scholarly journals PTPN14 Degradation by High-Risk Human Papillomavirus E7 Limits Keratinocyte Differentiation and Contributes to HPV-Mediated Oncogenesis

2018 ◽  
Author(s):  
Joshua Hatterschide ◽  
Amelia E. Bohidar ◽  
Miranda Grace ◽  
Tara J. Nulton ◽  
Brad Windle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Papillomavirus ◽  
High Risk ◽  
Human Keratinocytes ◽  
Keratinocyte Differentiation ◽  
Oncogenic Transformation ◽  
Hpv E7 ◽  
Hpv Oncoproteins ◽  
E7 Proteins ◽  
High Risk Hpv

AbstractHigh-risk human papillomavirus (HPV) E7 proteins enable oncogenic transformation of HPV-infected cells by inactivating host cellular proteins. High-risk but not low-risk HPV E7 target PTPN14 for proteolytic degradation, suggesting that PTPN14 degradation may be related to their oncogenic activity. HPV infects human keratinocytes but the role of PTPN14 in keratinocytes and the consequences of PTPN14 degradation are unknown. Using an HPV16 E7 variant that can inactivate RB1 but cannot degrade PTPN14 we found that high-risk HPV E7-mediated PTPN14 degradation impairs keratinocyte differentiation. Deletion ofPTPN14from primary human keratinocytes decreased keratinocyte differentiation gene expression. Related to oncogenic transformation, both HPV16 E7-mediated PTPN14 degradation andPTPN14deletion promoted keratinocyte survival following detachment from a substrate. PTPN14 degradation contributed to high-risk HPV E6/E7-mediated immortalization of primary keratinocytes and HPV-positive but not HPV-negative cancers exhibit a gene expression signature consistent with PTPN14 inactivation. We find that PTPN14 degradation impairs keratinocyte differentiation and propose that this contributes to high-risk HPV E7-mediated oncogenic activity independent of RB1 inactivation.Significance StatementHuman papillomaviruses uncouple proliferation from differentiation in order to enable virus replication in epithelial cells. HPV E7 proteins are well established to promote proliferation by binding to and inactivating retinoblastoma family proteins and other cell cycle inhibitors. However, mechanisms by which high-risk HPV oncoproteins inhibit differentiation have not been defined. This paper identifies the first mechanism by which high-risk HPV E7 inhibit keratinocyte differentiation. The inhibition of differentiation requires degradation of the cellular protein PTPN14 by high-risk HPV E7 and this degradation is related to the ability of high-risk HPV oncoproteins to immortalize keratinocytes and to cause cancer.

scholarly journals PTPN14 degradation by high-risk human papillomavirus E7 limits keratinocyte differentiation and contributes to HPV-mediated oncogenesis

2019 ◽  
Vol 116 (14) ◽  
pp. 7033-7042 ◽  
Author(s):  
Joshua Hatterschide ◽  
Amelia E. Bohidar ◽  
Miranda Grace ◽  
Tara J. Nulton ◽  
Hee Won Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Papillomavirus ◽  
High Risk ◽  
Gene Expression Signature ◽  
Human Keratinocytes ◽  
Keratinocyte Differentiation ◽  
Oncogenic Transformation ◽  
Hpv E7 ◽  
Oncogenic Activity ◽  
High Risk Hpv

High-risk human papillomavirus (HPV) E7 proteins enable oncogenic transformation of HPV-infected cells by inactivating host cellular proteins. High-risk but not low-risk HPV E7 target PTPN14 for proteolytic degradation, suggesting that PTPN14 degradation may be related to their oncogenic activity. HPV infects human keratinocytes but the role of PTPN14 in keratinocytes and the consequences of PTPN14 degradation are unknown. Using an HPV16 E7 variant that can inactivate retinoblastoma tumor suppressor (RB1) but cannot degrade PTPN14, we found that high-risk HPV E7-mediated PTPN14 degradation impairs keratinocyte differentiation. Deletion ofPTPN14from primary human keratinocytes decreased keratinocyte differentiation gene expression. Related to oncogenic transformation, both HPV16 E7-mediated PTPN14 degradation andPTPN14deletion promoted keratinocyte survival following detachment from a substrate. PTPN14 degradation contributed to high-risk HPV E6/E7-mediated immortalization of primary keratinocytes and HPV+but not HPV−cancers exhibit a gene-expression signature consistent with PTPN14 inactivation. We find that PTPN14 degradation impairs keratinocyte differentiation and propose that this contributes to high-risk HPV E7-mediated oncogenic activity independent of RB1 inactivation.

