in the presence of a DDR that ordinarily arrests the cell cycle? This raises a further question: why do HPV activate the DDR? The answers to these questions are only partially understood; a full understanding could identify novel therapeutic strategies to target HPV cancers. Here, we propose that the rapid replication of an 8 kb double stranded circular genome during infection creates aberrant DNA structures that attract and activate DDR proteins. Therefore, HPV replication in the presence of an active DDR is a necessity for a successful viral life cycle in order to resolve these DNA structures on viral genomes; without an active DDR, successful replication of the viral genome would not proceed. We discuss the essential role of TopBP1 in this process and also how viral and cellular replication proceeds in HPV infected cells in the presence of DDR signals.
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Morgan IM, DiNardo LJ, Windle B. Integration of Human Papillomavirus Genomes in Head and Neck Cancer: Is It Time to Consider a Paradigm Shift? Viruses 2017; 9:v9080208. [PMID: 28771189 PMCID: PMC5580465 DOI: 10.3390/v9080208] [Citation(s) in RCA: 51] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2017] [Revised: 07/28/2017] [Accepted: 07/31/2017] [Indexed: 12/14/2022] Open
Abstract
Human papillomaviruses (HPV) are detected in 70–80% of oropharyngeal cancers in the developed world, the incidence of which has reached epidemic proportions. The current paradigm regarding the status of the viral genome in these cancers is that there are three situations: one where the viral genome remains episomal, one where the viral genome integrates into the host genome and a third where there is a mixture of both integrated and episomal HPV genomes. Our recent work suggests that this third category has been mischaracterized as having integrated HPV genomes; evidence indicates that this category consists of virus–human hybrid episomes. Most of these hybrid episomes are consistent with being maintained by replication from HPV origin. We discuss our evidence to support this new paradigm, how such genomes can arise, and more importantly the implications for the clinical management of HPV positive head and neck cancers following accurate determination of the viral genome status.
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Affiliation(s)
- Iain M Morgan
- Philips Institute for Oral Health Research, Virginia Commonwealth University (VCU) School of Dentistry, Department of Oral and Craniofacial Molecular Biology, Richmond, VA 23298, USA.
- VCU Massey Cancer Center, Richmond, VA 23298, USA.
| | - Laurence J DiNardo
- VCU Massey Cancer Center, Richmond, VA 23298, USA.
- VCU Department of Otolaryngology, Richmond, VA 23298, USA.
| | - Brad Windle
- Philips Institute for Oral Health Research, Virginia Commonwealth University (VCU) School of Dentistry, Department of Oral and Craniofacial Molecular Biology, Richmond, VA 23298, USA.
- VCU Massey Cancer Center, Richmond, VA 23298, USA.
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The Deacetylase SIRT1 Regulates the Replication Properties of Human Papillomavirus 16 E1 and E2. J Virol 2017; 91:JVI.00102-17. [PMID: 28275188 DOI: 10.1128/jvi.00102-17] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2017] [Accepted: 02/23/2017] [Indexed: 12/23/2022] Open
Abstract
Human papillomaviruses (HPV) replicate their genomes in differentiating epithelium using the viral proteins E1 and E2 in association with host proteins. While the roles of E1 and E2 in this process are understood, the host factors involved and how they interact with and regulate E1-E2 are not. Our previous work identified the host replication and repair factor TopBP1 as an E2 partner protein essential for optimal E1-E2 replication and for the viral life cycle. The role of TopBP1 in host DNA replication is regulated by the class III deacetylase SIRT1; activation of the DNA damage response prevents SIRT1 deacetylation of TopBP1, resulting in a switch from DNA replication to repair functions for this protein and cell cycle arrest. Others have demonstrated an essential role for SIRT1 in regulation of the HPV31 life cycle; here, we report that SIRT1 can directly regulate HPV16 E1-E2-mediated DNA replication. SIRT1 is part of the E1-E2 DNA replication complex and is recruited to the viral origin of replication in an E1-E2-dependent manner. CRISPR/Cas9 was used to generate C33a clones with undetectable SIRT1 expression and lack of SIRT1 elevated E1-E2 DNA replication, in part due to increased acetylation and stabilization of the E2 protein in the absence of SIRT1. The results demonstrate that SIRT1 is a member of, and can regulate, the HPV16 replication complex. We discuss the potential role of this protein in the viral life cycle.IMPORTANCE HPV are causative agents in a number of human diseases, and currently only the symptoms of these diseases are treated. To identify novel therapeutic approaches for combating these diseases, the viral life cycle must be understood in more detail. This report demonstrates that a cellular enzyme, SIRT1, is part of the HPV16 DNA replication complex and is brought to the viral genome by the viral proteins E1 and E2. Using gene editing technology (CRISPR/Cas9), the SIRT1 gene was removed from cervical cancer cells. The consequence of this was that viral replication was elevated, probably due to a stabilization of the viral replication factor E2. The overall results demonstrate that an enzyme with known inhibitors, SIRT1, plays an important role in controlling how HPV16 makes copies of itself. Targeting this enzyme could be a new therapeutic approach for combating HPV spread and disease.
