Gulfaraz Khan

A Synthetic Lethal Interaction between K-Ras Oncogenes and Cdk4 Unveils a Therapeutic Strategy for Non-small Cell Lung Carcinoma

We have unveiled a synthetic lethal interaction between K-Ras oncogenes and Cdk4 in a mouse tumor model that closely recapitulates human non-small cell lung carcinoma (NSCLC). Ablation of Cdk4, but not Cdk2 or Cdk6, induces an immediate senescence response only in lung cells that express an endogenous K-Ras oncogene. No such response occurs in lungs expressing a single Cdk4 allele or in other K-Ras-expressing tissues. More importantly, targeting Cdk4 alleles in advanced tumors detectable by computed tomography scanning also induces senescence and prevents tumor progression. These observations suggest that robust and selective pharmacological inhibition of Cdk4 may provide therapeutic benefit for NSCLC patients carrying K-RAS oncogenes.

Simplified procedures for applying the polymerase chain reaction to routinely fixed paraffin wax sections.

The polymerase chain reaction was applied to the analysis of DNA contained in archival paraffin wax embedded material. DNA suitable for the reaction was obtained from these tissues by simple extraction methods, without previous dewaxing of tissue sections. When compared with unfixed material, the reaction efficiency was compromised, so that an increased number of amplification cycles were required to produce equivalent amounts of amplified product. This in turn led to an increase in amplification artefacts, which can be minimised by a simple modification of the standard reaction. Amplification of relatively large DNA fragments was not always successful, and it seems prudent to bear this in mind when designing oligonucleotide primers which are to be used for the amplification of archival material. The efficiency of the procedure can be improved by dividing the amplification cycles into two parts: this reduces the amount of reagent needed, is relatively simple and inexpensive, and can be performed in one working day.

Epstein Barr virus (EBV) encoded small RNAs: targets for detection by in situ hybridisation with oligonucleotide probes.

AIMS: To develop a rapid, sensitive, and specific non-isotopic in situ hybridisation (NISH) procedure for the detection of Epstein-Barr virus in formalin fixed, paraffin wax embedded tissues. METHODS: Two low molecular weight RNAs, designated EBER-1 and EBER-2 (Epstein-Barr encoded RNA), were used: cells latently infected with EBV secrete large amounts of EBERs. The method uses digoxigenin labelled anti-sense oligonucleotides, corresponding to sequences in EBER-1 and EBER-2. RESULTS: The use of these probes, in conjunction with high temperature microwave denaturation, ensured that the technique was considerably more sensitive than other in situ hybridisation techniques for detecting EBV. Furthermore, the hybridisation signal was morphologically distinct in that only the nucleus and not the nucleolus give a positive signal. No cross-hybridisation was observed with cells infected with other lymphotropic herpes viruses. CONCLUSION: The sensitivity, simplicity, and rapidity of this technique make it ideal for diagnostic use, and for studies investigating the role of this virus in neoplastic disease.

Global burden of deaths from Epstein-Barr virus attributable malignancies 1990-2010

BackgroundEpstein-Barr virus (EBV) is an oncogenic virus implicated in the pathogenesis of a number of human malignancies of both lymphoid and epithelial origin. Thus, a comprehensive and up-to-date analysis focused on the global burden of EBV-attributable malignancies is of significant interest.MethodsBased on published studies, we estimated the proportion of Burkitt’s lymphoma (BL), Hodgkin’s lymphoma (HL), nasopharyngeal carcinoma NPC), gastric carcinoma (GC) and post-transplant lymphoproliferative disease (PTLD) attributable to EBV, taking into consideration age, sex and geographical variations. This proportion was then imputed into the Global Burden of Disease 2010 dataset to determine the global burden of each EBV-attributable malignancy in males and females in 20 different age groups and 21 world regions from 1990 to 2010.ResultsThe analysis showed that the combined global burden of deaths in 2010 from all EBV-attributable malignancies was 142,979, representing 1.8% of all cancer deaths. This burden has increased by 14.6% over a period of 20 years. All 5 EBV-attributable malignancies were more common in males in all geographical regions (ratio of 2.6:1). Gastric cancer and NPC accounted for 92% of all EBV-attributable cancer deaths. Almost 50% of EBV-attributed malignancies occurred in East Asia. This region also had the highest age-standardized death rates for both NPC and GC.ConclusionsApproximately 143,000 deaths in 2010 were attributed to EBV-associated malignancies. This figure is likely to be an underestimate since some of the less prevalent EBV-associated malignancies have not been included. Moreover, the global increase in population and life-expectancy will further increase the overall burden of EBV-associated cancer deaths. Development of a suitable vaccine could have a substantial impact on reducing this burden.

