Cells Expressing HPV16 E6 Research Articles

HPV upregulates MARCHF8 ubiquitin ligase and inhibits apoptosis by degrading the death receptors in head and neck cancer.

The membrane-associated RING-CH-type finger ubiquitin ligase MARCHF8 is a human homolog of the viral ubiquitin ligases Kaposi's sarcoma herpesvirus K3 and K5 that promote host immune evasion. Previous studies have shown that MARCHF8 ubiquitinates several immune receptors, such as the major histocompatibility complex II and CD86. While human papillomavirus (HPV) does not encode any ubiquitin ligase, the viral oncoproteins E6 and E7 are known to regulate host ubiquitin ligases. Here, we report that MARCHF8 expression is upregulated in HPV-positive head and neck cancer (HNC) patients but not in HPV-negative HNC patients compared to normal individuals. The MARCHF8 promoter is highly activated by HPV oncoprotein E6-induced MYC/MAX transcriptional activation. The knockdown of MARCHF8 expression in human HPV-positive HNC cells restores cell surface expression of the tumor necrosis factor receptor superfamily (TNFRSF) death receptors, FAS, TRAIL-R1, and TRAIL-R2, and enhances apoptosis. MARCHF8 protein directly interacts with and ubiquitinates the TNFRSF death receptors. Further, MARCHF8 knockout in mouse oral cancer cells expressing HPV16 E6 and E7 augments cancer cell apoptosis and suppresses tumor growth in vivo. Our findings suggest that HPV inhibits host cell apoptosis by upregulating MARCHF8 and degrading TNFRSF death receptors in HPV-positive HNC cells.

Liposomal TLR9 Agonist Combined with TLR2 Agonist-Fused Antigen Can Modulate Tumor Microenvironment through Dendritic Cells.

Dendritic cells (DCs) are antigen-presenting cells involved in T cell activation and differentiation to regulate immune responses. Lipoimmunogens can be developed as pharmaceutical lipoproteins for cancer immunotherapy to target DCs via toll-like receptor 2 (TLR2) signaling. Previously, we constructed a lipoimmunogen, a lipidated human papillomavirus (HPV) E7 inactive mutant (rlipoE7m), to inhibit the growth of HPV16 E7-expressing tumor cells in a murine model. Moreover, this antitumor effect could be enhanced by a combinatory treatment with CpG oligodeoxynucleotides (ODN). To improve safety, we developed a rlipoE7m plus DOTAP liposome-encapsulated native phosphodiester CpG (POCpG/DOTAP) treatment to target DCs to enhance antitumor immunity. We optimized the formulation of rlipoE7m and POCpG/DOTAP liposomes to promote conventional DC and plasmacytoid DC maturation in vitro and in vivo. Combination of rlipoE7m plus POCpG/DOTAP could activate conventional DCs and plasmacytoid DCs to augment IL-12 production to promote antitumor responses by intravenous injection. In addition, the combination of rlipoE7m plus POCpG/DOTAP could elicit robust cytotoxic T lymphocytes (CTLs) by intravenous immunization. Interestingly, the combination of rlipoE7m plus POCpG/DOTAP could efficiently inhibit tumor growth via intravenous immunization. Moreover, rlipoE7m plus POCpG/DOTAP combined reduced the number of tumor-infiltrating regulatory T cells dramatically due to downregulation of IL-10 production by DCs. These results showed that the combination of rlipoE7m plus POCpG/DOTAP could target DCs via intravenous delivery to enhance antitumor immunity and reduce the number of immunosuppressive cells in the tumor microenvironment.