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Affiliation(s)
- Alison A. McBride
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Alix Warburton
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
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Association of Human Papillomavirus 16 E2 with Rad50-Interacting Protein 1 Enhances Viral DNA Replication. J Virol 2017; 91:JVI.02305-16. [PMID: 28031358 PMCID: PMC5309968 DOI: 10.1128/jvi.02305-16] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2016] [Accepted: 12/15/2016] [Indexed: 12/19/2022] Open
Abstract
Rad50-interacting protein 1 (Rint1) associates with the DNA damage response protein Rad50 during the transition from the S phase to the G2/M phase and functions in radiation-induced G2 checkpoint control. It has also been demonstrated that Rint1 is essential in vesicle trafficking from the Golgi apparatus to the endoplasmic reticulum (ER) through an interaction with Zeste-White 10 (ZW10). We have isolated a novel interaction between Rint1 and the human papillomavirus 16 (HPV16) transcription and replication factor E2. E2 binds to Rint1 within its ZW10 interaction domain, and we show that in the absence of E2, Rint1 is localized to the ER and associates with ZW10. E2 expression results in a disruption of the Rint1-ZW10 interaction and an accumulation of nuclear Rint1, coincident with a significant reduction in vesicle movement from the ER to the Golgi apparatus. Interestingly, nuclear Rint1 and members of the Mre11/Rad50/Nbs1 (MRN) complex were found in distinct E2 nuclear foci, which peaked during mid-S phase, indicating that the recruitment of Rint1 to E2 foci within the nucleus may also result in the recruitment of this DNA damage-sensing protein complex. We show that exogenous Rint1 expression enhances E2-dependent virus replication. Conversely, the overexpression of a truncated Rint1 protein that retains the E2 binding domain but not the Rad50 binding domain acts as a dominant negative inhibitor of E2-dependent HPV replication. Put together, these experiments demonstrate that the interaction between Rint1 and E2 has an important function in HPV replication. IMPORTANCE HPV infections are an important driver of many epithelial cancers, including those within the anogenital and oropharyngeal tracts. The HPV life cycle is tightly regulated and intimately linked to the differentiation of the epithelial cells that it infects. HPV replication factories formed in the nucleus are locations where viral DNA is copied to support virus persistence and amplification of infection. The recruitment of specific cellular protein complexes to these factories aids efficient and controlled viral replication. We have identified a novel HPV-host interaction that functions in the cellular response to DNA damage and cell cycle control. We show that the HPV E2 protein targets Rad50-interacting protein 1 (Rint1) to facilitate virus genome replication. These findings add to our understanding of how HPV replicates and the host cell pathways that are targeted by HPV to support virus replication. Understanding these pathways will allow further research into novel inhibitors of HPV genome replication.
