High-risk Human Papillomavirus E6 Proteins Research Articles

HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14.

High-risk human papillomavirus (HPV) oncoproteins inactivate cellular tumor suppressors to reprogram host cell signaling pathways. HPV E7 proteins bind and degrade the tumor suppressor PTPN14, thereby promoting the nuclear localization of the YAP1 oncoprotein and inhibiting keratinocyte differentiation. YAP1 is a transcriptional coactivator that drives epithelial cell stemness and self-renewal. YAP1 activity is inhibited by the highly conserved Hippo pathway, which is frequently inactivated in human cancers. MST1/2 and LATS1/2 kinases form the core of the Hippo kinase cascade. Active LATS1 kinase is phosphorylated on threonine 1079 and inhibits YAP1 by phosphorylating it on amino acids including serine 127. Here, we tested the effect of high-risk (carcinogenic) HPV18 E7 on Hippo pathway activity. We found that either PTPN14 knockout or PTPN14 degradation by HPV18 E7 decreased the phosphorylation of LATS1 T1079 and YAP1 S127 in human keratinocytes and inhibited keratinocyte differentiation. Conversely, PTPN14-dependent differentiation required LATS kinases and certain PPxY motifs in PTPN14. Neither MST1/2 kinases nor the putative PTPN14 phosphatase active sites were required for PTPN14 to promote differentiation. Together, these data support that PTPN14 inactivation or degradation of PTPN14 by HPV18 E7 reduce LATS1 activity, promoting active YAP1 and inhibiting keratinocyte differentiation.IMPORTANCEThe Hippo kinase cascade inhibits YAP1, an oncoprotein and driver of cell stemness and self-renewal. There is mounting evidence that the Hippo pathway is targeted by tumor viruses including human papillomavirus. The high-risk HPV E7 oncoprotein promotes YAP1 nuclear localization and the carcinogenic activity of high-risk HPV E7 requires YAP1 activity. Blocking HPV E7-dependent YAP1 activation could inhibit HPV-mediated carcinogenesis, but the mechanism by which HPV E7 activates YAP1 has not been elucidated. Here we report that by degrading the tumor suppressor PTPN14, HPV18 E7 inhibits LATS1 kinase, reducing inhibitory phosphorylation on YAP1. These data support that an HPV oncoprotein can inhibit Hippo signaling to activate YAP1 and strengthen the link between PTPN14 and Hippo signaling in human epithelial cells.

HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14.

High-risk human papillomavirus (HPV) oncoproteins inactivate cellular tumor suppressors to reprogram host cell signaling pathways. HPV E7 proteins bind and degrade the tumor suppressor PTPN14, thereby promoting the nuclear localization of the YAP1 oncoprotein and inhibiting keratinocyte differentiation. YAP1 is a transcriptional coactivator that drives epithelial cell stemness and self-renewal. YAP1 activity is inhibited by the highly conserved Hippo pathway, which is frequently inactivated in human cancers. MST1/2 and LATS1/2 kinases form the core of the Hippo kinase cascade. Active LATS1 kinase is phosphorylated on threonine 1079 and inhibits YAP1 by phosphorylating it on amino acids including serine 127. Here, we tested the effect of high-risk (carcinogenic) HPV18 E7 on Hippo pathway activity. We found that either PTPN14 knockout or PTPN14 degradation by HPV18 E7 decreased phosphorylation of LATS1 T1079 and YAP1 S127 in human keratinocytes and inhibited keratinocyte differentiation. Conversely, PTPN14-dependent differentiation required LATS kinases and certain PPxY motifs in PTPN14. Neither MST1/2 kinases nor the putative PTPN14 phosphatase active site were required for PTPN14 to promote differentiation. Taken together, these data support that PTPN14 inactivation or degradation of PTPN14 by HPV18 E7 reduce LATS1 activity, promoting active YAP1 and inhibiting keratinocyte differentiation.