Targeting interferon signaling and CTLA-4 enhance the therapeutic efficacy of anti-PD-1 immunotherapy in preclinical model of HPV+ oral cancer

BackgroundThe US is experiencing an epidemic of HPV+ oropharyngeal cancers (OPC), the rates and burden of which now exceed that for cervical cancer. Immunotherapy targeting programmed death 1 (PD-1) on tumor-infiltrating lymphocytes and/or its ligand PD-L1 on tumor cells, which was effective in several cancers has however, showed efficacy in only less than 15% of patients.MethodsWe used a preclinical HPV+ oral tumor model, mEER, consisting of mouse tonsil derived epithelial cells expressing HPV-16 E6 and E7 genes, along with the H-ras oncogene to test strategies for enhancing the efficacy of anti-PD-1 therapy.ResultsMonotherapy with PD-1 blocking antibody was ineffective against flank-implanted tumors, but induced regression in 54% of mice bearing orthotopic tongue tumors that correlated with higher CD8 T cell responses. Since the CD8+ T cells derived from tongue tumors also showed high levels of the immune checkpoint inhibitory receptor CTLA-4, we tested combination immunotherapy targeting both CTLA-4 and PD-1 together and observed 93.3% survival of mice bearing tumors in the tongue for the duration of our 100-day study. Protective immunity correlated with a significant decrease in immunosuppressive lymphoid and myeloid populations within the tumor microenvironment. Consistent with the reported capacity of interferon-driven PD-L1/PD-1 pathway induction to serve as a biomarker of response to PD-1 blockade, we observed elevated interferon signaling and significantly higher levels of PD-1/PD-L1 in tongue-implanted mEER tumors compared to those growing on the flank correlating with their preferential responsiveness to PD-1 blockade. More importantly, in a pseudometastasic mouse model bearing both flank and tongue tumors to represent metastatic disease, delivery of Stimulator of Interferon Induced Genes (STING) agonist into the flank tumors combined with systemic treatment with α-PD-1 and α-CTLA-4 antibodies resulted in sustained tumor regression in 71% of mice. In this case, productive abscopal anti-tumor immunity was associated with robust increases in the ratios of cytotoxic CD8+ T cells (CTL) versus regulatory T cells (Treg) and versus functional myeloid-derived suppressor cells (MDSC).ConclusionsThese results support combining α-PD-1 therapy with induction of IFN-α/β signaling via provision of STING agonist and/or through CTLA-4 blockade as potential treatment option for HNSCC patients, especially, those not responding to α-PD-1 monotherapy.

Abstract A62: In vivo electroporation improves the immune responses induced by a DNA vaccine and confers complete antitumor protection against HPV-associated tumors

Abstract Cervical cancer associated with persistent papilloma virus infections (HPV) is a public health problem and represents the third most frequent type of cancer in women. Our group developed a DNA vaccine against HPV-induced tumors based on the expression of the HPV-16 E7 oncoprotein genetically fused to the HSV-1 glycoprotein D (gD) (pgDE7h). This vaccine is capable of promoting antitumoral effects against tumor cells expressing HPV-16 E6 and E7 proteins (TC-1 cells) and induces specific cellular immune responses. In view of the low success rate of DNA vaccines in clinical trials, in vivo electroporation (EP) is a promising strategy for plasmid delivery, leading to increased cellular transfection and enhanced immune responses. In this study, C57BL/6 mice were inoculated with TC-1 cells subcutaneously and immunized 3 days later with pgDE7h intramuscularly, associated or not with EP. Immune responses were analyzed 14 days after vaccination and tumor growth was monitored for 60 days. Similar antitumor effects were obtained with 10-fold less DNA associated with EP when compared to the group that was inoculated only with DNA, reaching around 20% of tumor-free mice. Dose-response curves indicated that maximum protective immunity was achieved with 50 ug of vaccine inoculated with EP, promoting 100% of antitumor protection. This group also had enhanced frequencies of cytotoxic lymphocytes expressing the degranulation marker CD107a and high in vivo cytotoxic activities. Besides that, a delayed vaccination performed 10 days after the TC-1 injection led to 70% of tumor-free mice until the end of the observed period. Collectively, these data confirmed that EP can be used as an efficient method of DNA vaccine delivery and should be further studied to improve the chances of cancer vaccine success in clinical trials. Citation Format: Natiely Silva Sales, Mariana de Oliveira Diniz, Jamile Ramos Silva, Mariângela de Oliveira Silva, Bruna F. M. M. Porchia, Luana R. M. M. Aps, Luis Carlos Souza Ferreira. In vivo electroporation improves the immune responses induced by a DNA vaccine and confers complete antitumor protection against HPV-associated tumors [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A62.

