The human papillomavirus (HPV) E7 protein antagonises an Imiquimod-induced inflammatory pathway in primary human keratinocytes.
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ABSTRACT:
SUBMITTER: Richards KH
PROVIDER: S-EPMC4534800 | biostudies-other | 2015 Aug
REPOSITORIES: biostudies-other
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Project description:The productive program of human papillomaviruses occurs in differentiated squamous keratinocytes. We have previously shown that HPV-18 DNA amplification initiates in spinous cells in organotypic cultures of primary human keratinocytes during prolonged G(2) phase, as signified by abundant cytoplasmic cyclin B1 (Wang, H. K., Duffy, A. A., Broker, T. R., and Chow, L. T. (2009) Genes Dev. 23, 181-194). In this study, we demonstrated that the E7 protein, which induces S phase reentry in suprabasal cells by destabilizing the p130 pocket protein (Genovese, N. J., Banerjee, N. S., Broker, T. R., and Chow, L. T. (2008) J. Virol. 82, 4862-4873), also elicited extensive G(2) responses. Western blots and indirect immunofluorescence assays were used to probe for host proteins known to control G(2)/M progression. E7 expression induced cytoplasmic accumulation of cyclin B1 and cdc2 in the suprabasal cells. The elevated cdc2 had inactivating phosphorylation on Thr(14) or Tyr(15), and possibly both, due to an increase in the responsible Wee1 and Myt1 kinases. In cells that harbored cytoplasmic cyclin B1 or cdc2, there was also an accumulation of the phosphatase-inactive cdc25C phosphorylated on Ser(216), unable to activate cdc2. Moreover, E7 expression induced elevated expression of phosphorylated ATM (Ser(1981)) and the downstream phosphorylated Chk1, Chk2, and JNKs, kinases known to inactivate cdc25C. Similar results were observed in primary human keratinocyte raft cultures in which the productive program of HPV-18 took place. Collectively, this study has revealed the mechanisms by which E7 induces prolonged G(2) phase in the differentiated cells following S phase induction.
| S-EPMC3083224 | biostudies-literature
Project description:Human papillomavirus (HPV) can successfully evade the host immune response to establish a persistent infection. We show here that expression of the E7 oncoprotein in primary human keratinocytes results in increased production of interleukin-18 (IL-18) binding protein (IL-18BP). This anti-inflammatory cytokine binding protein is a natural antagonist of IL-18 and is necessary for skin homeostasis. We map increased IL-18BP production to the CR3 region of E7 and demonstrate that this ability is shared among E7 proteins from different HPV types. Furthermore, mutagenesis shows that increased IL-18BP production is mediated by a gamma-activated sequence (GAS) in the IL-18BP promoter. Importantly, the increased IL-18BP levels seen in E7-expressing keratinocytes are capable of diminishing IL-18-mediated CD4 lymphocyte activation. This study provides the first evidence for a virus protein that targets IL-18BP and further validates E7 as a key component of the HPV immune evasion armor.Infection with human papillomavirus is a leading cause of morbidity and mortality worldwide. This study demonstrates that the E7 protein increases production of the anti-inflammatory IL-18BP, a major regulator of epithelial homeostasis. A number of E7 proteins can increase IL-18BP production, and a region within the CR3 of E7 is necessary for mediating the increase. A consequence of increased IL-18BP production is a reduction in CD4-positive lymphocyte activation in response to IL-18 costimulation. These findings may shed light on the immune evasion abilities of HPV.
| S-EPMC3993750 | biostudies-literature
Project description:High-risk human papillomavirus (HPV) infections are the predominant cause of cervical cancer and its early gene E7 plays an important role in cellular proliferation and cell-cycle progression. While tremendous progress has been made in exploring the molecular mechanisms in late tumorigenesis, many pathways showing how HPV deregulates host gene expression in early inapparent infections and early tumorigenesis still remain undefined. Digital RNA sequencing was performed and a total of 195 differentially expressed genes were identified between the HPV16 E7-transfected NHEKs and control cells (p < 0.05, fold-change > 2). GO enrichment showed that HPV16 E7 primarily affected processes involved in anti-viral and immune responses, while KEGG pathway analysis showed enrichment of gene clusters of associated with HPV infection and MAPK signaling. Of the differentially expressed genes, IFI6, SLC39A9 and ZNF185 showed a strong correlation with tumor progression and patient survival in the OncoLnc database while roles for AKAP12 and DUSP5 in carcinogenesis and poor prognosis have previously been established for other cancer types. Our study identified several novel HPV16 E7-regulated candidate genes with putative functions in tumorigenesis, thus providing new insights into HPV persistence in keratinocytes and early onset of tumorigenesis.
