scholarly journals Papillomavirus Type 16 Oncogenes Downregulate Expression of Interferon-Responsive Genes and Upregulate Proliferation-Associated and NF-κB-Responsive Genes in Cervical Keratinocytes

2001 ◽  
Vol 75 (9) ◽  
pp. 4283-4296 ◽  
Author(s):  
Matthias Nees ◽  
Joel M. Geoghegan ◽  
Tehila Hyman ◽  
Stephan Frank ◽  
Lance Miller ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
P53 Protein ◽  
Human Papillomaviruses ◽  
Dominant Negative ◽  
Hpv 16 ◽  
Interacting Protein ◽  
Hpv E6 ◽  
E6 And E7 ◽  
E6 Protein ◽  
Functional Components

ABSTRACT Infection with high-risk human papillomaviruses (HPV) is a major risk factor for development of cervical cancer. Expression of the HPV E6 and E7 oncoproteins increases in differentiating keratinocytes, resulting in inactivation of the p53 and retinoblastoma proteins, two important transcriptional regulators. We used cDNA microarrays to examine global alterations in gene expression in differentiating cervical keratinocytes after infection with retroviruses encoding HPV type 16 (HPV-16) E6 and E7. Expression of 80 cellular genes (approximately 4% of the genes on the array) was altered reproducibly by E6 and/or E7. Cluster analysis classified these genes into three functional groups: (i) interferon (IFN)-responsive genes, (ii) genes stimulated by NF-κB, and (iii) genes regulated in cell cycle progression and DNA synthesis. HPV-16 E6 or a dominant negative p53 protein downregulated multiple IFN-responsive genes. E6 decreased expression of IFN-α and -β, downregulated nuclear STAT-1 protein, and decreased binding of STAT-1 to the IFN-stimulated response element. E7 alone was less effective; however, coexpression of E6 and E7 downregulated IFN-responsive genes more efficiently than E6. The HPV-16 E6 protein also stimulated expression of multiple genes known to be inducible by NF-κB and AP-1. E6 enhanced expression of functional components of the NF-κB signal pathway, including p50, NIK, and TRAF-interacting protein, and increased binding to NF-κB and AP-1 DNA consensus binding sites. Secretion of interleukin-8, RANTES, macrophage inflammatory protein 1α, and 10-κDa IFN-γ-inducible protein were increased in differentiating keratinocytes by E6. Thus, high-level expression of the HPV-16 E6 protein in differentiating keratinocytes directly alters expression of genes that influence host resistance to infection and immune function.

scholarly journals The E6 Oncoproteins of High-Risk Papillomaviruses Bind to a Novel Putative GAP Protein, E6TP1, and Target It for Degradation

1999 ◽  
Vol 19 (1) ◽  
pp. 733-744 ◽  
Author(s):  
Qingshen Gao ◽  
Seetha Srinivasan ◽  
Sarah N. Boyer ◽  
David E. Wazer ◽  
Vimla Band
Keyword(s):  
High Risk ◽  
P53 Protein ◽  
Single Gene ◽  
Human Papillomaviruses ◽  
Dominant Negative ◽  
Hpv16 E6 ◽  
Hpv E6 ◽  
E6 Oncoprotein ◽  
E6 Protein

ABSTRACT The high-risk human papillomaviruses (HPVs) are associated with carcinomas of the cervix and other genital tumors. Previous studies have identified two viral oncoproteins, E6 and E7, which are expressed in the majority of HPV-associated carcinomas. The ability of high-risk HPV E6 protein to immortalize human mammary epithelial cells (MECs) has provided a single-gene model to study the mechanisms of E6-induced oncogenic transformation. In this system, the E6 protein targets the p53 tumor suppressor protein for degradation, and mutational analyses have shown that E6-induced degradation of p53 protein is required for MEC immortalization. However, the inability of most dominant-negative p53 mutants to induce efficient immortalization of MECs suggests the existence of additional targets of the HPV E6 oncoprotein. Using the yeast two-hybrid system, we have isolated a novel E6-binding protein. This polypeptide, designated E6TP1 (E6-targeted protein 1), exhibits high homology to GTPase-activating proteins for Rap, including SPA-1, tuberin, and Rap1GAP. The mRNA for E6TP1 is widely expressed in tissues and in vitro-cultured cell lines. The gene for E6TP1 localizes to chromosome 14q23.2-14q24.3 within a locus that has been shown to undergo loss of heterozygosity in malignant meningiomas. Importantly, E6TP1 is targeted for degradation by the high-risk but not the low-risk HPV E6 proteins both in vitro and in vivo. Furthermore, the immortalization-competent but not the immortalization-incompetent HPV16 E6 mutants target the E6TP1 protein for degradation. Our results identify a novel target for the E6 oncoprotein and provide a potential link between HPV E6 oncogenesis and alteration of a small G protein signaling pathway.