MiR-2861 acts as a tumor suppressor via targeting EGFR/AKT2/CCND1 pathway in cervical cancer induced by human papillomavirus virus 16 E6.

Persistent infection with oncogenic human papillomavirus viruses (HPVs) is a casual factor for cervical cancer and its precursors, and the abnormal constitutive expression of viral oncoprotein E6 is a key event during the malignant transformation. Here, we performed miRNA microarray to identify changes of miRNAs following ectopic HPV16 E6 overexpression in HEK293T cells and found miR-2861 was greatly decreased in both HEK293T and HaCaT cells expressing HPV16 E6 compared to vector control. Further, we demonstrated a biological link among HPV16 E6, miR-2861, EGFR, AKT2, and CCND1 in cervical cancer cells. We showed that miR-2861 was downregulated in cervical cancer tissues and negatively correlated with advanced tumor stage and lymph node metastasis. Overexpression of miR-2861 suppressed cervical cancer cell proliferation and invasion and enhanced apoptosis. Subsequent investigation revealed that EGFR, AKT2, and CCND1 were all the direct targets of miR-2861. Importantly, silencing EGFR, AKT2, and/or CCND1 recapitulated the cellular effects seen upon miR-2861 overexpression. Restoration of EGFR, AKT2, and/or CCND1 counteracted the effects of miR-2861 expression. Thus, we identified a new pathway employing miR-2861, EGFR, AKT2, and CCND1 that may mediate HPV16 E6 induced initiation and progression of cervical cancer.

Transcriptional Suppression of E-Cadherin by HPV-16 E6 and E7 Oncogenes is Independent of Hypermethylation of E-Cadherin Promoter

Background: Cervical cancer is one of the most common cancers observed in women worldwide, and its development is related to E6 and E7 two viral oncoproteins of high-risk human papillomavirus (HPV) types. Aberrant expression of E-cadherin, which is associated with epithelial-to-mesenchymal transition (EMT), is frequently observed in cervical cancer. Objectives: The mechanisms underlying E-cadherin suppression in cervical cancer are not clear; therefore, this experimental study from Iran was designed to elucidate the relationship of DNA methyltransferase expression and E-cadherin promoter methylation with E-cadherin expression in HPV-16 E6- and E7-expressing cells. Materials and Methods: Real-time PCR and western blot were used to determine the effects of HPV-16 E6 and E7 on E-cadherin, DNMT1, DNMT3a, and DNMT3b expression in HCT-116 cell line. We also analyzed E-cadherin promoter methylation in cells expressing HPV-16 E6 and E7 oncoproteins by bisulfite sequencing. Results: HPV-16 E6 and E7 proteins reduced E-cadherin expression 3.7 and 2.2 times when compared with control cells (P = 0.0221 and P = 0.0461, respectively). This reduction was greater in HPV-16 E6-expressing cells than in HPV-16 E7-expressing cells. Although HPV-16 E6 and E7 increased DNA methyltransferase 1 expression 2.6 and 3.4 times, respectively (P = 0.0133 and P = 0.0113) when compared with control cells, they was no E-cadherin promoter methylation. Conclusions: Unlike other cancer-associated viruses (HBV, HCV, and EBV), reduction in E-cadherin expression in HPV-16 E6- and E7-expressing cells is not due to hypermethylation of the E-cadherin promoter.