scholarly journals A conserved amino acid in the C-terminus of HPV E7 mediates binding to PTPN14 and repression of epithelial differentiation

2020 ◽  
Author(s):  
Joshua Hatterschide ◽  
Alexis C. Brantly ◽  
Miranda Grace ◽  
Karl Munger ◽  
Elizabeth A. White
Keyword(s):  
Gene Expression ◽  
Human Papillomavirus ◽  
Life Cycle ◽  
Human Keratinocytes ◽  
Keratinocyte Differentiation ◽  
Carcinogenic Activity ◽  
Hpv E7 ◽  
Primary Driver ◽  
E7 Proteins ◽  
High Risk Hpv

ABSTRACTThe human papillomavirus (HPV) E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. The E7 proteins from diverse HPV bind to the host cellular non-receptor protein tyrosine phosphatase type 14 (PTPN14) and direct it for degradation through the activity of the E7-associated host E3 ubiquitin ligase UBR4. Herein we show that a highly conserved arginine residue in the C-terminal domain of diverse HPV E7 mediates interaction with PTPN14. We found that disruption of PTPN14 binding through mutation of the C-terminal arginine did not impact the ability of several high-risk HPV E7 proteins to bind and degrade the retinoblastoma tumor suppressor or activate E2F target gene expression. HPVs infect human keratinocytes and we previously reported that both PTPN14 degradation by HPV16 E7 and PTPN14 CRISPR knockout repress keratinocyte differentiation-related genes. Now we have found that blocking PTPN14 binding through mutation of the conserved C-terminal arginine rendered both HPV16 and HPV18 E7 unable to repress differentiation-related gene expression. We then confirmed that the HPV18 E7 variant that could not bind PTPN14 was also impaired in repressing differentiation when expressed from the complete HPV18 genome. Finally, we found that the ability of HPV18 E7 to extend the lifespan of primary human keratinocytes required PTPN14 binding. CRISPR/Cas9 knockout of PTPN14 rescued keratinocyte lifespan extension in the presence of the PTPN14 binding-deficient HPV18 E7 variant. These results support the model that PTPN14 degradation by high-risk HPV E7 leads to repression of differentiation and contributes to its carcinogenic activity.IMPORTANCEHuman papillomavirus (HPV)-positive carcinomas account for nearly 5% of the global human cancer burden. The E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. HPV E7 binds and degrades the putative tumor suppressor, PTPN14. However, the impact of PTPN14 binding by E7 on cellular processes is not well defined. Here, we show that PTPN14 binding is mediated by a conserved C-terminal arginine residue of HPV E7 in vivo. Additionally, we found that PTPN14 binding contributes to the carcinogenic activity of HPV18 E7 (the second most abundant HPV type in cancers). Finally, we determined that PTPN14 binding by HPV16 E7 and HPV18 E7 represses keratinocyte differentiation. HPV-positive cancers are frequently poorly differentiated and the HPV life cycle is dependent upon the keratinocyte differentiation program. The finding that PTPN14 binding by HPV E7 impairs differentiation has significant implications for both HPV-mediated carcinogenesis and the HPV life cycle.

scholarly journals A Conserved Amino Acid in the C Terminus of Human Papillomavirus E7 Mediates Binding to PTPN14 and Repression of Epithelial Differentiation