scholarly journals Human Papillomavirus Oncoprotein E6 Inactivates the Transcriptional Coactivator Human ADA3

2002 ◽  
Vol 22 (16) ◽  
pp. 5801-5812 ◽  
Author(s):  
Ajay Kumar ◽  
Yongtong Zhao ◽  
Gaoyuan Meng ◽  
Musheng Zeng ◽  
Seetha Srinivasan ◽  
...  
Keyword(s):  
High Risk ◽  
P53 Protein ◽  
Human Papillomaviruses ◽  
Transcriptional Coactivator ◽  
Interacting Protein ◽  
Hpv E6 ◽  
Cycle Arrest ◽  
Evolutionarily Conserved ◽  
Novel Strategy ◽  
High Risk Hpv

ABSTRACT High-risk human papillomaviruses (HPVs) are associated with carcinomas of the cervix and other genital tumors. The HPV oncoprotein E6 is essential for oncogenic transformation. We identify here hADA3, human homologue of the yeast transcriptional coactivator yADA3, as a novel E6-interacting protein and a target of E6-induced degradation. hADA3 binds selectively to the high-risk HPV E6 proteins and only to immortalization-competent E6 mutants. hADA3 functions as a coactivator for p53-mediated transactivation by stabilizing p53 protein. Notably, three immortalizing E6 mutants that do not induce direct p53 degradation but do interact with hADA3 induced the abrogation of p53-mediated transactivation and G1 cell cycle arrest after DNA damage, comparable to wild-type E6. These findings reveal a novel strategy of HPV E6-induced loss of p53 function that is independent of direct p53 degradation. Given the likely role of the evolutionarily conserved hADA3 in multiple coactivator complexes, inactivation of its function may allow E6 to perturb numerous cellular pathways during HPV oncogenesis.

scholarly journals Suppression of MicroRNA 424 Levels by Human Papillomaviruses Is Necessary for Differentiation-Dependent Genome Amplification

2017 ◽  
Vol 91 (24) ◽  
Author(s):  
Shiyuan Hong ◽  
Shouqiang Cheng ◽  
William Songock ◽  
Jason Bodily ◽  
Laimonis A. Laimins
Keyword(s):  
Dna Damage ◽  
Critical Role ◽  
Human Papillomaviruses ◽  
Expression Vectors ◽  
Hpv E6 ◽  
Damage Repair ◽  
Undifferentiated Cells ◽  
Efficient Replication ◽  
E6 And E7 ◽  
E7 Proteins

ABSTRACT High-risk human papillomaviruses (HPVs) link their life cycle to epithelial differentiation and require activation of DNA damage pathways for efficient replication. HPVs modulate the expression of cellular transcription factors, as well as cellular microRNAs (miRNAs) to control these activities. One miRNA that has been reported to be repressed in HPV-positive cancers of the cervix and oropharynx is miR-424. Our studies show that miR-424 levels are suppressed in cell lines that stably maintain HPV 31 or 16 episomes, as well as cervical cancer lines that contain integrated genomes such as SiHa. Introduction of expression vectors for miR-424 reduced both the levels of HPV genomes in undifferentiated cells and amplification upon differentiation. Our studies show that the levels of two putative targets of miR-424 that function in DNA damage repair, CHK1 and Wee1, are suppressed in HPV-positive cells, providing an explanation for why this microRNA is targeted in HPV-positive cells. IMPORTANCE We describe here for the first time a critical role for miR-424 in the regulation of HPV replication. HPV E6 and E7 proteins suppress the levels of miR-424, and this is important for controlling the levels of CHK1, which plays a central role in viral replication.

scholarly journals Two Polymorphic Variants of Wild-Type p53 Differ Biochemically and Biologically

1999 ◽  
Vol 19 (2) ◽  
pp. 1092-1100 ◽  
Author(s):  
Miranda Thomas ◽  
Ann Kalita ◽  
Sylvie Labrecque ◽  
David Pim ◽  
Lawrence Banks ◽  
...  
Keyword(s):  
P53 Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Hpv E6 ◽  
Induced Tumors ◽  
Wild Type P53 ◽  
The Difference ◽  
E6 Protein ◽  
Increased Susceptibility