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Abstract
As the ratio of the copy number of the most replicated to the unreplicated regions in the same chromosome, the definition of chromosomal replication complexity (CRC) appears to leave little room for variation, being either two during S-phase or one otherwise. However, bacteria dividing faster than they replicate their chromosome spike CRC to four and even eight. A recent experimental inquiry about the limits of CRC in Escherichia coli revealed two major reasons to avoid elevating it further: (i) increased chromosomal fragmentation and (ii) complications with subsequent double-strand break repair. Remarkably, examples of stable elevated CRC in eukaryotic chromosomes are well known under various terms like "differential replication," "underreplication," "DNA puffs," "onion-skin replication," or "re-replication" and highlight the phenomenon of static replication fork (sRF). To accurately describe the resulting "amplification by overinitiation," I propose a new term: "replification" (subchromosomal overreplication). In both prokaryotes and eukaryotes, replification, via sRF processing, causes double-strand DNA breaks and, with their repair elevating chromosomal rearrangements, represents a novel genome instability factor. I suggest how static replication bubbles could be stabilized and speculate that some tandem duplications represent such persistent static bubbles. Moreover, I propose how static replication bubbles could be transformed into tandem duplications, double minutes, or inverted triplications. Possible experimental tests of these models are discussed.
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Affiliation(s)
- Andrei Kuzminov
- Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America
- * E-mail:
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Bristol ML, Wang X, Smith NW, Son MP, Evans MR, Morgan IM. DNA Damage Reduces the Quality, but Not the Quantity of Human Papillomavirus 16 E1 and E2 DNA Replication. Viruses 2016; 8:v8060175. [PMID: 27338449 PMCID: PMC4926195 DOI: 10.3390/v8060175] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2016] [Revised: 06/14/2016] [Accepted: 06/16/2016] [Indexed: 12/19/2022] Open
Abstract
Human papillomaviruses (HPVs) are causative agents in almost all cervical carcinomas. HPVs are also causative agents in head and neck cancer, the cases of which are increasing rapidly. Viral replication activates the DNA damage response (DDR) pathway; associated proteins are recruited to replication foci, and this pathway may serve to allow for viral genome amplification. Likewise, HPV genome double-strand breaks (DSBs) could be produced during replication and could lead to linearization and viral integration. Many studies have shown that viral integration into the host genome results in unregulated expression of the viral oncogenes, E6 and E7, promoting HPV-induced carcinogenesis. Previously, we have demonstrated that DNA-damaging agents, such as etoposide, or knocking down viral replication partner proteins, such as topoisomerase II β binding protein I (TopBP1), does not reduce the level of DNA replication. Here, we investigated whether these treatments alter the quality of DNA replication by HPV16 E1 and E2. We confirm that knockdown of TopBP1 or treatment with etoposide does not reduce total levels of E1/E2-mediated DNA replication; however, the quality of replication is significantly reduced. The results demonstrate that E1 and E2 continue to replicate under genomically-stressed conditions and that this replication is mutagenic. This mutagenesis would promote the formation of substrates for integration of the viral genome into that of the host, a hallmark of cervical cancer.
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Affiliation(s)
- Molly L Bristol
- VCU Philips Institute for Oral Health Research, Department of Oral and Craniofacial Molecular Biology, Virginia Commonwealth University School of Dentistry, Richmond, VA 23298, USA.
| | - Xu Wang
- VCU Philips Institute for Oral Health Research, Department of Oral and Craniofacial Molecular Biology, Virginia Commonwealth University School of Dentistry, Richmond, VA 23298, USA.
| | - Nathan W Smith
- VCU Philips Institute for Oral Health Research, Department of Oral and Craniofacial Molecular Biology, Virginia Commonwealth University School of Dentistry, Richmond, VA 23298, USA.
| | - Minkyeong P Son
- VCU Philips Institute for Oral Health Research, Department of Oral and Craniofacial Molecular Biology, Virginia Commonwealth University School of Dentistry, Richmond, VA 23298, USA.
| | - Michael R Evans
- VCU Philips Institute for Oral Health Research, Department of Oral and Craniofacial Molecular Biology, Virginia Commonwealth University School of Dentistry, Richmond, VA 23298, USA.
| | - Iain M Morgan
- VCU Philips Institute for Oral Health Research, Department of Oral and Craniofacial Molecular Biology, Virginia Commonwealth University School of Dentistry, Richmond, VA 23298, USA.