Human papillomavirus type 16 E7 oncoprotein inhibits the anaphase promoting complex/cyclosome activity by dysregulating EMI1 expression in mitosis

The anaphase promoting complex/cyclosome (APC/C) is a ubiquitin ligase complex that orchestrates mitotic progression by targeting key mitotic regulators for proteasomal degradation. APC/C dysfunction is a frequent event during cancer development and can give rise to genomic instability. Here we report that the HPV16 E7 oncoprotein interferes with the degradation of APC/C substrates and that the APC/C inhibitor, EMI1, is expressed at higher levels in HPV16 E7-expressing mitotic cells. HPV16 E7 expression causes increased EMI1 mRNA expression and also inhibits EMI1 degradation. The resulting abnormally high EMI1 levels in HPV16 E7-expressing mitotic cells may inhibit degradation of APC/C substrates and cause the prometaphase delay that we have previously observed in such cells.

The human papillomavirus-16 E7 oncoprotein exerts antiapoptotic effects via its physical interaction with the actin-binding protein gelsolin

The oncoprotein E7 from human papillomavirus-16 (HPV-16 E7) plays a pivotal role in HPV postinfective carcinogenesis, and its physical interaction with host cell targets is essential to its activity. We identified a novel cellular partner for the viral oncoprotein: the actin-binding protein gelsolin (GSN), a key regulator of actin filament assembly and disassembly. In fact, biochemical analyses, generation of a 3D molecular interaction model and the use of specific HPV-16 E7 mutants provided clear cut evidence supporting the crucial role of HPV-16 E7 in affecting GSN integrity and function in human immortalized keratinocytes. Accordingly, functional analyses clearly suggested that stable HPV-16 E7 expression induced an imbalance between polymeric and monomeric actin in favor of the former. These events also lead to changes of cell cycle (increased S phase), to the inhibition of apoptosis and to the increase of cell survival. These results provide support to the hypotheses generated from the 3D molecular interaction model and encourage the design of small molecules hindering HPV-induced host cell reprogramming by specifically targeting HPV-16 E7-expressing cells.

Human Papillomavirus E7 Induces Rereplication in Response to DNA Damage

Human papillomavirus (HPV) infection is necessary but not sufficient for cervical carcinogenesis. Genomic instability caused by HPV allows cells to acquire additional mutations required for malignant transformation. Genomic instability in the form of polyploidy has been demonstrated to play an important role in cervical carcinogenesis. We have recently found that HPV-16 E7 oncogene induces polyploidy in response to DNA damage; however, the mechanism is not known. Here we present evidence demonstrating that HPV-16 E7-expressing cells have an intact G(2) checkpoint. Upon DNA damage, HPV-16 E7-expressing cells arrest at the G(2) checkpoint and then undergo rereplication, a process of successive rounds of host DNA replication without entering mitosis. Interestingly, the DNA replication initiation factor Cdt1, whose uncontrolled expression induces rereplication in human cancer cells, is upregulated in E7-expressing cells. Moreover, downregulation of Cdt1 impairs the ability of E7 to induce rereplication. These results demonstrate an important role for Cdt1 in HPV E7-induced rereplication and shed light on mechanisms by which HPV induces genomic instability.

Systematic identification of interactions between host cell proteins and E7 oncoproteins from diverse human papillomaviruses

More than 120 human papillomaviruses (HPVs) have now been identified and have been associated with a variety of clinical lesions. To understand the molecular differences among these viruses that result in lesions with distinct pathologies, we have begun a MS-based proteomic analysis of HPV-host cellular protein interactions and have created the plasmid and cell line libraries required for these studies. To validate our system, we have characterized the host cellular proteins that bind to the E7 proteins expressed from 17 different HPV types. These studies reveal a number of interactions, some of which are conserved across HPV types and others that are unique to a single HPV species or HPV genus. Binding of E7 to UBR4/p600 is conserved across all virus types, whereas the cellular protein ENC1 binds specifically to the E7s from HPV18 and HPV45, both members of genus alpha, species 7. We identify a specific interaction of HPV16 E7 with ZER1, a substrate specificity factor for a cullin 2 (CUL2)-RING ubiquitin ligase, and show that ZER1 is required for the binding of HPV16 E7 to CUL2. We further show that ZER1 is required for the destabilization of the retinoblastoma tumor suppressor RB1 in HPV16 E7-expressing cells and propose that a CUL2-ZER1 complex functions to target RB1 for degradation in HPV16 E7-expressing cells. These studies refine the current understanding of HPV E7 functions and establish a platform for the rapid identification of virus-host interactions.