ABSTRACT The wild-type p53 protein exhibits a common polymorphism at amino acid 72, resulting in either a proline residue (p53Pro) or an arginine residue (p53Arg) at this position. Despite the difference that this change makes in the primary structure of the protein resulting in a difference in migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no differences in the biochemical or biological characteristics of these wild-type p53 variants have been reported. We have recently shown that p53Arg is significantly more susceptible than p53Pro to the degradation induced by human papillomavirus (HPV) E6 protein. Moreover, this may result in an increased susceptibility to HPV-induced tumors in homozygous p53Argindividuals. In further investigating the characteristics of these p53 variants, we now show that both forms are morphologically wild type and do not differ in their ability to bind to DNA in a sequence-specific manner. However, there are a number of differences between the p53 variants in their abilities to bind components of the transcriptional machinery, to activate transcription, to induce apoptosis, and to repress the transformation of primary cells. These observations may have implications for the development of cancers which harbor wild-type p53 sequences and possibly for the ability of such tumors to respond to therapy, depending on their p53 genotype.

scholarly journals E6-mediated activation of JNK drives EGFR signalling to promote proliferation and viral oncoprotein expression in cervical cancer

2020 ◽  
Author(s):  
Ethan L. Morgan ◽  
James A. Scarth ◽  
Molly R. Patterson ◽  
Christopher W. Wasson ◽  
Georgia C. Hemingway ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cell Proliferation ◽  
Cancer Cells ◽  
Human Papillomaviruses ◽  
Signalling Pathway ◽  
Viral Oncoprotein ◽  
Hpv E6 ◽  
Cervical Cancer Cells ◽  
E6 And E7 ◽  
Novel Therapeutic

AbstractHuman papillomaviruses (HPV) are a major cause of malignancy worldwide, contributing to ~5% of all human cancers including almost all cases of cervical cancer and a growing number of ano-genital and oral cancers. HPV-induced malignancy is primarily driven by the viral oncogenes, E6 and E7, which manipulate host cellular pathways to increase cell proliferation and enhance cell survival, ultimately predisposing infected cells to malignant transformation. Consequently, a more detailed understanding of viral-host interactions in HPV-associated disease offers the potential to identify novel therapeutic targets. Here, we identify that the c-Jun N-terminal kinase (JNK) signalling pathway is activated in cervical disease and in cervical cancer. The HPV E6 oncogene induces JNK1/2 phosphorylation in a manner that requires the E6 PDZ binding motif. We show that blockade of JNK1/2 signalling using small molecule inhibitors, or knockdown of the canonical JNK substrate c-Jun, reduces cell proliferation and induces apoptosis in cervical cancer cells. We further demonstrate that this phenotype is at least partially driven by JNK-dependent activation of EGFR signalling via increased expression of EGFR and the EGFR ligands EGF and HB-EGF. JNK/c-Jun signalling promoted the invasive potential of cervical cancer cells and was required for the expression of the epithelial to mesenchymal transition (EMT)-associated transcription factor Slug and the mesenchymal marker Vimentin. Furthermore, JNK/c-Jun signalling is required for the constitutive expression of HPV E6 and E7, which are essential for cervical cancer cell growth and survival. Together, these data demonstrate a positive feedback loop between the EGFR signalling pathway and HPV E6/E7 expression, identifying a regulatory mechanism in which HPV drives EGFR signalling to promote proliferation, survival and EMT. Thus, our study has identified a novel therapeutic target that may be beneficial for the treatment of cervical cancer.

scholarly journals The Human Papillomavirus (HPV) E6* Proteins from High-Risk, Mucosal HPVs Can Direct Degradation of Cellular Proteins in the Absence of Full-Length E6 Protein

2009 ◽  
Vol 83 (19) ◽  
pp. 9863-9874 ◽  
Author(s):  
David Pim ◽  
Vjekoslav Tomaić ◽  
Lawrence Banks
Keyword(s):  
High Risk ◽  
Tumor Suppressor ◽  
Pdz Domain ◽  
Direct Interaction ◽  
Human Papillomaviruses ◽  
Full Length ◽  
Cellular Proteins ◽  
Hpv E6 ◽  
E6 Protein ◽  
Hpv 18