- VCU Massey Cancer Center, Richmond, VA 23298, USA.
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Chen Wongworawat Y, Filippova M, Williams VM, Filippov V, Duerksen-Hughes PJ. Chronic oxidative stress increases the integration frequency of foreign DNA and human papillomavirus 16 in human keratinocytes. Am J Cancer Res 2016; 6:764-80. [PMID: 27186429 PMCID: PMC4859882] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2016] [Accepted: 02/03/2016] [Indexed: 06/05/2023] Open
Abstract
Cervical cancer is the second most common cancer, and the fourth most common cause of cancer death in women worldwide. Nearly all of these cases are caused by high-risk HPVs (HR HPVs), of which HPV16 is the most prevalent type. In most cervical cancer specimens, HR HPVs are found integrated into the human genome, indicating that integration is a key event in cervical tumor development. An understanding of the mechanisms that promote integration may therefore represent a unique opportunity to intercept carcinogenesis. To begin identifying these mechanisms, we tested the hypothesis that chronic oxidative stress (OS) induced by virus- and environmentallymediated factors can induce DNA damage, and thereby increase the frequency with which HPV integrates into the host genome. We found that virus-mediated factors are likely involved, as expression of E6*, a splice isoform of HPV16 E6, increased the levels of reactive oxygen species (ROS), caused oxidative DNA damage, and increased the frequency of plasmid DNA integration as assessed by colony formation assays. To assess the influence of environmentally induced chronic OS, we used L-Buthionine-sulfoximine (BSO) to lower the level of the intracellular antioxidant glutathione. Similar to our observations with E6*, glutathione depletion by BSO also increased ROS levels, caused oxidative DNA damage and increased the integration frequency of plasmid DNA. Finally, under conditions of chronic OS, we were able to induce and characterize a few independent events in which episomal HPV16 integrated into the host genome of cervical keratinocytes. Our results support a chain of events leading from induction of oxidative stress, to DNA damage, to viral integration, and ultimately to carcinogenesis.
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Affiliation(s)
- Yan Chen Wongworawat
- Department of Basic Science, Loma Linda University School of Medicine Loma Linda, CA, USA
| | - Maria Filippova
- Department of Basic Science, Loma Linda University School of Medicine Loma Linda, CA, USA
| | - Vonetta M Williams
- Department of Basic Science, Loma Linda University School of Medicine Loma Linda, CA, USA
| | - Valery Filippov
- Department of Basic Science, Loma Linda University School of Medicine Loma Linda, CA, USA
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Mutagenic Potential ofBos taurus Papillomavirus Type 1 E6 Recombinant Protein: First Description. BIOMED RESEARCH INTERNATIONAL 2015; 2015:806361. [PMID: 26783529 PMCID: PMC4689895 DOI: 10.1155/2015/806361] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/16/2015] [Revised: 10/07/2015] [Accepted: 10/15/2015] [Indexed: 01/16/2023]
Abstract
Bovine papillomavirus (BPV) is considered a useful model to study HPV oncogenic process. BPV interacts with the host chromatin, resulting in DNA damage, which is attributed to E5, E6, and E7 viral oncoproteins activity. However, the oncogenic mechanisms of BPV E6 oncoprotein per se remain unknown. This study aimed to evaluate the mutagenic potential of Bos taurus papillomavirus type 1 (BPV-1) E6 recombinant oncoprotein by the cytokinesis-block micronucleus assay (CBMNA) and comet assay (CA). Peripheral blood samples of five calves were collected. Samples were subjected to molecular diagnosis, which did not reveal presence of BPV sequences. Samples were treated with 1 μg/mL of BPV-1 E6 oncoprotein and 50 μg/mL of cyclophosphamide (positive control). Negative controls were not submitted to any treatment. The samples were submitted to the CBMNA and CA. The results showed that BPV E6 oncoprotein induces clastogenesis per se, which is indicative of genomic instability. These results allowed better understanding the mechanism of cancer promotion associated with the BPV E6 oncoprotein and revealed that this oncoprotein can induce carcinogenesis per se. E6 recombinant oncoprotein has been suggested as a possible vaccine candidate. Results pointed out that BPV E6 recombinant oncoprotein modifications are required to use it as vaccine.