Structure-Based Discovery of Potential HPV E6 and EBNA1 Inhibitors: Implications for Cervical Cancer Treatment

Cervical cancer is the fourth most diagnosed cancer and the fourth leading cause of cancer death in women globally. Its onset and progression have been attributed to high-risk human papillomavirus (HPV) types, especially 16 and 18, while the Epstein–Barr virus (EBV) is believed to also significantly contribute to cervical cancer growth. The E6 protein associated with high-risk HPV strains, such as HPV16 and HPV18, is known for its role in promoting cervical cancer and other anogenital cancers. E6 proteins contribute to the malignant transformation of infected cells by targeting and degrading tumor suppressor proteins, especially p53. On the other hand, EBV nuclear antigen 1 (EBNA1) plays a crucial role in the maintenance and replication of the EBV genome in infected cells. EBNA1 is believed to increase HPV E6 and E7 levels, as well as c-MYC, and BIRC5 cellular genes in the HeLa cell line, implying that HPV/EBV co-infection accelerates cervical cancer onset and growth. Thus, the E6 and EBNA1 antigens of HPV and EBV, respectively, are attractive targets for cervical cancer immunotherapy. This study, therefore, virtually screened for potential drug candidates with good binding affinity to all three oncoviral proteins, HPV16 E6, HPV18 E6, and EBNA1. The compounds were further subjected to ADMET profiling, biological activity predictions, molecular dynamics (MD) simulations, and molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) calculations. A total of six compounds comprising ZINC000013380012, ZINC000070454124, ZINC000014588133, ZINC000085568136, ZINC000095909247, and ZINC000085597263 demonstrated very strong affinity (≤−60 kJ/mol) to the three oncoviral proteins (EBNA1, HPV16 E6, and HPV18 E6) after being subjected to docking, MD, and MM/PBSA. These compounds demonstrated relatively stronger binding than the controls used, inhibitors of EBNA1 (VK-1727) and HPV E6 (baicalein and gossypetin). Biological activity predictions also corroborated their antineoplastic, p53-enhancing, Pin1 inhibitory, and JAK2 inhibitory activities. Further experimental testing is required to validate the ability of the shortlisted compounds to silence the insidious effects of HPV E6 and EBNA1 proteins in cervical cancers.

Human papillomaviruses:Knowns, mysteries, and unchartered territories.

There has been an explosion in the number of papillomaviruses that have been identified and fully sequenced. Yet only a minute fraction of these has been studied in any detail. Most of our molecular research efforts have focused on the E6 and E7 proteins of "high-risk,"cancer-associated human papillomaviruses (HPVs). Interactions of the high-risk HPV E6 and E7 proteins with their respective cellular targets, the p53 and the retinoblastoma tumor suppressors, have been investigated in minute detail. Some have thus questioned if research on papillomaviruses remains an exciting and worthwhile area of investigation. However, fundamentally new insights on the biological activities and cellular targets of the high-risk HPV E6 and E7 proteins have been discovered and previously unstudied HPVs have been newly associated with human diseases. HPV infections continue to be an important cause of human morbidity and mortality and since there are no antivirals to combat HPV infections, research on HPVs should remain attractive to new investigators and biomedical funding agencies, alike.

HPV E7 affects the function of cervical cancer cells via the TAL1/lnc‑EBIC/KLHDC7B axis.