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Joshua Hatterschide ◽  
Alexis C. Brantly ◽  
Miranda Grace ◽  
Karl Munger ◽  
Elizabeth A. White
Keyword(s):  
High Risk ◽  
Life Span ◽  
Ubiquitin Ligase ◽  
Human Keratinocytes ◽  
Keratinocyte Differentiation ◽  
Carcinogenic Activity ◽  
Hpv E7 ◽  
Primary Driver ◽  
E7 Proteins ◽  
High Risk Hpv

ABSTRACT The human papillomavirus (HPV) E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. The E7 proteins from diverse HPVs bind to the host cellular nonreceptor protein tyrosine phosphatase type 14 (PTPN14) and direct it for degradation through the activity of the E7-associated host E3 ubiquitin ligase UBR4. Here, we show that a highly conserved arginine residue in the C-terminal domain of diverse HPV E7 mediates the interaction with PTPN14. We found that disruption of PTPN14 binding through mutation of the C-terminal arginine did not impact the ability of several high-risk HPV E7 proteins to bind and degrade the retinoblastoma tumor suppressor or activate E2F target gene expression. HPVs infect human keratinocytes, and we previously reported that both PTPN14 degradation by HPV16 E7 and PTPN14 CRISPR knockout repress keratinocyte differentiation-related genes. Now, we have found that blocking PTPN14 binding through mutation of the conserved C-terminal arginine rendered both HPV16 and HPV18 E7 unable to repress differentiation-related gene expression. We then confirmed that the HPV18 E7 variant that could not bind PTPN14 was also impaired in repressing differentiation when expressed from the complete HPV18 genome. Finally, we found that the ability of HPV18 E7 to extend the life span of primary human keratinocytes required PTPN14 binding. CRISPR/Cas9 knockout of PTPN14 rescued keratinocyte life span extension in the presence of the PTPN14 binding-deficient HPV18 E7 variant. These results support the model that PTPN14 degradation by high-risk HPV E7 leads to repression of differentiation and contributes to its carcinogenic activity. IMPORTANCE The E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. HPV E7 binds the putative tumor suppressor PTPN14 and targets it for degradation using the ubiquitin ligase UBR4. PTPN14 binds to a C-terminal arginine highly conserved in diverse HPV E7. Our previous efforts to understand how PTPN14 degradation contributes to the carcinogenic activity of high-risk HPV E7 used variants of E7 unable to bind to UBR4. Now, by directly manipulating E7 binding to PTPN14 and using a PTPN14 knockout rescue experiment, we demonstrate that the degradation of PTPN14 is required for high-risk HPV18 E7 to extend keratinocyte life span. Our data show that PTPN14 binding by HPV16 E7 and HPV18 E7 represses keratinocyte differentiation. HPV-positive cancers are frequently poorly differentiated, and the HPV life cycle depends upon keratinocyte differentiation. The finding that PTPN14 binding by HPV E7 impairs differentiation has significant implications for HPV-mediated carcinogenesis and the HPV life cycle.

scholarly journals Detection of human papillomavirus oncoprotein E7 in liquid-based cytology

2012 ◽  
Vol 93 (2) ◽  
pp. 356-363 ◽  
Author(s):  
Maria Lidqvist ◽  
Olle Nilsson ◽  
Jan Holmgren ◽  
Sebastian Hölters ◽  
Eva Röijer ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
High Risk ◽  
Low Risk ◽  
Transformed Cells ◽  
Specific Staining ◽  
Hpv E7 ◽  
Detection And Identification ◽  
Liquid Based Cytology ◽  
E7 Proteins ◽  
High Risk Hpv