The HPV-16 E7 oncoprotein induces centriole multiplication through deregulation of Polo-like kinase 4 expression

BackgroundInfection with high-risk human papillomaviruses (HPVs) such as HPV-16 is intimately associated with squamous cell carcinomas (SCCs) of the anogenital tract and a subset of oropharyngeal carcinomas. Such lesions, including pre-invasive precursors, frequently show multipolar mitoses and aneuploidy. The high-risk HPV-16-encoded E7 oncoprotein has been shown to rapidly induce centrosome abnormalities thereby causing the formation of supernumerary mitotic spindle poles and increasing the risk for chromosome missegregation. HPV-16 E7 has been found to rapidly induce centriole overduplication, in part, through the simultaneous formation of more than one daughter centriole at single maternal centrioles (centriole multiplication). The precise molecular mechanism that underlies HPV-16 E7-induced centriole multiplication, however, remains poorly understood.FindingsHere, we show that human keratinocytes engineered to stably express the HPV-16 E7 oncoprotein exhibit aberrant Polo-like kinase 4 (PLK4) protein expression at maternal centrioles. Real-time quantitative reverse transcriptase (qRT-PCR) analysis of these cells revealed an increase of PLK4 mRNA levels compared to control cells. Importantly, the ability of the HPV-16 E7 oncoprotein to induce centriole multiplication was found to correlate with its ability to activate the PLK4 promoter and to up-regulate PLK4 mRNA.ConclusionsThese results highlight the critical role of PLK4 transcriptional deregulation in centriole multiplication in HPV-16 E7-expressing cells. Our findings encourage further experiments to test transcriptional inhibitors or small molecules targeting PLK4 to prevent centriole abnormalities, mitotic infidelity and malignant progression in HPV-associated neoplasms and other tumors in which PLK4 regulation is disrupted.

Human papillomavirus E7 oncoprotein induces KDM6A and KDM6B histone demethylase expression and causes epigenetic reprogramming

Despite the availability of vaccines, human papillomavirus (HPV) infections remain a cause of significant cancer morbidity and mortality. We have previously shown that HPV16 E7 associates with and diminishes E2F6-containing polycomb repressive complexes. Here, we show that repressive trimethyl marks on lysine 27 of histone 3, which are necessary for binding of polycomb repressive complexes, are decreased in HPV16 E7-expressing cells and HPV16-positive cervical lesions. This is caused by transcriptional induction of the KDM6A and KDM6B histone 3 lysine 27-specific demethylases. HPV16 E7-mediated KDM6B induction accounts for expression of the cervical cancer biomarker, p16(INK4A). Moreover, KDM6A- and KDM6B-responsive Homeobox genes are expressed at significantly higher levels, suggesting that HPV16 E7 results in reprogramming of host epithelial cells. These effects are independent of the ability of E7 to inhibit the retinoblastoma tumor suppressor protein. Most importantly, these effects are reversed when E7 expression is silenced, indicating that this pathway may have prognostic and/or therapeutic significance.