High‑risk human papillomavirus (HPV)16 and 18 are the primary cause of cervical cancer (CC) and long non‑coding RNAs (lncRNAs/lncs) are often abnormally expressed in patients with CC. The authors' previous study indicated that oncogenic enhancer of zeste homolog2 (EZH2)‑binding lncRNA in cervical cancer (lnc‑EBIC) serves a role in the tumorigenic activity of the HPV E6 protein in patients with CC. However, whether HPV E7 affects the development of CC through lnc‑EBIC, and the potential mechanisms underlying this remains unclear. Therefore, the present study investigated the effects of lnc‑EBIC and HPV E7 in cervical cancer cell lines HeLa, CaSki and C33A invitro. CCK‑8, EdU and DAPI staining assays, flow cytometry, RT‑qPCR, western blotting and Transwell assay were performed on these cell lines. The results revealed that exogenous expression of HPV16/18 E7 significantly promoted lnc‑EBIC expression, and conversely, lnc‑EBIC was downregulated by silencing endogenous HPV16/18 E7 expression in corresponding CaSki and HeLa cells. Overexpression of lnc‑EBIC significantly increased cellular proliferation, migration and invasion, and inhibited apoptosis in HPV‑ C33A cells. The tumorigenic effects of HPV16/18 E7 in corresponding CaSki and HeLa cells were significantly blocked by the silencing of lnc‑EBIC expression. Molecular analysis revealed that HPV16/18 E7 depended on TAL BHLH transcription factor 1, erythroid differentiation factor inhibition to promote lnc‑EBIC expression, which also resulted in the upregulation of oncogenic Kelch domain‑containing 7B (KLHDC7B) in corresponding CaSki and HeLa cells. Additionally, KLHDC7B knockdown blocked the tumor‑promotive effects of lnc‑EBIC in HPV‑ C33A cells. Collectively, the results of the present study indicated that lnc‑EBIC acts as an oncogenic lncRNA by enhancing KLHDC7B expression in HPV+ and HPV‑ CC cells, and can be exploited by HPV16/18 E7 to accelerate tumorigenic activity in CC. These results further revealed that the lnc‑EBIC/KLHDC7B axis represents a novel molecular mechanism and potential therapeutic target for CC.

The high-risk HPV E6 proteins modify the activity of the eIF4E protein via the MEK/ERK and AKT/PKB pathways.

Previous studies have proposed that the human papillomavirus (HPV) E6 oncoproteins modify the transcriptional activity of eIF4E through mechanisms dependent on p53 degradation. However, the effect of these oncoproteins on pathways regulating the activity of the eIF4E protein remains poorly understood. Hence, we investigated the mechanisms whereby E6 proteins regulate the activity of the eIF4E protein and its effect on target genes. Overexpression of E6 constructs (HPV-6, HPV-16, HPV-18, and HPV52) showed that E6 oncoproteins increased phosphorylation of the eIF4E protein (Serine-209). This result was mainly mediated by phosphorylation of the 4EBP1 protein via the PI3K/AKT pathway. Additionally, the pharmacological inhibition of eIF4E phosphorylation in cervical cancer cell lines substantially reduced the protein levels of CCND1 and ODC1, indicating that E6 of the high-risk genotypes may modify protein synthesis of the eIF4E target genes by increasing the activity of the AKT and ERK pathways.

Beta Human Papillomavirus and Merkel Cell Polyomavirus in Skin Neoplasms

Beta Human Papillomavirus and Merkel Cell Polyomavirus in Skin Neoplasms

Complementary Targeting of Rb Phosphorylation and Growth in Cervical Cancer Cell Cultures and a Xenograft Mouse Model by SHetA2 and Palbociclib.

Cervical cancer is caused by high-risk human papillomavirus (HPV) types and treated with conventional chemotherapy with surgery and/or radiation. HPV E6 and E7 proteins increase phosphorylation of retinoblastoma (Rb) by cyclin D1/cyclin dependent kinase (CDK)4/6 complexes. We hypothesized that cyclin D1 degradation by the SHetA2 drug in combination with palbociclib inhibition of CDK4/6 activity synergistically reduces phosphorylated Rb (phospho-Rb) and inhibits cervical cancer growth. The effects of these drugs, alone, and in combination, were evaluated in SiHa and CaSki HPV-positive and C33A HPV-negative cervical cancer cell lines using cell culture, western blots and ELISA, and in a SiHa xenograft model. Endpoints were compared by isobolograms, ANOVA, and Chi-Square. In all cell lines, combination indexes documented synergistic interaction of SHetA2 and palbociclib in association SHetA2 reduction of cyclin D1 and phospho-Rb, palbociclib reduction of phospho-Rb, and enhanced phospho-Rb reduction upon drug combination. Both drugs significantly reduced phospho-Rb and growth of SiHa xenograft tumors as single agents and acted additively when combined, with no evidence of toxicity. Dilated CD31-negative blood vessels adjacent to, or within, areas of necrosis and apoptosis were observed in all drug-treated tumors. These results justify development of the SHetA2 and palbociclib combination for targeting phospho-Rb in cervical cancer treatment.

