Christopher W. Wasson

Neutrophil Elastase-mediated proteolysis activates the anti-inflammatory cytokine IL-36 Receptor antagonist

The interleukin-36 receptor antagonist (IL-36Ra) which regulates IL-36α, -β and -γ is linked to psoriatic inflammation, especially loss-of-function mutations in pustular psoriasis subtypes. As observed with other IL-1 superfamily proteins, the IL-36 members require N-terminal cleavage for full biological activity but the mechanisms of IL-36Ra activation remain poorly defined. Using different blood leukocyte and skin resident cell preparations, and recombinant proteins, we have identified that neutrophil elastase, but not other neutrophil derived proteases, cleaves IL-36Ra into its highly active antagonistic form. The activity of this processed form of IL-36Ra was confirmed in human primary dermal fibroblasts and keratinocytes and in skin equivalents. A significant dose dependent reduction of IL-36γ induced IL-8 and chemokine ligand 20 (CCL20) levels were detected following addition of the cleaved IL-36Ra compared to full length IL-36Ra. By activating IL-36Ra, the neutrophil derived protease can inhibit IL-36 induced chemokine production, including IL-8 and CCL20, and reduce further inflammatory cell infiltration. These findings strongly indicate neutrophil elastase to be a key enzyme in the biological function of IL-36Ra and that neutrophils can play a regulatory role in psoriatic inflammation with regard to balancing the pro-inflammatory activity of IL-36.

The human papillomavirus (HPV) E7 protein antagonises an Imiquimod-induced inflammatory pathway in primary human keratinocytes

High-risk human papillomaviruses (HPV) are the etiological pathogen of cervical and a number of ano-genital cancers. How HPVs overcome the significant barriers of the skin immune system has been the topic of intensive research. The E6 and E7 oncoproteins have emerged as key players in the deregulation of host innate immune pathways that are required for the recruitment of effector cells of the immune response. Here we demonstrate that E7, and to a lesser extend E6, strongly reduce NFκB activation in response to the inflammatory mediator imiquimod. Moreover, we establish that undifferentiated keratinocytes do not express the putative receptor for imiquimod, TLR7, and as such are stimulated by imiquimod through a novel pathway. Inhibition of imiquimod induced cytokine production required residues in the CR1 and CR3 regions of E7 and resulted in reduced nuclear translocation and acetylation of the p65 sub-unit of NFκB. The results provide further evidence for a TLR7-independent role of imiquimod in the epithelial immune response and reinforce the ability of the HPV oncoproteins to disrupt the innate immune response, which may have important consequences for establishment of a chronic infection.

Human Papillomavirus E7 Oncoprotein Increases Production of the Anti-Inflammatory Interleukin-18 Binding Protein in Keratinocytes

ABSTRACT 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. IMPORTANCE 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.

YIP1 family member 4 (YIPF4) is a novel cellular binding partner of the papillomavirus E5 proteins

E5 proteins are amongst the least understood of the Human Papillomavirus (HPV) encoded gene products. They are small, membrane-integrated proteins known to modulate a number of critical host pathways associated with pathogenesis including growth factor receptor signaling and immune evasion. Their role in the virus life cycle is less clear, indicating a role in the productive stages of the life cycle. However, a mechanism for this is currently lacking. Here we describe the identification of a novel binding partner of E5, YIPF4 using yeast two-hybrid analysis. YIPF4 is also a poorly characterized membrane spanning protein. Mutagenesis studies implicated the transmembrane regions of each protein as important for their interaction. Binding to YIPF4 was found for all E5 proteins tested suggesting that this interaction may mediate a conserved E5 function. In normal human keratinocytes YIPF4 expression was down-regulated upon differentiation and this reduction was partially rescued in cells harbouring HPV. Despite the conserved nature of the interaction with E5, siRNA mediated depletion of YIPF4 failed to impede two well-characterized functions of E5, namely EGFR trafficking or HLA class I presentation. Continued studies of YIPF4 are warranted to determine its role in the PV life cycle.

Inhibition of Plk1 and Cyclin B1 expression results in panobinostat-induced G2 delay and mitotic defects.

The development of clinically useful histone deacetylase inhibitors has expanded greatly. In a preclinical study, we showed that panobinostat (LBH589) inhibits cell cycle progression of human head and neck squamous cell carcinoma (HNSCC) cell lines at G2/M and an associated decrease in expression of particular genes required for passage through G2 and mitosis. In this study we sought to analyse the mechanistic underpinnings of panobinostat-induced growth arrest. HNSCC cell lines were synchronised and progression through mitosis monitored. We demonstrate that panobinostat causes a marked G2 delay and mitotic defects. A loss of G2-specific Plk1 and Cyclin B1 expression and co-incident increase in p21Waf1/Cip1 expression is also shown. Furthermore, we show a significant loss of E2F1 recruitment to the promoters of these genes in response to panobinostat treatment. These data provide mechanistic evidence of panobinostat-induced cell cycle arrest and highlight its potential as a chemotherapeutic agent for HNSCC.