The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42

Background:Guanidine exchange factor (GEF)-catalysed activation of Rho proteins such as Cdc42 has been shown to have a crucial role in cellular transformation, malignant progression and invasion. We have previously shown that the HPV16 E6 oncoprotein binds to the PDZ domain protein Tax-interacting-protein 1 (Tip-1) and we now report identification and functional analysis of a novel Tip-1 binding GEF.Methods:Yeast two-hybrid, in vitro pull-down, site-directed mutagenesis, semiquantitative PCR, co-immunoprecipitation and western blotting were used to identify/confirm novel Tip-1 binding partners and analyse cellular expression levels. In vitro kinetic analyses of recombinant proteins, siRNA gene silencing and in cell assays were used to measure Rho protein activation.Results:Tax-interacting-protein 1 was shown to interact with ARHGEF16 by its carboxyl PDZ binding motif. Levels of ARHGEF16 were increased in transformed and immortalised cells expressing ectopic HPV16 E6 and Cdc42 was co-immunoprecipitated by ARHGEF16 in the presence of high-risk HPV E6. In vitro kinetic analysis confirmed that recombinant ARHGEF16 activates Cdc42 and this was increased by the addition of recombinant Tip-1 and E6. Cells expressing HPV16 E6 had higher levels of Cdc42 activation, which was decreased by siRNA silencing of either Tip-1 or ARHGEF16.Conclusion:These data suggest that HPV16 E6, Tip-1 and ARHGEF16 may cooperate to activate Cdc42 and support a potential link between the expression of HPV16 E6 and Cdc42 activation.

Polo-like Kinase 1 Activated by the Hepatitis B Virus X Protein Attenuates Both the DNA Damage Checkpoint and DNA Repair Resulting in Partial Polyploidy

Hepatitis B virus X protein (pX), implicated in hepatocarcinogenesis, induces DNA damage because of re-replication and allows propagation of damaged DNA, resulting in partial polyploidy and oncogenic transformation. The mechanism by which pX allows cells with DNA damage to continue proliferating is unknown. Herein, we show pX activates Polo-like kinase 1 (Plk1) in the G(2) phase, thereby attenuating the DNA damage checkpoint. Specifically, in the G(2) phase of pX-expressing cells, the checkpoint kinase Chk1 was inactive despite DNA damage, and protein levels of claspin, an adaptor of ataxia telangiectasia-mutated and Rad3-related protein-mediated Chk1 phosphorylation, were reduced. Pharmacologic inhibition or knockdown of Plk1 restored claspin protein levels, Chk1 activation, and p53 stabilization. Also, protein levels of DNA repair protein Mre11 were decreased in the G(2) phase of pX-expressing cells but not with Plk1 knockdown. Interestingly, in pX-expressing cells, Mre11 co-immunoprecipitated with transfected Plk1 Polo-box domain, and inhibition of Plk1 increased Mre11 stability in cycloheximide-treated cells. These results suggest that pX-activated Plk1 by down-regulating Mre11 attenuates DNA repair. Importantly, concurrent inhibition of Plk1, p53, and Mre11 increased the number of pX-expressing cells with DNA damage entering mitosis, relative to Plk1 inhibition alone. By contrast, inhibition or knockdown of Plk1 reduced pX-induced polyploidy while increasing apoptosis. We conclude Plk1, activated by pX, allows propagation of DNA damage by concurrently attenuating the DNA damage checkpoint and DNA repair, resulting in polyploidy. We propose this novel Plk1 mechanism initiates pX-mediated hepatocyte transformation.

Human Papillomavirus-16 E7 Interacts with Glutathione S-Transferase P1 and Enhances Its Role in Cell Survival

BackgroundHuman Papillomavirus (HPV)-16 is a paradigm for “high-risk” HPVs, the causative agents of virtually all cervical carcinomas. HPV E6 and E7 viral genes are usually expressed in these tumors, suggesting key roles for their gene products, the E6 and E7 oncoproteins, in inducing malignant transformation.Methodology/Principal FindingsBy protein-protein interaction analysis, using mass spectrometry, we identified glutathione S-transferase P1-1 (GSTP1) as a novel cellular partner of the HPV-16 E7 oncoprotein. Following mapping of the region in the HPV-16 E7 sequence that is involved in the interaction, we generated a three-dimensional molecular model of the complex between HPV-16 E7 and GSTP1, and used this to engineer a mutant molecule of HPV-16 E7 with strongly reduced affinity for GSTP1.When expressed in HaCaT human keratinocytes, HPV-16 E7 modified the equilibrium between the oxidized and reduced forms of GSTP1, thereby inhibiting JNK phosphorylation and its ability to induce apoptosis. Using GSTP1-deficient MCF-7 cancer cells and siRNA interference targeting GSTP1 in HaCaT keratinocytes expressing either wild-type or mutant HPV-16 E7, we uncovered a pivotal role for GSTP1 in the pro-survival program elicited by its binding with HPV-16 E7.Conclusions/SignificanceThis study provides further evidence of the transforming abilities of this oncoprotein, setting the groundwork for devising unique molecular tools that can both interfere with the interaction between HPV-16 E7 and GSTP1 and minimize the survival of HPV-16 E7-expressing cancer cells.