HPV E6 and E7 oncoproteins cooperatively alter the expression of Disc Large 1 polarity protein in epithelial cells

BackgroundPersistent infection with high-risk Human Papillomavirus (HPVs) is associated with the development of cervical cancer. The transforming capacity of these viruses relies on the cooperative action of the E6 and E7 viral oncoproteins. Among the oncogenic activities of E6, the interaction and interference with cell polarity PDZ proteins have been well established. One of the most characterized PDZ targets of HPV E6 is human Disc large 1 (DLG1), a scaffolding protein involved in the control of cell polarity and proliferation. Interestingly, in cervical squamous intraepithelial lesions, alterations in DLG1 expression were observed in association to tumour progression. Moreover, the expression of both HPV E6 and E7 proteins may be responsible for the changes in DLG1 abundance and cell localization observed in the HPV-associated lesions.MethodsDue to the relevance of DLG1 deregulation in tumour development, we have performed an in-depth investigation of the expression of DLG1 in the presence of the HPV oncoproteins in epithelial cultured cells. The effects of HPV E6 and E7 proteins on DLG1 abundance and subcellular localization were assessed by western blot and confocal fluorescence microscopy, respectively.ResultsWe demonstrated that the relative abundance of HPV-18 E6 and DLG1 is a key factor that contributes to defining the expression abundance of both proteins. We also show here that a high expression level of DLG1 may negatively affect HPV-18 E6 nuclear expression. Moreover, the co-expression of HPV-18 E6 and E7 produces a striking effect on DLG1 subcellular localization and a co-distribution in the cytoplasmic region. Interestingly, HPV-18 E7 is also able to increase DLG1 levels, likely by rescuing it from the E6-mediated proteasomal degradation.ConclusionsIn general, the data suggest that HPV-18 E6 and E7 may have opposing activities in regards to the regulation of DLG1 levels and may cooperatively contribute to its subcellular redistribution in the HPV context. These findings constitute a step forward in understanding the differential expression of DLG1 during tumour progression in an HPV-associated model.

HPV-mediated nuclear export of HP1\u03b3 drives cervical tumorigenesis by downregulation of p53

E6 oncoprotein derived from high-risk human papillomavirus (HPV) drives the development of cervical cancer through p53 degradation. Because cervical cancer therapies to inactivate HPV or E6 protein are not available, alternative strategies are required. Here, we show that HPV-mediated nuclear export of human heterochromatin protein 1γ (HP1γ) reduces the stability of p53 through UBE2L3-mediated p53 polyubiquitination during cervical cancer progression. In general, HP1 plays a key role in heterochromatin formation and transcription in the nucleus. However, our immunostaining data showed that the majority of HP1γ is localized in the cytoplasm in HPV-mediated cervical cancer. We found that HPV E6 protein drives unusual nuclear export of HP1γ through the interaction between the NES sequence of HP1γ and exportin-1. The mutation of the NES sequence in HP1γ led to nuclear retention of HP1γ and reduced cervical cancer cell growth and tumor generation. We further discovered that HP1γ directly suppresses the expression of UBE2L3 which drives E6-mediated proteasomal degradation of p53 in cervical cancer. Downregulation of UBE2L3 by overexpression of HP1γ suppressed UBE2L3-dependent p53 degradation-promoting apoptosis of cervical cancer cells. Our findings propose a useful strategy to overcome p53 degradation in cervical cancer through the blockage of nuclear export of HP1γ.

