Evaluation of LMP1 of Epstein-Barr virus as a therapeutic target by its inhibition

BACKGROUND:The latent membrane protein-1 (LMP1) encoded by Epstein-Barr virus (EBV) is an oncoprotein which acts by constitutive activation of various signalling pathways, including NF-kappaB. In so doing it leads to deregulated cell growth intrinsic to the cancer cell as well as having extrinsic affects upon the tumour microenvironment. These properties and that it is a foreign antigen, lead to the proposition that LMP1 may be a good therapeutic target in the treatment of EBV associated disease. LMP1 is expressed in several EBV-associated malignancies, notably in Hodgkin's lymphoma and nasopharyngeal carcinoma (NPC). However, the viral protein is only detected in approximately 30%-50% of NPC samples, as such its role in carcinogenesis and tumour maintenance can be questioned and thus its relevance as a therapeutic target.RESULTS:In order to explore if LMP1 has a continuous function in established tumours, its activity was inhibited through expression of a dominant negative LMP1 mutant in tumour cell lines derived from transgenic mice. LMP1 is the tumour predisposing oncogene in two different series of transgenic mice which separately give rise to either B-cell lymphomas or carcinomas. Inhibition of LMP1 activity in the carcinoma cell lines lead to a reduction in clonagenicity and clone viability in all of the cell lines tested, even those with low or below detection levels of LMP1. Inhibition of LMP1 activity in the transgenic B-cell lines was incompatible with growth and survival of the cells and no clones expressing the dominant negative LMP1 mutant could be established.CONCLUSIONS:LMP1 continues to provide a tumour cell growth function in cell lines established from LMP1 transgenic mouse tumours, of both B-cell and epithelial cell origin. LMP1 can perform this function, even when expressed at such low levels as to be undetectable, whereby evidence of its expression can only be inferred by its inhibition being detrimental to the growth of the cell. This raises the possibility that LMP1 still performs a pro-oncogenic function in the 50% to 70% of NPC tumours wherein LMP1 protein expression cannot be detected. This reinforces the basis for pursuing LMP1 as a therapeutic target in EBV associated LMP1-expressing malignancie

Evaluation of cumulative cognitive deficits from electroconvulsive therapy

Background Electroconvulsive therapy (ECT) is the most effective acute treatment for severe depression, but widely held concerns about memory problems may limit its use. Aims To find out whether repeated or maintenance courses of ECT cause cumulative cognitive deterioration. Method Analysis of the results of 10 years of cognitive performance data collection from patients who have received ECT. The 199 patients had a total of 498 assessments, undertaken after a mean of 15.3 ECT sessions (range 0–186). A linear mixed-effect regression model was used, testing whether an increasing number of ECT sessions leads to deterioration in performance. Results The total number of previous ECT sessions had no effect on cognitive performance. The major factors affecting performance were age, followed by the severity of depression at the time of testing and the number of days since the last ECT session. Conclusions Repeated courses of ECT do not lead to cumulative cognitive deficits. This message is reassuring for patients, carers and prescribers who are concerned about memory problems and confusion during ECT. Electroconvulsive therapy (ECT) is the most effective acute treatment for severe depression,1 with reported remission rates above 50%.2,3 Although some reports demonstrate even higher remission rates (such as 75% in patients with psychotic depression4), these could be below 50% for treatment-resistant depression or in community settings.5,6 ECT is often portrayed in mainstream media as a barbaric treatment7 and its cognitive side-effects as profound and debilitating, leading to public, patient and carer concerns. ECT does cause retrograde amnesia and acute disorientation immediately following a treatment,8 however, research has suggested that this is only a short-lived side-effect. A meta-analysis by Semkovska & McLoughlin9 analysed the cognitive tests of 2981 patients from 84 studies, performed before and after single courses of ECT, and found that a decline in cognitive performance was limited to the first 3 days following a treatment. Patients showed no cognitive deterioration when tested 2 or more weeks after their last ECT session. This does not apply to retrograde amnesia, which was not part of this analysis, and it cannot be extended to cognitive functions that were not tested. Much less is known about the side-effects of long-term ECT, including maintenance ECT. A major concern of patients and some health professionals is that it could lead to progressive cognitive deficits, especially if given for prolonged periods of time. Small studies and case reports have addressed this question and have found no evidence to support this concern (see Discussion). Over the past 10 years we performed prospective cognitive tests on 199 patients, of whom 96 had >12 ECTsessions during their lifetime (the usual maximum duration of a single ECT course). We wanted to find out whether there was evidence that their cognitive performance deteriorated with the increasing number of ECT sessions

Lymphocyte deficiency limits Epstein-Barr virus latent membrane protein 1 induced chronic inflammation and carcinogenic pathology in vivo