HPV16 E7 oncogene expression in normal human epithelial cells causes molecular changes indicative of an epithelial to mesenchymal transition

Cancer-associated epithelial to mesenchymal transition (EMT) is crucial for invasion and metastasis. Molecular hallmarks of EMT include down-regulation of the epithelial adhesion protein E-cadherin and de-novo expression of N-cadherin and the mesenchymal intermediate filament proteins vimentin and fibronectin. Expression of HPV16 E7 in normal human epithelial cells caused increased levels of vimentin and fibronectin, whereas the epithelial adhesion protein E-cadherin was expressed at decreased levels. Similar expression patterns of vimentin, fibronectin and E-cadherin were also detected in cells expressing HPV16 E6 and E7 or the entire HPV16 early transcriptional unit. HPV16 E6 and E7 were each able to induce N-cadherin expression. Interestingly, these changes in expression levels of EMT-associated proteins are not similarly reflected at the level of mRNA expression, suggesting that HPV16 oncoproteins also modulate EMT through non-transcriptional mechanisms. Hence, HPV16 oncoproteins may contribute to malignant progression through EMT induction.

The expression of the soluble isoform of hFlt3 ligand by recombinant vaccinia virus enhances immunogenicity of the vector

Recombinant vaccinia viruses (rVACV) expressing various tumor-associated antigens have been shown to elicit anti-tumor effect in numerous experimental models and clinical trials. We tested the hypotheses that rVACV expressing biologically active fms-like tyrosine kinase 3 ligand (Flt3L) would show higher immunogenicity than control viruses expressing only model antigen and that coexpression of Flt3L would influence anti-tumor activity of rVACV in the preventive and therapeutic arrangements of the in vivo experiment. To answer these questions, we took advantage of the well-described model of transplanted tumor cells expressing HPV16 E6 and E7 oncoproteins. To determine the effects of hFlt3L on the induction of anti-tumor immunity, we generated live vaccinia viruses that express human Flt3L regulated by the early H5 or strong synthetic E/L promoter together with fusion protein SigE7LAMP, which is a highly immunogenic form of HPV E7 oncoprotein. We tested Flt3L production in vitro and in vivo. Despite higher expression of Flt3L from the synthetic E/L promoter in vitro, the P13-E/L-FL-SigE7LAMP induced lower levels of Flt3L in the serum of mice than P13-H5-FL-SigE7LAMP. The Flt3L expression under the strong early VACV H5 promoter is able to inhibit expansion of CD11b+Gr-1+ myeloid suppressor cells (MSC) and increase the amount of CD11b+ CD11c+ dendritic cells in the spleen of mice immunized with vaccinia virus. Determination of viral DNA isolated from the ovaries of infected animals did not reveal differences in replication between rVACVs in this organ. Coexpression of Flt3L by replication-competent virus P13-FL-SigE7LAMP induced enhancement of the cellular immune response against HPV16 E7 and VACV E3 proteins as well as increased anti-tumor efficacy in both the protective and therapeutic immunization schemes. On the other hand, the short-time Flt3L coexpression by MVA-H5-FL-SigE7LAMP was not sufficient to enhance anti-tumor effect of immunization.