Ubiquitination of the HPV Oncoprotein E6 Is Critical for E6/E6AP-Mediated p53 Degradation.

High-risk Human papillomaviruses (HPVs) types are associated with more than 90% of premalignant and malignant squamous lesions of the uterine cervix. The E6 oncoprotein of high-risk HPVs is a key determinant in cell transformation because it induces the degradation of the host pro-apoptotic tumor suppressor p53. E6 recruits the intracellular ubiquitin ligase E6AP and subsequently induces proteasome-dependent p53 degradation. Neither E6 nor E6AP alone interact with p53; however, the precise mechanism of the functional regulation of the E6/E6AP/p53 complex is unclear. Here, we showed that the high-risk HPV E6 proteins are ubiquitinated during E6/E6AP/p53 complex assembly and degraded by the proteasome system. Increasing p53 expression enhanced E6/E6AP/p53 assembly and facilitated E6 ubiquitination and degradation. The dominant negative mutant of p53 R175H, which does not efficiently bind E6, decreased E6 ubiquitination and increased stability. Furthermore, we showed that the ubiquitin ligase E6AP is essential for E6 ubiquitination, and downregulation of E6AP expression increased E6 stability. We also showed that p53 R175H inhibited E6-mediated p53 degradation. Consistently, the host deubiquitinating enzyme USP15 removed ubiquitin chains from E6 proteins and inhibited E6-mediated p53 degradation. Crucially, ectopic expression of either p53 R175H or USP15 promoted p53-triggered apoptosis in human cervical cancer cells. Considering the importance of ubiquitinated E6 on p53 degradation, the disruption of E6 ubiquitination represents an attractive pharmacological intervention against HPV-positive human cervical cancer.ImportanceVirtually 100% of cervical cancers are linked to HPV infection. Commercial HPV vaccines are estimated to prevent up to 90% of HPV-associated cancers, while they do not eliminate persistent HPV infections and have no effect on the progression to malignancy. Hence, the development of novel therapeutic interventions against HPV is urgently required. The HPV oncoprotein E6 binds to the intracellular E3 ubiquitin ligase E6AP and p53 resulting in the degradation of p53. In this study, we demonstrate that HPV E6 is ubiquitinated by E6AP in presence of p53. Crucially, ubiquitination of E6 is important for p53 degradation and blockage of E6 ubiquitination negatively interferes with E6-mediated p53 degradation and enhances the apoptotic effects of p53 and the cytotoxicity of DNA damage in HPV-positive cervical cancer cells. Importantly, our data suggest that the HPV oncogene E6 might be an optimal pharmacologic.

E6 proteins from high-risk HPV, low-risk HPV, and animal papillomaviruses activate the Wnt/β-catenin pathway through E6AP-dependent degradation of NHERF1.

High-risk human papillomavirus (HPV) E6 proteins associate with the cellular ubiquitin ligase E6-Associated Protein (E6AP), and then recruit both p53 and certain cellular PDZ proteins for ubiquitination and degradation by the proteasome. Low-risk HPV E6 proteins also associate with E6AP, yet fail to recruit p53 or PDZ proteins; their E6AP-dependent targets have so far been uncharacterized. We found a cellular PDZ protein called Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) is targeted for degradation by both high and low-risk HPV E6 proteins as well as E6 proteins from diverse non-primate mammalian species. NHERF1 was degraded by E6 in a manner dependent upon E6AP ubiquitin ligase activity but independent of PDZ interactions. A novel structural domain of E6, independent of the p53 recognition domain, was necessary to associate with and degrade NHERF1, and the NHERF1 EB domain was required for E6-mediated degradation. Degradation of NHERF1 by E6 activated canonical Wnt/β-catenin signaling, a key pathway that regulates cell growth and proliferation. Expression levels of NHERF1 increased with increasing cell confluency. This is the first study in which a cellular protein has been identified that is targeted for degradation by both high and low-risk HPV E6 as well as E6 proteins from diverse animal papillomaviruses. This suggests that NHERF1 plays a role in regulating squamous epithelial growth and further suggests that the interaction of E6 proteins with NHERF1 could be a common therapeutic target for multiple papillomavirus types.