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Ustav M, Castaneda FR, Reinson T, Männik A, Ustav M. Human Papillomavirus Type 18 cis-Elements Crucial for Segregation and Latency. PLoS One 2015; 10:e0135770. [PMID: 26288015 PMCID: PMC4545946 DOI: 10.1371/journal.pone.0135770] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2015] [Accepted: 07/24/2015] [Indexed: 11/29/2022] Open
Abstract
Stable maintenance replication is characteristic of the latency phase of HPV infection, during which the viral genomes are actively maintained as extrachromosomal genetic elements in infected proliferating basal keratinocytes. Active replication in the S-phase and segregation of the genome into daughter cells in mitosis are required for stable maintenance replication. Most of our knowledge about papillomavirus genome segregation has come from studies of bovine papillomavirus type 1 (BPV-1), which have demonstrated that the E2 protein cooperates with cellular trans-factors and that E2 binding sites act as cis-regulatory elements in the viral genome that are essential for the segregation process. However, the genomic organization of the regulatory region in HPVs, and the properties of the viral proteins are different from those of their BPV-1 counterparts. We have designed a segregation assay for HPV-18 and used it to demonstrate that the E2 protein performs segregation in combination with at least two E2 binding sites. The cooperative binding of the E2 protein to two E2 binding sites is a major determinant of HPV-18 genome segregation, as demonstrated by the change in spacing between adjacent binding sites #1 and #2 in the HPV-18 Upstream Regulatory Region (URR). Duplication or triplication of the natural 4 bp 5’-CGGG-3’ spacer between the E2 binding sites increased the cooperative binding of the E2 molecules as well as E2-dependent segregation. Removal of any spacing between these sites eliminated cooperative binding of the E2 protein and disabled segregation of the URR and HPV-18 genome. Transfer of these configurations of the E2 binding sites into viral genomes confirmed the role of the E2 protein and binding sites #1 and #2 in the segregation process. Additional analysis demonstrated that these sites also play an important role in the transcriptional regulation of viral gene expression from different HPV-18 promoters.
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Affiliation(s)
- Mart Ustav
- University of Tartu, Institute of Technology, Tartu, Estonia
| | | | - Tormi Reinson
- University of Tartu, Institute of Technology, Tartu, Estonia
| | | | - Mart Ustav
- University of Tartu, Institute of Technology, Tartu, Estonia
- Icosagen Cell Factory OÜ, Tartu, Estonia
- Estonian Academy of Sciences, Tallinn, Estonia
- * E-mail:
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Visalli G, Riso R, Facciolà A, Mondello P, Caruso C, Picerno I, Di Pietro A, Spataro P, Bertuccio MP. Higher levels of oxidative DNA damage in cervical cells are correlated with the grade of dysplasia and HPV infection. J Med Virol 2015; 88:336-44. [DOI: 10.1002/jmv.24327] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/04/2015] [Indexed: 01/08/2023]
Affiliation(s)
- Giuseppa Visalli
- Department of Biomedical Sciences and Morphological and Functional Images; University of Messina; Messina Italy
| | - Romana Riso
- Department of Biomedical Sciences and Morphological and Functional Images; University of Messina; Messina Italy
| | - Alessio Facciolà
- Department of Biomedical Sciences and Morphological and Functional Images; University of Messina; Messina Italy
| | | | - Carmela Caruso
- Department of Paediatric, Gynaecological, Microbiological and Biomedical Sciences; University of Messina; Messina Italy
| | - Isa Picerno
- Department of Biomedical Sciences and Morphological and Functional Images; University of Messina; Messina Italy
| | - Angela Di Pietro
- Department of Biomedical Sciences and Morphological and Functional Images; University of Messina; Messina Italy
| | - Pasquale Spataro
- Department of Biomedical Sciences and Morphological and Functional Images; University of Messina; Messina Italy
| | - Maria Paola Bertuccio
- Department of Biomedical Sciences and Morphological and Functional Images; University of Messina; Messina Italy
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Modulation of DNA damage and repair pathways by human tumour viruses. Viruses 2015; 7:2542-91. [PMID: 26008701 PMCID: PMC4452920 DOI: 10.3390/v7052542] [Citation(s) in RCA: 67] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2015] [Accepted: 05/12/2015] [Indexed: 02/07/2023] Open
Abstract