Human papillomavirus type 18 E5 oncogene supports cell cycle progression and impairs epithelial differentiation by modulating growth factor receptor signalling during the virus life cycle

Deregulation of proliferation and differentiation-dependent signalling pathways is a hallmark of human papillomavirus (HPV) infection. Although the manipulation of these pathways by E6 and E7 has been extensively studied, controversies surround the role of the E5 oncoprotein during a productive virus life cycle. By integrating primary keratinocytes harbouring wild type or E5 knockout HPV18 genomes with pharmacological and gain/loss of function models, this study aimed to provide molecular information about the role of E5 in epithelial proliferation and differentiation. We show that E5 contributes to cell cycle progression and unscheduled host DNA synthesis in differentiating keratinocytes. E5 function correlates with increased EGFR activation in differentiating cells and blockade of this pathway impairs differentiation-dependent cell cycle progression of HPV18 containing cells. Our findings provide a functional requirement of enhanced EGFR signalling for suprabasal cellular DNA synthesis during the virus life cycle. They also reveal an unrecognised contribution of E5 towards the impaired keratinocyte differentiation observed during a productive HPV infection. E5 suppresses a signalling axis consisting of the keratinocyte growth factor receptor (KGFR) pathway. Inhibition of this pathway compensates for the loss of E5 in knockout cells and re-instates the delay in differentiation. The negative regulation of KGFR involves suppression by the EGFR pathway. Thus our data reveal an unappreciated role for E5-mediated EGFR signalling in orchestrating the balance between proliferation and differentiation in suprabasal cells.

Merkel Cell Polyomavirus Small T Antigen Drives Cell Motility via Rho-GTPase-Induced Filopodium Formation

ABSTRACT Cell motility and migration is a complex, multistep, and multicomponent process intrinsic to progression and metastasis. Motility is dependent on the activities of integrin receptors and Rho family GTPases, resulting in the remodeling of the actin cytoskeleton and formation of various motile actin-based protrusions. Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high likelihood of recurrence and metastasis. Merkel cell polyomavirus (MCPyV) is associated with the majority of MCC cases, and MCPyV-induced tumorigenesis largely depends on the expression of the small tumor antigen (ST). Since the discovery of MCPyV, a number of mechanisms have been suggested to account for replication and tumorigenesis, but to date, little is known about potential links between MCPyV T antigen expression and the metastatic nature of MCC. Previously, we described the action of MCPyV ST on the microtubule network and how it impacts cell motility and migration. Here, we demonstrate that MCPyV ST affects the actin cytoskeleton to promote the formation of filopodia through a mechanism involving the catalytic subunit of protein phosphatase 4 (PP4C). We also show that MCPyV ST-induced cell motility is dependent upon the activities of the Rho family GTPases Cdc42 and RhoA. In addition, our results indicate that the MCPyV ST-PP4C interaction results in the dephosphorylation of β1 integrin, likely driving the cell motility pathway. These findings describe a novel mechanism by which a tumor virus induces cell motility, which may ultimately lead to cancer metastasis, and provides opportunities and strategies for targeted interventions for disseminated MCC. IMPORTANCE Merkel cell polyomavirus (MCPyV) is the most recently discovered human tumor virus. It causes the majority of cases of Merkel cell carcinoma (MCC), an aggressive skin cancer. However, the molecular mechanisms implicating MCPyV-encoded proteins in cancer development are yet to be fully elucidated. This study builds upon our previous observations, which demonstrated that the MCPyV ST antigen enhances cell motility, providing a potential link between MCPyV protein expression and the highly metastatic nature of MCC. Here, we show that MCPyV ST remodels the actin cytoskeleton, promoting the formation of filopodia, which is essential for MCPyV ST-induced cell motility, and we also implicate the activity of specific Rho family GTPases, Cdc42 and RhoA, in these processes. Moreover, we describe a novel mechanism for the activation of Rho-GTPases and the cell motility pathway due to the interaction between MCPyV ST and the cellular phosphatase catalytic subunit PP4C, which leads to the specific dephosphorylation of β1 integrin. These findings may therefore provide novel strategies for therapeutic intervention for disseminated MCC.