Human papillomavirus E7 induces p63 expression to modulate DNA damage response

Cervical cancer is the third most common malignancy diagnosed in women worldwide. The major aetiological factor underlying the malignant transformation of cervical cells is the persistent infection with high-risk human papillomaviruses (HR-HPV), with more than 99% of cases expressing viral sequences. Here, we report a previously unknown mechanism driven by high-risk human papillomavirus E7 protein to modulate response to DNA damage in cervical cancer cells. Our data shows that HR-HPV E7 oncoprotein induces the transcription of the p53-family member p63, which modulates DNA damage response pathways, to facilitate repair of DNA damage. Based on our findings, we proposed a model, where HR-HPV could interfere with the sensitivity of transformed cells to radiation therapy by modulating DNA damage repair efficiency. Importantly, we have shown for the first time a critical role for p63 in response to DNA damage in cervical cancer cells.

Understanding the Role of Intrinsic Disorder of Viral Proteins in the Oncogenicity of Different Types of HPV.

Intrinsic disorder is very important in the biological function of several proteins, and is directly linked to their foldability during interaction with their targets. There is a close relationship between the intrinsically disordered proteins and the process of carcinogenesis involving viral pathogens. Among these pathogens, we have highlighted the human papillomavirus (HPV) in this study. HPV is currently among the most common sexually transmitted infections, besides being the cause of several types of cancer. HPVs are divided into two groups, called high- and low-risk, based on their oncogenic potential. The high-risk HPV E6 protein has been the target of much research, in seeking treatments against HPV, due to its direct involvement in the process of cell cycle control. To understand the role of intrinsic disorder of the viral proteins in the oncogenic potential of different HPV types, the structural characteristics of intrinsically disordered regions of high and low-risk HPV E6 proteins were analyzed. In silico analyses of primary sequences, prediction of tertiary structures, and analyses of molecular dynamics allowed the observation of the behavior of such disordered regions in these proteins, thereby proving a direct relationship of structural variation with the degree of oncogenicity of HPVs. The results obtained may contribute to the development of new therapies, targeting the E6 oncoprotein, for the treatment of HPV-associated diseases.

A Mathematical Model of Cell Cycle Dysregulation Due to Human Papillomavirus Infection.

Human papillomaviruses (HPVs) that infect mucosal epithelium can be classified as high risk or low risk based on their propensity to cause lesions that can undergo malignant progression. HPVs produce the E7 protein that binds to cell cycle regulatory proteins including the retinoblastoma tumor suppressor protein (RB) to modulate cell cycle control. Generally, high-risk HPV E7 proteins bind to RB with a higher affinity than low-risk HPV E7s, but both are able to deactivate RB and trigger S phase progression. In uninfected cells, RB inactivation is a tightly controlled process that must coincide with growth factor stimulation to commit cells to division. High-risk HPV E7 proteins short-circuit this control by decreasing growth factor requirement for cell division. We develop a mathematical model to examine the role that RB binding affinity, growth factor concentration, and E7 concentration have on cell cycle progression. Our model predicts that high RB binding affinity and E7 concentration accelerate the [Formula: see text] to S phase transition and weaken the dependence on growth factor. This model thus captures a key step in high-risk HPV oncogenesis.

Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 Oncoproteins.

ABSTRACTThe E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs). While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR) levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs) with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation.

High-Risk Human Papillomavirus E7 Proteins Target PTPN14 for Degradation.

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.