| S-EPMC7419603 | biostudies-literature
Project description:It is suggested that HPV-18 variants from the A lineage have higher oncogenic potential compared to B variants. Some studies show uneven distribution of HPV-18 variants in cervical adenocarcinomas and squamous cell carcinomas. Regarding HPV-18 variants' functions, the few studies reported focus on E6, and none were performed using natural host cells. Here, we immortalized primary human keratinocytes (PHKs) with E6/E7 of HPV-18 A1 and B1 sublineages and functionally characterized these cells. PHK18A1 reached immortalization significantly faster than PHK18B1 and formed a higher number of colonies in monolayer and 3D cultures. Moreover, PHK18A1 showed greater invasion ability and higher resistance to apoptosis induced by actinomycin-D. Nevertheless, no differences were observed regarding morphology, proliferation after immortalization, migration, or epithelial development in raft cultures. Noteworthy, our study highlights qualitative differences among HPV-18 A1 and B1 immortalized PHKs: in contrast to PHK18A1, which formed more compact colonies and spheroids of firmly grouped cells and tended to invade and migrate as clustered cells, morphologically, PHK18B1 colonies and spheroids were looser, and migration and invasion of single cells were observed. Although these observations may be relevant for the association of these variants with cervical cancer of different histological subtypes, further studies are warranted to elucidate the mechanisms behind these findings.
| S-EPMC8228617 | biostudies-literature
Project description:Background: Human papillomavirus type 8 (HPV8) has been implicated in the progress of non-melanoma skin cancers and their precursor lesions. The HPV8 E7 oncoprotein plays a key role in the tumorigenesis of HPV-associated cutaneous tumors. However, the exact role of HPV8 E7 in human epidermal carcinogenesis has not been fully elucidated. Methods: To investigate the potential carcinogenic effects of HPV8 E7 on epithelial cells, we used RNA-sequencing technology to analyze the gene expression profile of HPV8 E7-overexpressed normal human epidermal keratinocytes (NHEKs). Results: RNA-sequencing revealed 831 differentially expressed genes (DEGs) between HPV8 E7-expressing NHEKs and control cells, among which, 631 genes were significantly upregulated, and 200 were downregulated. Gene ontology annotation enrichment analysis showed that HPV8 E7 mainly affected the expression of genes associated with protein heterodimerization activity, DNA binding, nucleosomes, and nucleosome assembly. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that overexpression of HPV8 E7 affected the expression of gene clusters associated with viral carcinogenesis and transcriptional misregulation in cancer and necroptosis signaling pathways that reportedly play crucial roles in HPV infection promotion and cancer progression. We also found the DEGs, such as HKDC1 and TNFAIP3, were associated with epigenetic modifications, immune regulation, and metabolic pathways. Conclusion: Our results demonstrate that the pro-carcinogenic effect of HPV8 expression in epithelial cells may be attributed to the regulatory effect of oncogene E7 on gene expression associated with epigenetic modifications and immune and metabolic status-associated gene expression. Although our data are based on an in vitro experiment, it provides the theoretical evidence that the development of squamous cell carcinoma can be caused by HPV.