The selection and characterization of a set of mouse mAbs against high-risk human papillomavirus (HPV) E7 oncoprotein and the development of protocols for immunocytochemistry (ICC) are described here. A large number of antibodies raised towards HPV16 and 18 E7 were tested for high-risk specificity by ELISA using a panel of HPV E7 proteins. Antibodies detecting low-risk E7 were discarded, resulting in 38 high-risk HPV E7-specific antibodies. The corresponding epitopes were mapped using overlapping HPV E7 fragments displayed on phage particles. Functionality in ICC against formalin-fixed cervical cancer cell lines was demonstrated for ten mAbs; their high-risk specificity was confirmed by Western blot analysis and ICC on transiently transformed cells expressing high- or low-risk HPV E7. These mAbs were specific for one or several of the high-risk strains HPV16, 18, 31, 35 and 45. Specific E7 staining of liquid-based cytology (LBC) samples was demonstrated for seven mAbs and optimized protocols were established. The E716-41 and E718-79 mAbs demonstrated particularly strong and specific staining of cells stored in LBC fluid for at least 6 months. It is proposed that the high-risk HPV E7 staining protocols established in this study may have the potential to be included in a complementary test for the detection and identification of malignantly transformed cells, in for example atypical squamous cells of undetermined significance samples.

scholarly journals High-Risk Human Papillomavirus E7 Proteins Target PTPN14 for Degradation

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Elizabeth A. White ◽  
Karl Münger ◽  
Peter M. Howley
Keyword(s):  
Cervical Cancer ◽  
High Risk ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Human Papillomaviruses ◽  
E7 Oncoprotein ◽  
Hpv E7 ◽  
Transforming Activity ◽  
E7 Proteins ◽  
High Risk Hpv

ABSTRACTThe major transformation activity of the high-risk human papillomaviruses (HPV) is associated with the E7 oncoprotein. The interaction of HPV E7 with retinoblastoma family proteins is important for several E7 activities; however, this interaction does not fully account for the high-risk E7-specific cellular immortalization and transformation activities. We have determined that the cellular non-receptor protein tyrosine phosphatase PTPN14 interacts with HPV E7 from many genus alpha and beta HPV types. We find that high-risk genus alpha HPV E7, but not low-risk genus alpha or beta HPV E7, is necessary and sufficient to reduce the steady-state level of PTPN14 in cells. High-risk E7 proteins target PTPN14 for proteasome-mediated degradation, which requires the ubiquitin ligase UBR4, and PTPN14 is degraded by the proteasome in HPV-positive cervical cancer cell lines. Residues in the C terminus of E7 interact with the C-terminal phosphatase domain of PTPN14, and interference with the E7-PTPN14 interaction restores PTPN14 levels in cells. Finally, PTPN14 degradation correlates with the retinoblastoma-independent transforming activity of high-risk HPV E7.IMPORTANCEHigh-risk human papillomaviruses (HPV) are the cause of cervical cancer, some other anogenital cancers, and a growing fraction of oropharyngeal carcinomas. The high-risk HPV E6 and E7 oncoproteins enable these viruses to cause cancer, and the mechanistic basis of their carcinogenic activity has been the subject of intense study. The high-risk E7 oncoprotein is especially important in the immortalization and transformation of human cells, which makes it a central component of HPV-associated cancer development. E7 oncoproteins interact with retinoblastoma family proteins, but for several decades, it has been recognized that high-risk HPV E7 oncoproteins have additional cancer-associated activities. We have determined that high-risk E7 proteins target the proteolysis of the cellular protein tyrosine phosphatase PTPN14 and find that this activity is correlated with the retinoblastoma-independent transforming activity of E7.

scholarly journals 2-APB arrests human keratinocyte proliferation and inhibits cutaneous squamous cell carcinomain vitro

2018 ◽  
Author(s):  
Aislyn M. Nelson ◽  
Yalda Moayedi ◽  
Sophie A. Greenberg ◽  
Marlon E. Ruiz ◽  
Uffe B. Jensen ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Risk ◽  
Cell Lines ◽  
Squamous Cell ◽  
Transient Receptor Potential ◽  
Human Keratinocytes ◽  
Keratinocyte Differentiation ◽  
Differentiation Markers ◽  
Keratinocyte Proliferation ◽  
Human Keratinocyte