High-risk human papillomavirus E6 protein promotes reprogramming of Fanconi anemia patient cells through repression of p53 but does not allow for sustained growth of induced pluripotent stem cells.

DNA repair plays a crucial role in embryonic and somatic stem cell biology and cell reprogramming. The Fanconi anemia (FA) pathway, which promotes error-free repair of DNA double-strand breaks, is required for somatic cell reprogramming to induced pluripotent stem cells (iPSC). Thus, cells from Fanconi anemia patients, which lack this critical pathway, fail to be reprogrammed to iPSC under standard conditions unless the defective FA gene is complemented. In this study, we utilized the oncogenes of high-risk human papillomavirus 16 (HPV16) to overcome the resistance of FA patient cells to reprogramming. We found that E6, but not E7, recovers FA iPSC colony formation and, furthermore, that p53 inhibition is necessary and sufficient for this activity. The iPSC colonies resulting from each of these approaches stained positive for alkaline phosphatase, NANOG, and Tra-1-60, indicating that they were fully reprogrammed into pluripotent cells. However, FA iPSC were incapable of outgrowth into stable iPSC lines regardless of p53 suppression, whereas their FA-complemented counterparts grew efficiently. Thus, we conclude that the FA pathway is required for the growth of iPSC beyond reprogramming and that p53-independent mechanisms are involved. A novel approach is described whereby HPV oncogenes are used as tools to uncover DNA repair-related molecular mechanisms affecting somatic cell reprogramming. The findings indicate that p53-dependent mechanisms block FA cells from reprogramming but also uncover a previously unrecognized defect in FA iPSC proliferation independent of p53.

P16 in HPV-associated cancers

Infections with high-risk human papillomaviruses (HPV) account for approximately 5% of all human cancers. Cervical cancer, in particular, is almost universally caused by “high-risk” HPV infections and these HPVs also cause a large fraction of other anogenital tract cancers as well as oral carcinomas. The viral E6 and E7 proteins are the universal oncogenic drivers of HPV-associated cancers, and persistent E6/E7 expression is necessary for the maintenance of the transformed state. Hence the HPV E6 and E7 proteins should represent excellent therapeutic targets. The HPV E6 and E7 proteins lack intrinsic enzymatic activities and attempts to discover small molecule inhibitors of E6 or E7 containing cellular protein complexes have been futile. HPV E6 and E7 function by associating with host cellular proteins, thereby functionally re-programming cellular signal transduction pathways. The resulting rewiring of cellular circuitry causes “addictions” to certain signaling pathways that are non-essential and/or redundant in normal cells. This in turn generates specific cellular vulnerabilities that may be more readily druggable. The article (McLaughlin-Drubin ME et al., Proc. Natl. Acad. Sci. USA. 2013; 110:16175-16180) identifies a surprising and potentially druggable vulnerability in the retinoblastoma tumor suppressor pathway caused by the high-risk HPV E7 protein. High-risk HPV E7 protein expression triggers a cellular defense response referred to as oncogene induced senescence (OIS), which is mediated by the p16INK4A (p16) tumor suppressor and executed by the retinoblastoma tumor suppressor protein, Rb. In normal cells, p16 expression is epigenetically silenced by polycomb repressive complexes. p16 inhibits cyclin dependent kinase 4 and 6 (CDK4/6). This causes accumulation of hypophosphorylated Rb, which in turn signals cell cycle arrest (Fig. ​(Fig.11 upper panel). E7 protein triggers p16. High-level p16 expression is an excellent biomarker for high-risk HPV-associated lesions and cancers. Cell cycle arrest and senescence in these tumors, however, is subverted by E7 targeting Rb for proteasomal degradation (Fig. ​(Fig.11 lower panel). p16 induction, in the absence of Rb (through Rb degradation by E7) causes a unique and surprising addiction to p16INK4A expression. Even though p16 is a tumor suppressor in most cellular contexts, it is essential for survival of cervical carcinoma lines. The iconoclastic “oncogenic” activity of p16 in HPV E7 expressing cells, like its more familiar tumor suppressor activity, is based on the ability to inhibit CDK4/6. Indeed, a human melanoma derived p16-insensitive CDK4 mutant also causes cell death when expressed in HPV E7-expressing cells. Hence, this oncogenic CDK4 mutant exhibits biological activities that are akin to those of a tumor suppressor in the context of a p16-expressing, Rb defective cell. These results imply that CDK4/6 activity is not tolerated in cells that lack Rb activity. One may hypothesize that there must be CDK4/6 substrates that cause cell death when they are phosphorylated in cells that lack Rb. Tumorigenic activities of the Rb pathway including CDK4/6 and p16 are context dependent. CDK4/CDK6 inhibition may be efficacious only in tumors that retain functional Rb. In contrast, CDK4/CDK6 inhibition in cancers that have suffered Rb mutations is necessary for tumor growth and survival. In such cases it may be therapeutically useful to activate rather than to inhibit CDK4/CDK6 activity. One way CDK4/6 activation may be achieved is by epigenetic silencing of p16 through inhibition of the KDM6B enzyme. Indeed the KDM6-selective small molecule inhibitor GSK-J4 induced cell death specifically in KDM6B/p16-addicted cell lines. This shows that the addiction of HPV-associated lesions and cancer to p16 expression creates a cellular vulnerability to KDM6B inhibition. De-repression of polycomb-silenced genes, such as p16, is a complex, multistep process. Hence, “epigenetic therapies” targeting KDM6B and potentially other enzymes that may also be rate limiting for maintaining the transcriptional competence of the p16 promoter may be efficacious in p16-addicted cancers. Figure 1 The retinoblastoma (Rb) tumor suppressor pathway It has been noted early on that p16 expression is confined to those tumors that contain pRB mutations, and a number of non HPV-associated cancer types, including some breast, prostate, lung and high-grade serous ovarian carcinomas express p16. It will be important to determine whether some of these tumors are similarly addicted to p16 expression and whether they are vulnerable to inhibition of KDM6B and/or other epigenetic enzymes that are necessary for p16 expression.