With between 10% and 15% of human cancers attributable to viral infection, there is great interest, from both a scientific and clinical viewpoint, as to how these pathogens modulate host cell functions. Seven human tumour viruses have been identified as being involved in the development of specific malignancies. It has long been known that the introduction of chromosomal aberrations is a common feature of viral infections. Intensive research over the past two decades has subsequently revealed that viruses specifically interact with cellular mechanisms responsible for the recognition and repair of DNA lesions, collectively known as the DNA damage response (DDR). These interactions can involve activation and deactivation of individual DDR pathways as well as the recruitment of specific proteins to sites of viral replication. Since the DDR has evolved to protect the genome from the accumulation of deleterious mutations, deregulation is inevitably associated with an increased risk of tumour formation. This review summarises the current literature regarding the complex relationship between known human tumour viruses and the DDR and aims to shed light on how these interactions can contribute to genomic instability and ultimately the development of human cancers.
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McKinney CC, Hussmann KL, McBride AA. The Role of the DNA Damage Response throughout the Papillomavirus Life Cycle. Viruses 2015; 7:2450-69. [PMID: 26008695 PMCID: PMC4452914 DOI: 10.3390/v7052450] [Citation(s) in RCA: 61] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2015] [Accepted: 05/13/2015] [Indexed: 12/25/2022] Open
Abstract
The DNA damage response (DDR) maintains genomic integrity through an elaborate network of signaling pathways that sense DNA damage and recruit effector factors to repair damaged DNA. DDR signaling pathways are usurped and manipulated by the replication programs of many viruses. Here, we review the papillomavirus (PV) life cycle, highlighting current knowledge of how PVs recruit and engage the DDR to facilitate productive infection.
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Affiliation(s)
- Caleb C McKinney
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
| | - Katherine L Hussmann
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
| | - Alison A McBride
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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The transcription map of HPV11 in U2OS cells adequately reflects the initial and stable replication phases of the viral genome. Virol J 2015; 12:59. [PMID: 25890000 PMCID: PMC4414447 DOI: 10.1186/s12985-015-0292-6] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2015] [Accepted: 04/01/2015] [Indexed: 12/20/2022] Open
Abstract
BACKGROUND Although prophylactic vaccines have been developed against HPV6, HPV11, HPV16 and HPV18 there is the clear unmet medical need in order to justify the development of drugs targeting human papillomavirus replication. The native host cells of HPVs are human primary keratinocytes which can be cultivated in raft cultures. However, this method is difficult to use in high-throughput screening assays and the need for a cost-effective cellular system for screening potential anti-HPV drug candidates during all stages of HPV genome replication remains. METHODS U2OS cells were transfected with HPV11 wt or E8- minicircle genomes and their gene expression was studied via 3' RACE, 5' RACE or via real time PCR methods. The DNA replication of these genomes was detected by Southern blot methods. RESULTS The analysis of HPV11 transcripts in U2OS cells showed that the patterns of promoter use, splice sites and polyadenylation cleavage sites are identical to those previously characterized in human HPV-related lesions, human squamous carcinoma cell lines (e.g., SSC-4) and laryngeal papillomas. Transcriptional initiation from the three previously described HPV11 promoters in the E6 and E7 ORFs (P90, P264, and P674-714) were functional, and these promoters were used together with two promoter regions in the E1 ORF (P1092 and P1372). Mutating the E8 ORF ATG start codon to ACG eliminated the translation of fusion proteins from the E8 ORF coupled to E1 and E2 proteins C-terminal sequences, leading to the de-repression of gene expression (particularly from the P1092 promoter) and to the activation of genome replication. These data suggested that the expression of the functional E8^E2 protein is used to control viral gene expression and copy number of the HPV11 genome. The analysis of HPV11 E1 expression plasmids showed that the E6/E7 region, together with the E1 coding region, is crucial for the production of functionally active E1 protein. CONCLUSIONS The data presented in this paper suggest that in human osteosarcoma cell line U2OS the gene expression pattern of the HPV11 truly reflect the expression profile of the replicating HPV genome and therefore this cellular system is suitable for drug development program targeting HPV replication.