AbstractBackgroundThe epidermis is a stratified epithelium whose differentiation program is triggered in part by calcium. Dysregulation of keratinocyte differentiation may lead to non-melanoma skin cancers, including cutaneous squamous cell carcinoma (cSCC). The compound 2-aminoethoxydiphenyl borate (2-APB) modulates calcium signaling by altering activity of calcium-permeable channels of the transient receptor potential (TRP) and ORAI families, and is therefore poised to govern signaling pathways that control the balance of keratinocyte proliferation and differentiation.ObjectiveWe sought to determine whether 2-APB alters differentiation of normal human keratinocytes and progression of human cSCCs modelsin vitro.MethodsPrimary human keratinocyte cultures were treated with 2-APB and levels of proliferation (EdU incorporation) and differentiation markers [quantitative PCR (qPCR)] were assessed. Human cSCC biopsies and cell lines were analyzed for TRP and ORAI gene expression via qPCR. cSCC cell lines were cultured in organtypic cultures and analyzed for growth and invasiveness after 2-APB or vehicle treatment.ResultsCulturing human keratinocytes with 2-APB arrested cell proliferation, triggered differentiation-gene expression and altered epidermal stratification, indicating that 2-APB application is sufficient to promote differentiation. In human organotypic cSCC cultures, 2-APB attenuated tumor growth and invasiveness. Finally, expression of a panel of 2-APB-targeted ion channels (TRPV3, TRPV1, TRPC1, OraI1, OraI2 and OraI3) was dysregulated in high-risk cSCC biopsies.ConclusionsCollectively, these findings identify 2-APB as a potential therapeutic for high-risk cSCCs.

scholarly journals Human Papillomavirus E7 Protein Deregulates Mitosis via an Association with Nuclear Mitotic Apparatus Protein 1

2008 ◽  
Vol 83 (4) ◽  
pp. 1700-1707 ◽  
Author(s):  
Christine L. Nguyen ◽  
Karl Münger
Keyword(s):  
Human Papillomavirus ◽  
High Risk ◽  
Mitotic Spindles ◽  
Mitotic Apparatus ◽  
Human Papillomavirus Type 16 ◽  
Hpv E6 ◽  
Hpv E7 ◽  
Nuclear Mitotic Apparatus ◽  
E6 And E7 ◽  
High Risk Hpv

ABSTRACT We previously observed that high-risk human papillomavirus type 16 (HPV16) E7 expression leads to the delocalization of dynein from mitotic spindles (C. L. Nguyen, M. E. McLaughlin-Drubin, and K. Munger, Cancer Res. 68:8715-8722, 2008). Here, we show that HPV16 E7 associates with nuclear mitotic apparatus protein 1 (NuMA) and that NuMA binding and the ability to induce dynein delocalization map to similar carboxyl-terminal sequences of E7. Additionally, we show that the delocalization of dynein from mitotic spindles by HPV16 E7 and the interaction between HPV16 E7 and NuMA correlate with the induction of defects in chromosome alignment during prometaphase even in cells with normal centrosome numbers. Furthermore, low-risk HPV6b and HPV11 E7s also associate with NuMA and also induce a similar mitotic defect. It is possible that the disruption of mitotic events by HPV E7, via targeting of the NuMA/dynein complex and potentially other NuMA-containing complexes, contributes to viral maintenance and propagation potentially through abrogating the differentiation program of the infected epithelium. Furthermore, in concert with activities specific to high-risk HPV E6 and E7, such as the inactivation of the p53 and pRB tumor suppressors, respectively, the disruption of the NuMA/dynein network may result in mitotic errors that would make an infected cell more prone to the accumulation of aneuploidy even in the absence of supernumerary centrosomes.

scholarly journals The Human DEK Proto-Oncogene Is a Senescence Inhibitor and an Upregulated Target of High-Risk Human Papillomavirus E7