| S-EPMC9156622 | biostudies-literature
Project description:It is established that the host cell transcriptomes of natural lesions, organotypic rafts, and human papillomavirus (HPV)-immortalized keratinocytes are altered in the presence of HPV genomes. However, the establishment of HPV-harboring cell lines requires selection and immortalization, which makes it impossible to distinguish between alterations directly induced by HPV or indirectly by the need for immortalization or selection. To address direct effects of HPV infection on the host cell transcriptome, we have used our recently established infection model that allows efficient infection of primary keratinocytes with HPV16 virions. We observed only a small set of genes to be deregulated at the transcriptional level at 7 days postinfection (dpi), most of which fall into the category regulated by pocket proteins pRb, p107, and p130. Furthermore, cell cycle genes were not deregulated in cells infected with a virus lacking E7 despite the presence of episomal genome and viral transcripts. These findings imply that the majority of transcriptional changes are due to the E7 protein impairing pocket protein function. Additional pathways, such as the Fanconi anemia-BRCA pathway, became perturbed only after long-term culturing of infected cells. When grown as organotypic raft cultures, keratinocytes infected with wild-type but not E7 mutant virus had perturbed transcriptional regulation of pathways previously identified in natural lesions and in rafts derived from immortalized keratinocytes. We conclude that the HPV infection model provides a valuable tool to distinguish immediate transcriptional alterations from those induced by persistent infection and the need for selection and immortalization.IMPORTANCE To establish infection and complete the viral life cycle, human papillomavirus (HPV) needs to alter the transcriptional program of host cells. Until recently, studies were restricted to keratinocyte-derived cell lines immortalized by HPV due to the lack of experimental systems to efficiently infect primary keratinocytes. Need for selection and immortalization made it impossible to distinguish between alterations induced by HPV and secondary adaptation due to selection and immortalization. With our recent establishment of an extracellular matrix (ECM)-to-cell transfer system allowing efficient infection of primary keratinocytes, we were able to identify transcriptional changes attributable to HPV16 infection. Most perturbed genes fall into the class of S-phase genes, which are regulated by pocket proteins. Indeed, infection with viruses lacking E7 abrogated most transcriptional changes. It is important to note that many transcriptional alterations thought to be important for the HPV life cycle are actually late events that may reflect immortalization and, possibly, disease progression.
| S-EPMC7000963 | biostudies-literature
Project description:BackgroundmiRNAs and lncRNAs can regulate cellular biological processes both under physiological and pathological conditions including tumour initiation and progression. Interactions between differentially expressed diverse RNA species, as a part of a complex intracellular regulatory network (ceRNA network), may contribute also to the pathogenesis of HPV-associated cancer. The purpose of this study was to investigate the global expression changes of miRNAs, lncRNAs and mRNAs driven by the E6 and E7 oncoproteins of HPV16, and construct a corresponding ceRNA regulatory network of coding and non-coding genes to suggest a regulatory network associated with high-risk HPV16 infections. Furthermore, additional GO and KEGG analyses were performed to understand the consequences of mRNA expression alterations on biological processes.MethodsSmall and large RNA deep sequencing were performed to detect expression changes of miRNAs, lncRNAs and mRNAs in primary human keratinocytes expressing HPV16 E6, E7 or both oncoproteins. The relationships between lncRNAs, miRNAs and mRNAs were predicted by using StarBase v2.0, DianaTools-LncBase v.2 and miRTarBase. The lncRNA-miRNA-mRNA regulatory network was visualized with Cytoscape v3.4.0. GO and KEEG pathway enrichment analysis was performed using DAVID v6.8.ResultsWe revealed that 85 miRNAs in 21 genomic clusters and 41 lncRNAs were abnormally expressed in HPV E6/E7 expressing cells compared with controls. We constructed a ceRNA network with members of 15 lncRNAs - 43 miRNAs - 358 mRNAs with significantly altered expressions. GO and KEGG functional enrichment analyses identified numerous cancer related genes, furthermore we recognized common miRNAs as key regulatory elements in biological pathways associated with tumorigenesis driven by HPV16.ConclusionsThe multiple molecular changes driven by E6 and E7 oncoproteins resulting in the malignant transformation of HPV16 host cells occur, at least in part, due to the abnormal alteration in expression and function of non-coding RNA molecules through their intracellular competing network.