High-risk human papillomaviruses down-regulate expression of the Ste20 family kinase MST1 to inhibit the Hippo pathway and promote transformation

Human papillomaviruses (HPV) are a major cause of malignancy worldwide They are the aetiological agent of almost all cervical cancers and an increasing number of head and neck carcinomas. Deregulation of the Hippo pathway component YAP1 has recently been demonstrated to play a role in HPV-mediated cervical cancer, but whether other components of this pathway are implicated in the pathogenesis of this disease remains poorly understood. The expression level and activation status of critical Hippo pathway components were analysed across multiple cytology samples from patients with cervical disease, as well as HPV positive (HPV+) and HPV negative (HPV-) cervical cancer cell lines using real time qPCR, western blot and immunohistochemistry. In parallel, we assessed the effects of MST1 and MST2 overexpression upon cervical cancer cell proliferation, migration and invasion. Finally, we interrogated the consequences of interrupted MST1 and MST2 function using a targeted small molecule inhibitor in tandem with kinase inactive MST mutants. Our analysis found that expression of the Ste20 kinase MST1 was decreased within both HPV+ primary patient samples and cervical cancer cell lines. This effect was mediated by the virus-coded oncoproteins E6 and E7, which impair MST1 transcription. Reintroduction of MST1, or its paralogue MST2, into HPV positive cervical cancer cells re-activated the Hippo pathway, leading to a reduction in cell proliferation, migration and invasion. Finally, using a small molecule inhibitor of MST1/2 or kinase inactive mutants of either protein, we demonstrated that this effect required the kinase function of MST1/2. Our results reveal that HPV down regulates MST1 expression to inactivate the Hippo pathway and so drive cells towards transformation.

STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle

Human papillomaviruses (HPV) activate a number of host factors to control their differentiation-dependent life cycles. The transcription factor signal transducer and activator of transcription (STAT)-3 is important for cell cycle progression and cell survival in response to cytokines and growth factors. STAT3 requires phosphorylation on Ser727, in addition to phosphorylation on Tyr705 to be transcriptionally active. In this study, we show that STAT3 is essential for the HPV life cycle in undifferentiated and differentiated keratinocytes. Primary human keratinocytes containing high-risk HPV18 genomes display enhanced STAT3 phosphorylation compared to normal keratinocytes. Expression of the E6 oncoprotein is sufficient to induce the dual phosphorylation of STAT3 at Ser727 and Tyr705 by a mechanism requiring Janus kinases and members of the MAPK family. E6-mediated activation of STAT3 induces the transcription of STAT3 responsive genes including cyclin D1 and Bcl-xL. Silencing of STAT3 protein expression by siRNA or inhibition of STAT3 activation by small molecule inhibitors, or by expression of dominant negative STAT3 phosphorylation site mutants, results in blockade of cell cycle progression. Loss of active STAT3 impairs HPV gene expression and prevents episome maintenance in undifferentiated keratinocytes and upon differentiation, lack of active STAT3 abolishes virus genome amplification and late gene expression. Organotypic raft cultures of HPV18 containing keratinocytes expressing a phosphorylation site STAT3 mutant display a profound reduction in suprabasal hyperplasia, which correlates with a loss of cyclin B1 expression and increased differentiation. Finally, increased STAT3 expression and phosphorylation is observed in HPV positive cervical disease biopsies compared to control samples, highlighting a role for STAT3 activation in cervical carcinogenesis. In summary, our data provides evidence of a critical role for STAT3 in the HPV18 life cycle.

Alkyl-imino sugars inhibit the pro-oncogenic ion channel function of human papillomavirus (HPV) E5

Abstract Despite the availability of prophylactic vaccines the burden of human papillomavirus (HPV) associated malignancy remains high and there is a need to develop additional therapeutic strategies to complement vaccination. We have previously shown that the poorly characterised E5 oncoprotein forms a virus‐coded ion channel or viroporin that was sensitive to the amantadine derivative rimantadine. We now demonstrate that alkylated imino sugars, which have antiviral activity against a number of viruses, inhibit E5 channel activity in vitro. Using molecular modelling we predict that imino sugars intercalate between E5 protomers to prevent channel oligomerisation. We explored the ability of these viroporin inhibitors to block E5‐mediated activation of mitogenic signalling in keratinocytes. Treatment with either rimantadine or imino sugars prevented ERK‐MAPK phosphorylation and reduced cyclin B1 expression in cells expressing E5 from a number of high‐risk HPV types. Moreover, viroporin inhibitors also reduced ERK‐MAPK activation and cyclin B1 expression in differentiating primary human keratinocytes containing high‐risk HPV18. These observations provide evidence of a key role for E5 viroporin function during the HPV life cycle. Viroporin inhibitors could be utilised for stratified treatment of HPV associated tumours prior to virus integration, or as true antiviral therapies to eliminate virus prior to malignant transformation. HighlightsImino sugars inhibit the viroporin activity of the E5 oncoprotein.Imino sugars likely interact at E5 protomer interfaces within a channel to prevent oligomerisation.Imino sugars and adamantanes block mitogenic signalling mediated by E5 from a range of high‐risk HPV types.Viroporin inhibitors reduce mitogenic signalling in differentiating primary keratinocytes containing high‐risk HPV18.