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Evidence supporting a role for TopBP1 and Brd4 in the initiation but not continuation of human papillomavirus 16 E1/E2-mediated DNA replication. J Virol 2015; 89:4980-91. [PMID: 25694599 DOI: 10.1128/jvi.00335-15] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2015] [Accepted: 02/12/2015] [Indexed: 12/17/2022] Open
Abstract
UNLABELLED To replicate the double-stranded human papillomavirus 16 (HPV16) DNA genome, viral proteins E1 and E2 associate with the viral origin of replication, and E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated the Brd4 requirement for the transactivation properties of E2, while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More-recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here, we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2-containing nuclear foci, and the viral origin of replication is required for the efficient formation of these foci. Short hairpin RNA (shRNA) against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2-mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall, the results suggest that interactions between TopBP1 and E2 and between Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination. IMPORTANCE Human papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers, and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations, but antivirals targeting HPV16 are required for the alleviation of disease burden on the current, and future, generations. Targeting viral DNA replication that is mediated by two viral proteins, E1 and E2, in association with cellular proteins such as TopBP1 and Brd4 would have therapeutic benefits. This report suggests a role for these cellular proteins in the initiation of viral DNA replication by HPV16 E1-E2 but not for continuing replication. This is important if viral replication is to be effectively targeted; we need to understand the viral and cellular proteins required at each phase of viral DNA replication so that it can be effectively disrupted.
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Wendzicki JA, Moore PS, Chang Y. Large T and small T antigens of Merkel cell polyomavirus. Curr Opin Virol 2015; 11:38-43. [PMID: 25681708 DOI: 10.1016/j.coviro.2015.01.009] [Citation(s) in RCA: 81] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2014] [Accepted: 01/19/2015] [Indexed: 12/16/2022]
Abstract
Merkel cell polyomavirus (MCV) is the etiological agent of Merkel cell carcinoma (MCC), a rare and highly lethal human skin cancer. A natural component of skin flora, MCV becomes tumorigenic only after integration into the host DNA together with specific mutations to the viral genome. Research on MCV large T (LT) and small T (sT) antigens, the only viral products expressed in MCC, shows that these major oncoproteins not only possess biochemical functions found in common with other polyomavirus T antigens, but also demonstrate new cellular targets not described in previous polyomavirus models. This review provides a map of the relevant functional motifs and domains in MCV T antigens that have been identified, highlighting their roles in tumorigenesis.
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Affiliation(s)
- Justin A Wendzicki
- Cancer Virology Program, University of Pittsburgh Cancer Institute, Suite 1.8, 5117 Centre Avenue, Pittsburgh, PA 15213, United States
| | - Patrick S Moore
- Cancer Virology Program, University of Pittsburgh Cancer Institute, Suite 1.8, 5117 Centre Avenue, Pittsburgh, PA 15213, United States.
| | - Yuan Chang
- Cancer Virology Program, University of Pittsburgh Cancer Institute, Suite 1.8, 5117 Centre Avenue, Pittsburgh, PA 15213, United States.
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