ABSTRACT The E6 oncoproteins from high-risk mucosotrophic human papillomaviruses (HPVs) target a range of cellular proteins for proteasome-mediated degradation. Apart from the tumor suppressor p53 and proapoptotic Bcl-2 family member Bak, many targets contain class 1 PDZ domains and are involved in cell junction stability and signaling. The targeting mechanism is considered to function by the E6 protein acting as an adaptor molecule linking a cellular ubiquitin ligase to the target protein. In each case, whether the target is the p53 tumor suppressor or a member of the group of PDZ domain-containing targets, this mechanism relies on a direct interaction between E6 and its cellular target. This study focuses on the impact of the HPV type 18 (HPV-18) E6*I protein on the stability of Akt, Dlg, MAGI-1, MAGI-2, and Scribble. We show that HPV-18 E6* expression can downregulate the expression levels of Akt, Dlg, and Scribble in the absence of full-length HPV-18 E6 protein. The reduction in Dlg levels by E6* is independent of transcription and does not require a direct interaction between the two proteins although the proteasome pathway is involved. Further, we provide evidence that activation of certain signal transduction pathways has a profound effect on the targeting of Dlg by E6* and suggest that high-risk HPV E6 oncoproteins can target certain substrates both directly and indirectly through the E6* proteins and may cooperate in their degradation.

scholarly journals Disruption of repressive p130–DREAM complexes by human papillomavirus 16 E6/E7 oncoproteins is required for cell-cycle progression in cervical cancer cells

2011 ◽  
Vol 92 (11) ◽  
pp. 2620-2627 ◽  
Author(s):  
Nurshamimi Nor Rashid ◽  
Rohana Yusof ◽  
Roger J. Watson
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Cancer Cells ◽  
Cell Cycle Progression ◽  
Human Papillomaviruses ◽  
G1 Arrest ◽  
Cycle Progression ◽  
Hpv16 E6 ◽  
Cervical Cancer Cells ◽  
E6 And E7

Human papillomaviruses (HPVs) with tropism for mucosal epithelia are the major aetiological factors in cervical cancer. Most cancers are associated with so-called high-risk HPV types, in particular HPV16, and constitutive expression of the HPV16 E6 and E7 oncoproteins is critical for malignant transformation in infected keratinocytes. E6 and E7 bind to and inactivate the cellular tumour suppressors p53 and Rb, respectively, thus delaying differentiation and inducing proliferation in suprabasal keratinocytes to enable HPV replication. One member of the Rb family, p130, appears to be a particularly important target for E7 in promoting S-phase entry. Recent evidence indicates that p130 regulates cell-cycle progression as part of a large protein complex termed DREAM. The composition of DREAM is cell cycle-regulated, associating with E2F4 and p130 in G0/G1 and with the B-myb transcription factor in S/G2. In this study, we addressed whether p130–DREAM is disrupted in HPV16-transformed cervical cancer cells and whether this is a critical function for E6/E7. We found that p130–DREAM was greatly diminished in HPV16-transformed cervical carcinoma cells (CaSki and SiHa) compared with control cell lines; however, when E6/E7 expression was targeted by specific small hairpin RNAs, p130–DREAM was reformed and the cell cycle was arrested. We further demonstrated that the profound G1 arrest in E7-depleted CaSki cells was dependent on p130–DREAM reformation by also targeting the expression of the DREAM component Lin-54 and p130. The results show that continued HPV16 E6/E7 expression is necessary in cervical cancer cells to prevent cell-cycle arrest by a repressive p130–DREAM complex.

scholarly journals Spindle assembly checkpoint signaling and sister chromatid cohesion are disrupted by HPV E6-mediated transformation

2017 ◽  
Vol 28 (15) ◽  
pp. 2035-2041 ◽  
Author(s):  
Hazheen K. Shirnekhi ◽  
Erin P. Kelley ◽  
Jennifer G. DeLuca ◽  
Jacob A. Herman
Keyword(s):  
Cancer Progression ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Mitotic Exit ◽  
Human Papillomaviruses ◽  
Checkpoint Signaling ◽  
Hpv E6 ◽  
Chromatid Cohesion ◽  
E6 And E7 ◽  
Transforming Proteins

Aneuploidy, a condition that results from unequal partitioning of chromosomes during mitosis, is a hallmark of many cancers, including those caused by human papillomaviruses (HPVs). E6 and E7 are the primary transforming proteins in HPV that drive tumor progression. In this study, we stably expressed E6 and E7 in noncancerous RPE1 cells and analyzed the specific mitotic defects that contribute to aneuploidy in each cell line. We find that E6 expression results in multiple chromosomes associated with one or both spindle poles, causing a significant mitotic delay. In most cells, the misaligned chromosomes eventually migrated to the spindle equator, leading to mitotic exit. In some cells, however, mitotic exit occurred in the presence of pole-associated chromosomes. We determined that this premature mitotic exit is due to defects in spindle assembly checkpoint (SAC) signaling, such that cells are unable to maintain a prolonged mitotic arrest in the presence of unaligned chromosomes. This SAC defect is caused in part by a loss of kinetochore-associated Mad2 in E6-expressing cells. Our results demonstrate that E6-expressing cells exhibit previously unappreciated mitotic defects that likely contribute to HPV-mediated cancer progression.