Epstein-Barr virus-encoded small RNAs (EBERs) are present in fractions related to exosomes released by EBV-transformed cells.

Epstein-Barr virus (EBV) is an oncogenic herpesvirus associated with a number of human malignancies of epithelial and lymphoid origin. However, the mechanism of oncogenesis is unclear. A number of viral products, including EBV latent proteins and non-protein coding RNAs have been implicated. Recently it was reported that EBV-encoded small RNAs (EBERs) are released from EBV infected cells and they can induce biological changes in cells via signaling from toll-like receptor 3. Here, we investigated if these abundantly expressed non-protein coding EBV RNAs (EBER-1 and EBER-2) are excreted from infected cells in exosomal fractions. Using differential ultracentrifugation we isolated exosomes from three EBV positive cell lines (B95-8, EBV-LCL, BL30-B95-8), one EBER-1 transfected cell line (293T-pHEBo-E1) and two EBV-negative cell lines (BL30, 293T-pHEBo). The identity of purified exosomes was determined by electron microscopy and western blotting for CD63. The presence of EBERs in cells, culture supernatants and purified exosomal fractions was determined using RT-PCR and confirmed by sequencing. Purified exosomal fractions were also tested for the presence of the EBER-1-binding protein La, using western blotting. Both EBER-1 and EBER-2 were found to be present not only in the culture supernatants, but also in the purified exosome fractions of all EBV-infected cell lines. EBER-1 could also be detected in exosomal fractions from EBER-1 transfected 293T cells whilst the fractions from vector only transfectants were clearly negative. Furthermore, purified exosomal fractions also contained the EBER-binding protein (La), supporting the notion that EBERs are most probably released from EBV infected cells in the form of EBER-La complex in exosomes.

A novel coronavirus capable of lethal human infections: an emerging picture

SummaryIn September 2012, a novel coronavirus was isolated from a patient in Saudi Arabia who had died of an acute respiratory illness and renal failure. The clinical presentation was reminiscent of the outbreak caused by the SARS-coronavirus (SARS-CoV) exactly ten years ago that resulted in over 8000 cases. Sequence analysis of the new virus revealed that it was indeed a member of the same genus as SARS-CoV. By mid-February 2013, 12 laboratory-confirmed cases had been reported with 6 fatalities. The first 9 cases were in individuals resident in the Middle East, while the most recent 3 cases were in family members resident in the UK. The index case in the UK family cluster had travel history to Pakistan and Saudi Arabia. Although the current evidence suggests that this virus is not highly transmissible among humans, there is a real danger that it may spread to other parts of the world. Here, a brief review of the events is provided to summarize the rapidly emerging picture of this new virus.

Localization of Epstein-Barr virus to infiltrating lymphocytes in breast carcinomas and not malignant cells

The pathogenesis of breast cancer is unknown. In recent years, a number of studies have implicated a role for Epstein-Barr virus (EBV) in a subset of cases. However, these findings are controversial and others have failed to find any link between the virus and this malignancy. We hypothesized that technical differences and the different type and ethnic origin of the cases may be the cause of the disparities reported. Using a highly sensitive EBER-in situ hybridization and immunohistochemistry, we examined 219 samples (158 malignant and 61 non-malignant) from 61 Emirati breast cancer cases to determine if EBV was etiologically associated with Emirati cases and if there was any correlation with other established prognostic factors such as age, histological type, lymph node metastasis, estrogen, progesterone and HER2 expression. We found 47.5% of the cases to be EBV positive, but the virus was localized to occasional infiltrating lymphocytes and not in the malignant cells. EBV lymphocytes were more commonly observed in lymph nodes than in breast tissues, but there was no correlation with malignancy or hormone status. The mean age of our patients was 48years and hormone receptor staining revealed 20% of the cases to be triple negative (ER-/PR-/HER2-). We conclude that although EBV can be detected in breast cancer cases, it is not directly associated with the disease. Thus, a PCR-based approach cannot be used to link this ubiquitous virus to the pathogenesis of breast cancer. Furthermore, we do not find any correlation between the presence of EBV in infiltrating lymphocytes and ER, PR, HER2 expression. We believe our findings will help explain some of the controversies relating to the role of EBV in the pathogenesis of breast cancer.

Epstein-Barr virus-infected cells release Fas ligand in exosomal fractions and induce apoptosis in recipient cells via the extrinsic pathway.