2005 ◽  
Vol 79 (22) ◽  
pp. 14309-14317 ◽  
Author(s):  
Trisha M. Wise-Draper ◽  
Hillary V. Allen ◽  
Megan N. Thobe ◽  
Elizabeth E. Jones ◽  
Kristen B. Habash ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Human Papillomavirus ◽  
High Risk ◽  
Cancer Cell ◽  
Replicative Senescence ◽  
Human Keratinocytes ◽  
Primary Cells ◽  
Nucleic Acid Binding ◽  
Human Carcinogenesis ◽  
Hpv E7

ABSTRACT The human DEK proto-oncogene is a nucleic acid binding protein with suspected roles in human carcinogenesis, autoimmune disease, and viral infection. Intracellular DEK functions, however, are poorly understood. In papillomavirus-positive cervical cancer cells, downregulation of viral E6/E7 oncogene expression results in cellular senescence. We report here the specific repression of DEK message and protein levels in senescing human papillomavirus type 16- (HPV16-) and HPV18-positive cancer cell lines as well as in primary cells undergoing replicative senescence. Cervical cancer cell senescence was partially overcome by DEK overexpression, and DEK overexpression was sufficient for extending the life span of primary keratinocytes, supporting critical roles for this molecule as a senescence regulator. In order to determine whether DEK is a bona fide HPV oncogene target in primary cells, DEK expression was monitored in human keratinocytes transduced with HPV E6 and/or E7. The results identify high-risk HPV E7 as a positive DEK regulator, an activity that is not shared by low-risk HPV E7 protein. Experiments in mouse embryo fibroblasts recapitulated the observed E7-mediated DEK induction and demonstrated that both basal and E7-induced regulation of DEK expression are controlled by the retinoblastoma protein family. Taken together, our results suggest that DEK upregulation may be a common event in human carcinogenesis and may reflect its senescence inhibitory function.

scholarly journals Roles of the E6 and E7 Proteins in the Life Cycle of Low-Risk Human Papillomavirus Type 11

2004 ◽  
Vol 78 (5) ◽  
pp. 2620-2626 ◽  
Author(s):  
Stephen T. Oh ◽  
Michelle S. Longworth ◽  
Laimonis A. Laimins
Keyword(s):  
Human Papillomavirus ◽  
Life Cycle ◽  
High Risk ◽  
Translation Termination ◽  
Human Keratinocytes ◽  
Low Risk ◽  
Hpv E6 ◽  
E6 And E7 ◽  
Substitution Mutations ◽  
E7 Proteins

ABSTRACT Many important functions have been attributed to the high-risk human papillomavirus (HPV) E6 and E7 proteins, including binding and degradation of p53 as well as interacting with Rb proteins. In contrast, the physiological roles of the low-risk E6 and E7 proteins remain unclear. Previous studies demonstrated that the high-risk E6 and E7 proteins also play roles in the productive life cycle by facilitating the maintenance of viral episomes (J. T. Thomas, W. G. Hubert, M. N. Ruesch, and L. A. Laimins, Proc. Natl. Acad. Sci. USA 96:8449-8454, 1999). In order to determine whether low-risk E6 or E7 is similarly necessary for the stable maintenance of episomes, HPV type 11 (HPV-11) genomes that contained translation termination mutations in E6 or E7 were constructed. Upon transfection into normal human keratinocytes, genomes in which E6 function was abolished were unable to be maintained episomally. Transfection of genomes containing substitution mutations in amino acids conserved in high- and low-risk HPV types suggested that multiple protein domains are involved in this process. Examination of cells transfected with HPV-11 genomes in which E7 function was inhibited were found to exhibit a more complex phenotype. At the second passage following transfection, mutant genomes were maintained as episomes but at significantly reduced levels than in cells transfected with the wild-type HPV-11 genome. Upon further passage in culture, however, the episomal forms of these E7 mutant genomes quickly disappeared. These findings identify important new functions for the low-risk E6 and E7 proteins in the episomal maintenance of low-risk HPV-11 genomes and suggest that they may act in a manner similar to that observed for the high-risk proteins.

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