scholarly journals Human Papillomavirus Type 16 E6/E7 Upregulation of Nucleophosmin Is Important for Proliferation and Inhibition of Differentiation

2010 ◽  
Vol 84 (10) ◽  
pp. 5131-5139 ◽  
Author(s):  
Rachel McCloskey ◽  
Craig Menges ◽  
Alan Friedman ◽  
Daksha Patel ◽  
Dennis J. McCance
Keyword(s):  
Cellular Transformation ◽  
Human Papillomaviruses ◽  
Two Dimensional ◽  
Primary Cells ◽  
Human Papillomavirus Type 16 ◽  
Hpv E6 ◽  
Elevated Expression ◽  
E6 And E7 ◽  
First Time ◽  
Inhibition Of Differentiation

ABSTRACT The E6 and E7 oncoproteins of high-risk human papillomaviruses (HPVs) are together sufficient to cause cellular transformation. Nucleophosmin (NPM) was identified as a protein with increased levels in two-dimensional (2-D) gel analysis of human foreskin keratinocytes (HFKs) expressing E7 following methylcellulose-induced differentiation. Analysis of NPM expression in E7-expressing cells and E6- and E7-expressing cells in culture and in organotypic rafts confirmed the increased levels observed in 2-D gel analysis. The elevated expression of NPM was determined to be posttranscriptional and was attributed to increased v-akt murine thymoma viral oncogene (AKT) activity in the E6- and E7-expressing cells. Depletion of NPM caused a reduction in the replicative capacity of E7- and E6/E7-expressing HFKs and an increase in markers of differentiation. Also, the p53 and pRb tumor suppressor levels are increased with the knockdown of NPM in E6/E7-expressing cells, and, interestingly, p14ARF is relocalized from the nucleolus to the nucleoplasm and cytoplasm in these cells. The results show for the first time that NPM is required for the proliferation and inhibition of differentiation observed in HPV E6- and E7-expressing primary cells.

scholarly journals Leaky Scanning Is the Predominant Mechanism for Translation of Human Papillomavirus Type 16 E7 Oncoprotein from E6/E7 Bicistronic mRNA

2000 ◽  
Vol 74 (16) ◽  
pp. 7284-7297 ◽  
Author(s):  
Simon N. Stacey ◽  
Deborah Jordan ◽  
Andrew J. K. Williamson ◽  
Michael Brown ◽  
Joanna H. Coote ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Human Papillomaviruses ◽  
Start Codon ◽  
Leaky Scanning ◽  
Structural Element ◽  
Entry Site ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
E6 And E7 ◽  
Bicistronic Mrna

ABSTRACT Human papillomaviruses (HPV) are unique in that they generate mRNAs that apparently can express multiple proteins from tandemly arranged open reading frames. The mechanisms by which this is achieved are uncertain and are at odds with the basic predictions of the scanning model for translation initiation. We investigated the unorthodox mechanism by which the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV-16) can be translated from a single, bicistronic mRNA. The short E6 5′ untranslated region (UTR) was shown to promote translation as efficiently as a UTR from Xenopusβ-globin. Insertion of a secondary structural element into the UTR inhibited both E6 and E7 expression, suggesting that E7 expression depends on ribosomal scanning from the 5′ end of the mRNA. E7 translation was found to be cap dependent, but E6 was more dependent on capping and eIF4F activity than E7. Insertion of secondary structural elements at various points in the region upstream of E7 profoundly inhibited translation, indicating that scanning was probably continuous. Insertion of the E6 region between Renilla and firefly luciferase genes revealed little or no internal ribosomal entry site activity. However when E6 was located at the 5′ end of the mRNA, it permitted over 100-fold-higher levels of downstream cistron translation than did the Renilla open reading frame. Internal AUGs in the E6 region with strong or intermediate Kozak sequence contexts were unable to inhibit E7 translation, but initiation at the E7 AUG was efficient and accurate. These data support a model in which E7 translation is facilitated by an extreme degree of leaky scanning, requiring the negotiation of 13 upstream AUGs. Ribosomal initiation complexes which fail to initiate at the E6 start codon can scan through to the E7 AUG without initiating translation, but competence to initiate is achieved once the E7 AUG is reached. These findings suggest that the E6 region of HPV-16 comprises features that sponsor both translation of the E6 protein and enhancement of translation at a downstream site.

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