Background: The importance of the malignant cell environment to its growth and survival is becoming increasingly apparent, with dynamic cross talk between the neoplastic cell, the leukocyte infiltrate and the stroma. Most cancers are accompanied by leukocyte infiltration which, contrary to an anticipated immuno-protective role, could be contributing to tumour development and cancer progression. Epstein-Barr virus (EBV) associated cancers, including nasopharyngeal carcinoma and Hodgkin's Disease, show a considerable leukocyte infiltration which surrounds the neoplastic cells, raising the questions as to what role these cells play in either restricting or supporting the tumour and what draws the cells into the tumour. In order to begin to address this we have studied a transgenic model of multistage carcinogenesis with epithelial expression of the EBV primary oncoprotein, latent membrane protein 1 (LMP1). LMP1 is expressed particularly in the skin, which develops a hyperplastic pathology soon after birth. Results: The pathology advances with time leading to erosive dermatitis which is inflamed with a mixed infiltrate involving activated CD8+ T-cells, CD4+ T-cells including CD4+/CD25+/FoxP3+ Treg cells, mast cells and neutrophils. Also significant dermal deposition of immunoglobulin-G (IgG) is observed as the pathology advances. Along with NF-kappaB activation, STAT3, a central factor in inflammation regulation, is activated in the transgenic tissue. Several inflammatory factors are subsequently upregulated, notably CD30 and its ligand CD153, also leukocyte trafficking factors including CXCL10, CXCL13, L-selectin and TGF beta 1, and inflammatory cytokines including IL-1 beta, IL-3 and the murine IL-8 analogues CXCL1, CXCL2 and CXCL5-6, amongst others. The crucial role of mature T- and/or B-lymphocytes in the advancing pathology is demonstrated by their elimination, which precludes mast cell infiltration and limits the pathology to an early, benign stage. Conclusions: LMP1 can lead to the activation of several key factors mediating proliferation, angiogenesis and inflammation in vivo. With the initiation of an inflammatory programme, leukocyte recruitment follows which then itself contributes to the progressing pathology in these transgenic mice, with a pivotal role for B-and/or T-cells in the process. The model suggests a basis for the leukocyte infiltrate observed in EBV-associated cancer and its supporting role, as well as potential points for therapeutic intervention

Lymphomas driven by Epstein-Barr virus nuclear antigen-1 (EBNA1) are dependant upon Mdm2

Epstein-Barr virus (EBV)-associated Burkitt's lymphoma is characterised by the deregulation of c-Myc expression and a restricted viral gene expression pattern in which the EBV nuclear antigen-1 (EBNA1) is the only viral protein to be consistently expressed. EBNA1 is required for viral genome propagation and segregation during latency. However, it has been much debated whether the protein plays a role in viral-associated tumourigenesis. We show that the lymphomas which arise in EµEBNA1 transgenic mice are unequivocally linked to EBNA1 expression and that both C-Myc and Mdm2 deregulation are central to this process. Tumour cell survival is supported by IL-2 and there is a skew towards CD8-positive T cells in the tumour environment, while the immune check-point protein PD-L1 is upregulated in the tumours. Additionally, several isoforms of Mdm2 are upregulated in the EµEBNA1 tumours, with increased phosphorylation at ser166, an expression pattern not seen in Eµc-Myc transgenic tumours. Concomitantly, E2F1, Xiap, Mta1, C-Fos and Stat1 are upregulated in the tumours. Using four independent inhibitors of Mdm2 we demonstrate that the EµEBNA1 tumour cells are dependant upon Mdm2 for survival (as they are upon c-Myc) and that Mdm2 inhibition is not accompanied by upregulation of p53, instead cell death is linked to loss of E2F1 expression, providing new insight into the underlying tumourigenic mechanism. This opens a new path to combat EBV-associated disease

Role of Inflammation and Oxidative Stress in Epstein-Barr Virus Oncogene Induced Carcinogenesis

We have been studying the role of inflammation and oxidative stress in a transgenic mouse model of carcinogenesis. In our model, we have used the primary oncogene of Epstein-Barr virus (EBV) encoding the latent membrane protein-1 (LMP1) (Stevenson et al 2005, Cancer Research 65, 8826-8835). EBV is tightly associated with nasopharyngeal carcinoma (NPC), a tumour which is so highly infiltrated with lymphocytes that it is frequently referred to as a lymphoepithelioma. In our transgenic mice (L2LMP1 mice), the transgene is expressed in the epidermis. The skin of these mice (most notably in hairless regions such as ears) develops a progressive pathology from birth, initiating with hyperplasia and hypervascularization, dramatically worsening with time, increasing inflammation, necrosis, ulceration, leading to keratocanthoma and papilloma formation, which can progress on to carcinoma development. We have shown that the preneoplastic (inflamed) skin of the transgenic mice is heavily infiltrated with T-cells, mast cells and neutrophils. Affected tissues also show upregulation of several chemokines and cytokines including CD30, CD30L, CD40, L-Selectin, IL-3, IL-1β and macrophage inflammatory proteins. We have also shown increased deposition of immunoglobulins and increased levels of complement component 3 (C-3) in the inflamed preneoplastic skin. By proteomic analysis, we have demonstrated massive upregulation of an enzymatically inactive chitinase-like protein, called chitinase-3-like-4 (chi3l4 or YM2) in the transgenic epidermis (Hannigan et al 2007, Journal of Proteome Research, 6,3422-3432). Such massive upregulation of this protein may suggest a role in inflammation induced carcinogenesis. We have found a decrease in SOD1 levels and increase in H2O2 in the affected skin suggesting that the tissue is also under oxidative stress. Furthermore our preliminary data reveals evidence of DNA damage in the affected tissues which could result from excess of reactive oxygen species. These data will be presented and discussed. Dissecting the role of inflammation and oxidative stress in this model of carcinogenesis may provide new insights for therapeutic designs