Increased tumorigenicity and metastasis of cervical cancer cells by HPV 16 E6-mediated blockade of lymphotoxin signaling pathway (88.14)

Abstract To elucidate the regulatory role of LT on human papillomavirus (HPV) E6-mediated tumorigenecity in cervical cancer, HPV 16 E6-overexpressing HeLa (HE6F) and suppressing Caski cervical cancer cells (CE6R) were established. LT gene expression was markedly suppressed in HE6F cells compared to HeLa cells, which led to a significant augmentation of HE6F cell growth via the reduction of apoptosis-associated gene expression. HPV 16 E6-induced HE6F metastasis was further amplified by silencing of LTβR using small interference RNAs. The blockade of LT signaling by LTβR-Ig treatment in HE6F cells significantly suppressed cytotoxicity mediated by NK cells. Recovery of LT by administration of LT DNA in mice challenged with TC-1 cells expressing HPV16 E6 and E7 resulted in the suppression of tumor mass, and the prolonged survival rate. Furthermore, IgA production in both fecal extracts and vaginal washes of mice administrated with LT DNA was significantly higher than that of control mice. Taken together, these results demonstrated that progress rate of cervical cancer could be promoted by HPV E6-mediated blockade of LT signaling pathway. Regulation of LT might be an effective way to achieve stable suppression of HPV E6 oncogenesis in cervical cancer. This work was supported by the SRC/ERC program of MOST/KOSEF (RESEARCH CENTER FOR WOMEN'S DISEASES) and by a grant from Biogreen 21 project, Rural Development Administration.

Inhibition of cervical cancer cell growth by human papillomavirus virus–like particles packaged with human papillomavirus oncoprotein short hairpin RNAs

Overexpression of human papillomavirus (HPV E6 and HPV E7) oncogenes in human cervical cells results in the development of cancer, and E6 and E7 proteins are therefore targets for preventing cervical cancer progression. Here, we describe the silencing of E6 and E7 expression in cervical carcinoma cells by RNA interference. In order to increase the efficacy of the RNA interference, HPV pseudovirions coding for a short hairpin RNA (shRNA) sequence were produced. The results indicated the degradation of E6 and E7 mRNAs when shRNA against E6 or E7 were delivered by pseudovirions in HPV-positive cells (CaSki and TC1 cells). E6 silencing resulted in the accumulation of cellular p53 and reduced cell viability. More significant cell death was observed when E7 expression was suppressed. Silencing E6 and E7 and the consequences for cancer cell growth were also investigated in vivo in mice using the capacity of murine TC1 cells expressing HPV-16 E6 and E7 oncogenes to induce fast-growing tumors. Treatment with lentiviruses and HPV virus-like particle vectors coding for an E7 shRNA sequence both resulted in dramatic inhibition of tumor growth. These results show the ability of pseudovirion-delivered shRNA to produce specific gene suppression and provide an effective means of reducing HPV-positive tumor growth.

Human Papillomavirus Type 16 E7 Oncoprotein Associates with the Cullin 2 Ubiquitin Ligase Complex, Which Contributes to Degradation of the Retinoblastoma Tumor Suppressor

Human papillomavirus type 16 (HPV16) and other high-risk HPVs are etiologically linked to the development of cervical carcinomas and contribute to a number of other tumors of the anogenital tract, as well as oral cancers. The high-risk HPV E6 and E7 oncoproteins are consistently expressed in cervical cancer cells and are necessary for the induction and maintenance of the transformed phenotype. An important aspect of HPV16 E7's oncogenic activities is destabilization of the retinoblastoma tumor suppressor (pRB) through a ubiquitin/proteasome-dependent mechanism, although the exact molecular mechanism is unknown. Here, we report that HPV16 E7 is associated with an enzymatically active cullin 2 ubiquitin ligase complex and that the HPV16 E7/pRB complex contains cullin 2. Depletion of cullin 2 by RNA interference causes increased steady-state levels and stability of pRB in HPV16 E7-expressing cells, and ectopic expression of HPV16 E7 and the cullin 2 complex leads to pRB ubiquitination in vivo. Hence, we propose that the HPV16 E7-associated cullin 2 ubiquitin ligase complex contributes to aberrant degradation of the pRB tumor suppressor in HPV16 E7-expressing cells.