Epstein-Barr virus (EBV; human herpesvirus 4) is an oncogenic herpesvirus implicated in the pathogenesis of several human malignancies. A number of recent studies indicate that EBV can manipulate the local microenvironment by excreting viral and cellular components in nanovesicles called exosomes. In this study, we investigated the impact of EBV-derived exosomes on apoptosis of recipient cells and the molecular pathway involved in this process. Exosomes from EBV-infected but not from non-infected cells induced apoptosis in a number of different cell types, including B-cells, T-cells and epithelial cells. However, this phenomenon was not universal and the Burkitts lymphoma-derived B-cell line BJAB was found to be resistant to apoptosis. Exosomes from both type I and type III EBV latently infected cells induced apoptosis in a dose- and time-dependent manner. Moreover, cells exposed to EBV exosomes did not form colonies in soft agar assays. We further show that fluorescently labelled exosomes derived from EBV-infected cells are taken up by non-infected cells and induce apoptosis via the extrinsic pathway. Inhibition of caspase-3/7/8 blocks EBV exosome-mediated apoptosis. Furthermore, our data indicate that EBV exosomes trigger apoptosis through the Fas ligand (FasL)-mediated extrinsic pathway, as FasL was present in EBV exosomal fractions and anti-FasL antibodies could block EBV exosome-mediated apoptosis. Together, these data support the notion that EBV hijacks the exosome pathway to excrete viral and cellular components that can modulate its microenvironment.

The labyrinth of interactions of Epstein–Barr virus-encoded small RNAs

Epstein–Barr Virus (EBV) is an oncogenic herpesvirus implicated in the pathogenesis of a number of human malignancies. However, the mechanism by which EBV leads to malignant transformation is not clear. A number of viral latent gene products, including non‐protein coding small RNAs, are believed to be involved. Epstein–Barr virus‐encoded RNA 1 (EBER1) and EBER2 are two such RNA molecules that are abundantly expressed (up to 107 copies) in all EBV‐infected cells, but their function remains poorly understood. These polymerase III transcripts have extensive secondary structure and exist as ribonucleoproteins. An accumulating body of evidence suggests that EBERs play an important role, directly or indirectly, in EBV‐induced oncogenesis. Here, we summarize the current understanding of the complex interactions of EBERs with various cellular factors and the potential pathways by which these small RNAs are able to influence EBV‐infected cells to proliferate and to induce tumorigenesis. The exosome pathway is probably involved in the cellular excretion of EBERs and facilitating some of their biological effects. Copyright

Is Epstein–Barr virus associated with aggressive forms of breast cancer?

Sir, We read with great interest the paper by Mazouni et al (2011) recently published in the British Journal of Cancer linking Epstein–Barr virus (EBV) to aggressive forms of breast cancer. EBV is a lymphotropic herpesvirus aetiologically associated with a number of human malignancies of both epithelial and lymphoid origin. Although numerous studies have been published over the last 10– 15 years looking at the possible link between EBV and the pathogenesis of breast cancer, the association remains controversial and reports from both corners of the arena continue to be published (reviewed in Amarante and Watanabe, 2009). It is possible that the discrepancies between the different reports are due to the differing methodologies used for the detection of EBV, the histological types of tumours examined, and the ethnic/ geographical background of the cases studied. For example, using PCR-based techniques, a number of studies have reported a positive correlation between EBV and breast cancer, with up to 50% of cases giving a positive signal (Murray et al, 2003; Preciado et al, 2005; Fawzy et al, 2008). However, owing to the fact that EBV is a ubiquitous virus present asymptomatically in over 90% of the world population, its mere detection in tumour tissue cannot be used to imply disease association. In this context, the findings reported by Marzouni et al have to be interpreted with caution. Indeed, several studies that have used the EBER-in situ hybridisation (EBER-ISH) approach have failed to show an association, even in cases that were EBV PCR positive (Deshpande et al, 2002; Herrmann and Niedobitek, 2003; Murray et al, 2003; Thorne et al, 2005). Similarly, PCR studies on microdissected tumour cells have also led to contradictory findings. While Fina et al (2001) reported the presence of EBV in microdissected tumour cells, Murray et al (2003) did not find any evidence of the presence of EBV in the microdissected tumour cells of their cases, even though 21% of the cases were EBV positive by quantitative real-time PCR. We too have recently examined the association of EBV with the pathogenesis of breast cancer in a large series of cases in the Arab population from the United Arab Emirates. A total of 219 samples from 61 cases were examined using an EBER-ISH method capable of detecting as little as a few EBV-infected lymphocytes in an entire tissue section (Khan et al, 1992). All but four cases had multiple tissues (both benign and malignant) that were studied. We found that, although EBV can be detected in approximately 50% of breast cancer cases, the virus is not present in the malignant cells. Rather, the virus is localised to occasional infiltrating lymphocytes (Figure 1), which could give rise to ‘false’ interpretations