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Structure based identification and characterization of flavonoids that disrupt human papillomavirus-16 E6 function.

ABSTRACT: Expression and function of the human papillomavirus (HPV) early protein 6 (E6) is necessary for viral replication and oncogenesis in cervical cancers. HPV E6 targets the tumor suppressor protein p53 for degradation. To achieve this, "high-risk" HPV E6 proteins bind to and modify the target specificity of the ubiquitin ligase E6AP (E6 associated protein). This E6-dependent loss of p53 enables the virus to bypass host cell defenses and facilitates virally induced activation of the cell cycle progression during viral replication. Disruption of the interaction between E6 and E6AP and stabilization of p53 should decrease viability and proliferation of HPV positive cells. A new in vitro high-throughput binding assay was developed to assay binding between HPV-16 E6 and E6AP and to identify compounds that inhibit this interaction. The compound luteolin emerged from the screen and a library of novel flavones based on its structure was synthesized and characterized using this in vitro binding assay. The compounds identified in this study disrupt the E6/E6AP interaction, increase the levels of p53 and p21(Cip1/Waf1), and decrease proliferation of HPV positive cell lines. The new class of flavonoid E6 inhibitors displays a high degree of specificity for HPV positive cells. Docking analyses suggest that these compounds bind in a hydrophobic pocket at the interface between E6 and E6AP and mimic the leucines in the conserved ?-helical motif of E6AP. The activity and specificity of these compounds represent a promising new lead for development as an antiviral therapy in the treatment of HPV infection and cervical cancer.

SUBMITTER: Cherry JJ

PROVIDER: S-EPMC3871595 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Structure based identification and characterization of flavonoids that disrupt human papillomavirus-16 E6 function.

Cherry Jonathan J JJ Rietz Anne A Malinkevich Anna A Liu Yuqi Y Xie Meng M Bartolowits Matthew M Davisson V Jo VJ Baleja James D JD Androphy Elliot J EJ

PloS one 20131223 12

Expression and function of the human papillomavirus (HPV) early protein 6 (E6) is necessary for viral replication and oncogenesis in cervical cancers. HPV E6 targets the tumor suppressor protein p53 for degradation. To achieve this, "high-risk" HPV E6 proteins bind to and modify the target specificity of the ubiquitin ligase E6AP (E6 associated protein). This E6-dependent loss of p53 enables the virus to bypass host cell defenses and facilitates virally induced activation of the cell cycle progr ...[more]

PMID: 24376816

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Project description:High-risk human papillomavirus (HPV) is a major risk factor for oral and pharyngeal cancers (OPCs), yet the detailed mechanisms by which HPV promotes OPCs are not understood. Forkhead box M1B (FoxM1B) is an oncogene essential for cell cycle progression and tumorigenesis, and it is aberrantly overexpressed in many tumors. We previously showed that FoxM1B was the putative target of an epithelial-specific transcription factor, Grainyhead-like 2 (GRHL2). In the current study, we demonstrate that HPV type 16 (HPV-16) E6 induces FoxM1B in human oral keratinocytes (HOKs) and tonsillar epithelial cells (TECs) in part through GRHL2. FoxM1B was barely detectable in cultured normal human oral keratinocytes (NHOKs) and progressively increased in immortalized HOKs harboring HPV-16 genome (HOK-16B) and tumorigenic HOK-16B/BaP-T cells. Retroviral expression of HPV-16 E6 and/or E7 in NHOKs, TECs, and hypopharyngeal carcinoma cells (FaDu) revealed induction of FoxM1B and GRHL2 by the E6 protein but not E7. Both GRHL2 and FoxM1B were strongly induced in the epidermis of HPV-16 E6 transgenic mice and HPV+ oral squamous cell carcinomas. Ectopic expression of FoxM1B led to acquisition of transformed phenotype in HOK-16B cells. Loss of FoxM1B by lentiviral short hairpin RNA vector or chemical inhibitor led to elimination of tumorigenic characteristics of HOK-16B/BaP-T cells. Luciferase reporter assay revealed that GRHL2 directly bound and regulated the FoxM1B gene promoter activity. Using epithelial-specific Grhl2 conditional knockout mice, we exposed wild-type (WT) and Grhl2 KO mice to 4-nitroquinolin 1-oxide (4-NQO), which led to induction of FoxM1B in the tongue tissues and rampant oral tumor development in the WT mice. However, 4-NQO exposure failed to induce tongue tumors or induction of FoxM1B expression in Grhl2 KO mice. Collectively, these results indicate that HPV-16 induces FoxM1B in part through GRHL2 transcriptional activity and that elevated FoxM1B level is required for oropharyngeal cancer development.

Project description:BACKGROUND:Human papillomavirus 16 (HPV16) E6 antibodies may be an early marker of the diagnosis and recurrence of human papillomavirus-driven oropharyngeal cancer (HPV-OPC). METHODS:This study identified 161 incident oropharyngeal cancer (OPC) cases diagnosed at the University of Pittsburgh (2003-2013) with pretreatment serum. One hundred twelve had preexisting clinical HPV testing with p16 immunohistochemistry and HPV in situ hybridization (87 were dual-positive [HPV-OPC], and 25 were dual-negative [HPV-negative]); 62 had at least 1 posttreatment serum sample. Eighty-six of the 161 tumors were available for additional HPV16 DNA/RNA testing (45 were dual-positive [HPV16-OPC], and 19 were dual-negative [HPV16-negative). HPV16 E6 antibody testing was conducted with multiplex serology. The following were evaluated: 1) the sensitivity and specificity of HPV16 E6 serology for distinguishing HPV-OPC and HPV16-OPC from HPV-negative OPC, 2) HPV16 E6 antibody decay after treatment with linear models accommodating correlations in variance estimates, and 3) pre- and posttreatment HPV16 E6 levels and the risk of recurrence with Cox proportional hazards models. RESULTS:Seventy-eight of 87 HPV-OPCs were HPV16 E6-seropositive (sensitivity, 89.7%; 95% confidence interval [CI], 81.3%-95.2%), and 24 of 25 HPV-negative OPCs were HPV16 E6-seronegative (specificity, 96.0%; 95% CI, 79.6%-99.9%). Forty-two of 45 HPV16-OPCs were HPV16 E6-seropositive (sensitivity, 93.3%; 95% CI, 81.7%-98.6%), and 18 of 19 HPV16-negative OPCs were HPV16 E6-seronegative (specificity, 94.7%; 95% CI, 74.0%-99.9%). Posttreatment HPV16 E6 antibody levels did not decrease significantly from the baseline (P = .575; median follow-up, 307 days) and were not associated with the risk of recurrence. However, pretreatment HPV16 E6 seropositivity was associated with an 86% reduced risk of local/regional recurrence (hazard ratio, 0.14; 95% CI, 0.03-0.68; P = .015). CONCLUSIONS:HPV16 E6 antibodies may have potential clinical utility for the diagnosis and/or prognosis of HPV-OPC. Cancer 2017;123:4382-90. © 2017 American Cancer Society.

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

Dataset Information

Structure based identification and characterization of flavonoids that disrupt human papillomavirus-16 E6 function.

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Structure based identification and characterization of flavonoids that disrupt human papillomavirus-16 E6 function.

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