Identification of miRNAs dysregulated in human foreskin keratinocytes (HFKs) expressing the human papillomavirus (HPV) Type 16 E6 and E7 oncoproteins.

Human papillomaviruses (HPVs) are associated with the pathogenesis of a variety of human cancers, including cervical and oropharyngeal cancers. The HPV E6 and E7 oncogenes are usually expressed to high levels in these cancers. Previous studies have shown dysregulation of cellular microRNAs (miRNAs) in HPV-positive cell lines and cancer tissues and recent studies have identified a few miRNAs whose levels are altered in the presence of the viral E6 and E7 proteins. In order to identify all the cellular miRNAs whose expression may be affected by these oncoproteins, we carried out microarray analysis using human foreskin keratinocytes (HFKs) expressing either or both of these two proteins. These studies showed that 90 and 60 miRNAs were dysregulated in the presence of the E6 or the E7 protein, respectively. Of these, 43 miRNAs were similarly affected in HFK-E6 and/or HFK-E7 when compared to control cells. The joint expression of E6 and E7 proteins in HFKs caused changes in the levels of 64 miRNAs, of which 24 were similarly affected in HFK-E6 and/or HFK-E7 cells relative to controls. The microarray experiments were validated by quantitative real-time RT-PCR analysis of several differentially expressed miRNAs. Several miRNAs dysregulated by the E6 and/or E7 proteins are known to be altered in a variety of human cancers. Furthermore, previously known cellular targets of these miRNAs are involved in processes such as cell cycle regulation, apoptosis, cell-cell adhesion, cell mobility and proliferation, and alterations in their levels may contribute to HPV-associated carcinogenesis. Taken together, the results of our studies suggest that high risk HPV E6 and E7 proteins share the ability to regulate a subset of cellular miRNAs.