| S-EPMC8185923 | biostudies-literature
Project description:Several lines of evidence indicate that cutaneous human papillomavirus (HPV) types belonging to the beta genus of the HPV phylogenetic tree synergize with UV radiation in the development of skin cancer. Accordingly, the E6 and E7 oncoproteins from some beta HPV types are able to deregulate pathways related to immune response and cellular transformation. Toll-like receptor 9 (TLR9), in addition to playing a role in innate immunity, has been shown to be involved in the cellular stress response. Using primary human keratinocytes as experimental models, we have shown that UV irradiation (and other cellular stresses) activates TLR9 expression. This event is closely linked to p53 activation. Silencing the expression of p53 or deleting its encoding gene affected the activation of TLR9 expression after UV irradiation. Using various strategies, we have also shown that the transcription factors p53 and c-Jun are recruited onto a specific region of the TLR9 promoter after UV irradiation. Importantly, the E6 and E7 oncoproteins from beta HPV38, by inducing the accumulation of the p53 antagonist ΔNp73α, prevent the UV-mediated recruitment of these transcription factors onto the TLR9 promoter, with subsequent impairment of TLR9 gene expression. This study provides new insight into the mechanism that mediates TLR9 upregulation in response to cellular stresses. In addition, we show that HPV38 E6 and E7 are able to interfere with this mechanism, providing another explanation for the possible cooperation of beta HPV types with UV radiation in skin carcinogenesis.IMPORTANCE Beta HPV types have been suggested to act as cofactors in UV-induced skin carcinogenesis by altering several cellular mechanisms activated by UV radiation. We show that the expression of TLR9, a sensor of damage-associated molecular patterns produced during cellular stress, is activated by UV radiation in primary human keratinocytes (PHKs). Two transcription factors known to be activated by UV radiation, p53 and c-Jun, play key roles in UV-activated TLR9 expression. The E6 and E7 oncoproteins from beta HPV38 strongly inhibit UV-activated TLR9 expression by preventing the recruitment of p53 and c-Jun to the TLR9 promoter. Our findings provide additional support for the role that beta HPV types play in skin carcinogenesis by preventing activation of specific pathways upon exposure of PHKs to UV radiation.
| S-EPMC5599736 | biostudies-literature
Project description:Octamer binding transcription factor-4 (Oct4), is highly expressed in stem cells and has indispensable roles in pluripotency and cellular reprogramming. In contrast to other factors used for cellular reprogramming, a role for Oct4 outside embryonic stem cells has been elusive and highly controversial. Emerging evidence implicates Oct4 in the carcinogenic process, but the mechanism through which Oct4 may be functioning in cancers is not fully appreciated. Here, we provide evidence that Oct4 is expressed in human cervical cancer and this expression correlates with the presence of the human papillomavirus (HPV) oncogenes E6 and E7. Surprisingly, the viral oncogenes can complement exogenously provided Oct4 in reprogramming assays, providing functional validation for their ability to activate Oct4 transcription in Mouse Embryonic Fibroblasts (MEFs). To interrogate potential roles of Oct4 in cervical cancers we knocked-down Oct4 in HPV(+) (HeLa & CaSki) and HPV(-) (C33A) cervical cancer cell lines and found that Oct4 knockdown attenuated clonogenesis, only in the HPV(+) cells. More unexpectedly, cell proliferation and migration, were differentially affected in HPV(+) and HPV(-) cell lines. We provide evidence that Oct4 interacts with HPV E7 specifically at the CR3 region of the E7 protein and that introduction of the HPV oncogenes in C33A cells and human immortalised keratinocytes generates Oct4-associated transcriptional and phenotypic patterns, which mimic those seen in HPV(+) cells. We propose that a physical interaction of Oct4 with E7 regulates its activity in HPV(+) cervical cancers in a manner not seen in other cancer types.
| S-EPMC7228134 | biostudies-literature
Project description:Human papillomavirus (HPV) infection distinctly alters methylation patterns in HPV-associated cancer. We have recently reported that HPV E7-dependent promoter hypermethylation leads to downregulation of the chemokine CXCL14 and suppression of antitumor immune responses. To investigate the extent of gene expression dysregulated by HPV E7-induced DNA methylation, we analyzed parallel global gene expression and DNA methylation using normal immortalized keratinocyte lines, NIKS, NIKS-16, NIKS-18, and NIKS-16?E7. We show that expression of the MHC class I genes is downregulated in HPV-positive keratinocytes in an E7-dependent manner. Methylome analysis revealed hypermethylation at a distal CpG island (CGI) near the HLA-E gene in NIKS-16 cells compared to either NIKS cells or NIKS-16?E7 cells, which lack E7 expression. The HLA-E CGI functions as an active promoter element which is dramatically repressed by DNA methylation. HLA-E protein expression on cell surface is downregulated by high-risk HPV16 and HPV18 E7 expression, but not by low-risk HPV6 and HPV11 E7 expression. Conversely, demethylation at the HLA-E CGI restores HLA-E protein expression in HPV-positive keratinocytes. Because HLA-E plays an important role in antiviral immunity by regulating natural killer and CD8+ T cells, epigenetic downregulation of HLA-E by high-risk HPV E7 may contribute to virus-induced immune evasion during HPV persistence.
| S-EPMC5473897 | biostudies-literature