Role of Inflammation and Oxidative Stress in Epstein-Barr Virus Oncogene Induced Carcinogenesis

We have been studying the role of inflammation and oxidative stress in a transgenic mouse model of carcinogenesis. In our model, we have used the primary oncogene of Epstein-Barr virus (EBV) encoding the latent membrane protein-1 (LMP1) (Stevenson et al 2005, Cancer Research 65, 8826-8835). EBV is tightly associated with nasopharyngeal carcinoma (NPC), a tumour which is so highly infiltrated with lymphocytes that it is frequently referred to as a lymphoepithelioma. In our transgenic mice (L2LMP1 mice), the transgene is expressed in the epidermis. The skin of these mice (most notably in hairless regions such as ears) develops a progressive pathology from birth, initiating with hyperplasia and hypervascularization, dramatically worsening with time, increasing inflammation, necrosis, ulceration, leading to keratocanthoma and papilloma formation, which can progress on to carcinoma development. We have shown that the preneoplastic (inflamed) skin of the transgenic mice is heavily infiltrated with T-cells, mast cells and neutrophils. Affected tissues also show upregulation of several chemokines and cytokines including CD30, CD30L, CD40, L-Selectin, IL-3, IL-1β and macrophage inflammatory proteins. We have also shown increased deposition of immunoglobulins and increased levels of complement component 3 (C-3) in the inflamed preneoplastic skin. By proteomic analysis, we have demonstrated massive upregulation of an enzymatically inactive chitinase-like protein, called chitinase-3-like-4 (chi3l4 or YM2) in the transgenic epidermis (Hannigan et al 2007, Journal of Proteome Research, 6,3422-3432). Such massive upregulation of this protein may suggest a role in inflammation induced carcinogenesis. We have found a decrease in SOD1 levels and increase in H2O2 in the affected skin suggesting that the tissue is also under oxidative stress. Furthermore our preliminary data reveals evidence of DNA damage in the affected tissues which could result from excess of reactive oxygen species. These data will be presented and discussed. Dissecting the role of inflammation and oxidative stress in this model of carcinogenesis may provide new insights for therapeutic designs

Chitinase-Like Proteins Are Autoantigens in a Model of Inflammation-Promoted Incipient Neoplasia

An important role for B cells and immunoglobulin deposition in the inflammatory tumor cell environment has been recognized in several cancers, and this is recapitulated in our murine model of inflammation-associated carcinogenesis: transgenic mice expressing the Epstein-Barr virus oncogene LMP1 in epithelia. Similarly in several autoimmune disorders, immunoglobulin deposition represents a key underlying event in the disease process. However, the autoantigens in most cases are not known. In other studies, overexpression of the enzymatically inactive mammalian chitinase-like proteins (CLPs) has been observed in a number of autoimmune disorders and numerous cancers, with expression correlated with poor prognosis, although the function of these proteins is largely unknown. We have now linked these observations demonstrating that overexpression of the CLPs renders them the targets for autoantigenicity during carcinogenic progression. We show that the CLPs, Chi3L1, Chi3L3 /YM1, and Chi3L4/YM2, are abundantly overexpressed in the transgenic epidermis at an early, preneoplastic stage and secreted into the serum. Immunoglobulin G reactive to the CLPs is detected in the serum and deposited in the hyperplastic tissue, which goes on to become inflamed and progressively displastic. The CLPs are also upregulated in chemical carcinogen-promoted lesions in both transgenic and wild-type mice. Expression of the related, active chitinases, Chit1 and AMCase, increases following infiltration of inflammatory cells. In this model, the 3 CLPs are autoantigens for the tissue-deposited immunoglobulin, which we propose plays a causative role in promoting the inflammation-associated carcinogenesis. This may reflect their normal, benign function to promote tissue remodeling and to amplify immune responses. Their induction during carcinogenesis and consequent autoantigenicity provides a missing link between the oncogenic event and subsequent inflammation. This study identifies the CLPs as important and novel therapeutic targets to limit inflammation in cancer and potentially also autoimmune disorders