Epstein-Barr virus latent membrane protein inhibits human epithelial cell differentiation

Epstein-Barr virus (EBV), a human herpesvirus, is strongly linked with two relatively rare forms of B-cell lymphoma and with a much more prevalent epithelial malignancy, undifferentiated nasopharyngeal carcinoma (NPC). The availability of suitable culture systems has allowed detailed analysis of EBV-induced growth transformation in B lymphocytes, but little is known about the virus--epithelial cell interaction or about the possible effector role of viral proteins in the pathogenesis of NPC. Here we describe an experimental system to monitor the effects of introduced viral or cellular genes upon human epithelial cell growth and differentiation. We transfected a human epithelial cell line, which retains several features of normal keratinocyte behaviour in vitro, with the EBV gene encoding latent membrane protein (LMP), one of only two viral proteins known to be expressed in NPC cells in vivo. LMP expression was accompanied by changes in the epithelial cell surface phenotype, mimicking surface changes observed in NPC cells, and by severe impairment of the cellular response to differentiation signals. The ability of LMP to inhibit terminal differentiation indicates a mechanism whereby EBV infection of squamous epithelium could contribute to the multi-step pathogenesis of NPC.     

Phorbol ester and Epstein-Barr virus dependent transformation of normal primary human skin epithelial cells

Substantial evidence has implicated Epstein-Barr virus (EBV) in the aetiology of two human neoplasms, nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma. This is supported by the presence of high antibody titres to EBV early antigen and virus capsid antigen, as well as antibody to two viral-associated enzymes, DNase and DNA polymerase. Patients with NPC, particularly the undifferentiated form, are commonly found to have EBV DNA in the tumour. Ito and others have presented strong epidemiological evidence that phorbol esters are related to the unusual geographic distribution of NPC in southeastern regions of China. There appears to be a close link between the widespread EBV infection of the Asian population and the distinct regional distribution in China of plants that produce diterpene ester. Naturally occurring phorbol esters are produced by plants of the Euphorbiaceae and Thymelaeaceae, which are used as traditional herbal medicines. Although it has been established that EBV can infect epithelial cells isolated from NPC as well as certain normal epithelial cells, there has been no in vitro evidence that EBV induces neoplastic transformation in normal human epithelial cells with or without exposure to phorbol esters. We report here evidence that transformation of normal human epithelial cells results from exposure to infectious EBV and that transformation is dependent on the presence of phorbol esters.     

Epstein--Barr virus-induced cell fusion

Serological and molecular biological studies have shown an association between Epstein--Barr virus (EBV) and nasopharyngeal carcinoma. Although it has been shown that the epithelioid tumour cells carry EBV genomes, they are apparently devoid of receptors for EBV (H.W., unpublished observations). Other have suggested that fusion of EBV carrying cells with epithelial cells may be the mode of entry of the virus into cells unable to absorb the virus and that this may be mediated by one of the known syncytium-forming viruses which inhabit the respiratory tract (for example, members of the paramyxovirus group). de Thé and colleagues suggested that intercellular bridges could be seen in NPC tumour material. We have developed a technique which permits the preparation of stable monolayers of viable human lymphoblastoid cell lines. Using this technique we have now demonstrated that EBV can induce fusion between EBV-superinfected lymphoblastoid cells and cells devoid of EBV receptors.     

Replication of Epstein-Barr virus in human epithelial cells infected in vitro

Epstein-Barr virus (EBV), a member of the herpes group of viruses and the aetiological agent of infectious mononucleosis, is usually thought of as a lymphotrophic virus with the ability to transform B lymphocytes. So the association of EBV with nasopharyngeal carcinoma is puzzling, especially given the lack of success of attempts to infect epithelial cells with EBV in culture and the apparent lack of EBV receptors on epithelial cells. Circumvention of the apparent requirement for membrane receptors by techniques of transfection, microinjection and receptor transplantation has clearly demonstrated that there is no inherent barrier to EBV replication in nonlymphoid cells, including epithelial cell types. Our ability routinely to detect EBV DNA by in situ hybridization in epithelial cells of the oropharynx from persons with acute infectious mononucleosis suggests that, in vivo, EBV regularly gains access to and replicates lytically in epithelial cells. We report here in vitro evidence for direct infection by EBV and replication of the virus in cultured normal human epithelial cells.     

Human N-myc gene contributes to neoplastic transformation of mammalian cells in culture

Proto-oncogenes represent a group of eukaryotic genes whose activated forms are implicated in the development of cancer. We have recently identified a human gene, N-myc, that is distantly related to the proto-oncogene c-myc. N-myc is expressed at abnormally high levels consequent to amplification in numerous human neuroblastoma cell lines and metastatic neuroblastoma tumours. In addition, enhanced expression of N-myc, often a result of amplification, has been found in retinoblastoma cell lines and tumours (refs 5, 7 and M.S., unpublished data) and in cell lines derived from small-cell carcinomas of the lung. Here, we show that enhanced expression of N-myc subsequent to co-transfections of an N-myc expression vector and the mutant c-Ha-ras-1(EJ) (from the human bladder carcinoma cell line EJ) is a factor in tumorigenic conversion of secondary rat embryo cells. The transformed cells elicit tumours in athymic mice and isogeneic rats. The ability of N-myc to contribute to neoplastic transformation of cultured mammalian cells raises the possibility that enhanced expression consequent to amplification of N-myc may be a factor in the aetiology of human neuroblastoma.     

Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin

Epidermal growth factor (EGF), through interaction with specific cell surface receptors, generates a pleiotropic response that, by a poorly defined mechanism, can induce proliferation of target cells. Subversion of the EGF mitogenic signal through expression of a truncated receptor may be involved in transformation by the avian erythroblastosis virus (AEV) oncogene v-erb-B, suggesting that similar EGF receptor defects may be found in human neoplasias. Overexpression of EGF receptors has been reported on the epidermoid carcinoma cell line A431, in various primary brain tumours and in squamous carcinomas. In A431 cells the receptor gene is amplified. Here we show that 4 of 10 primary brain tumours of glial origin which express levels of EGF receptors that are higher than normal also have amplified EGF receptor genes. Amplified receptor genes were not detected in the other brain tumours examined. Further analysis of EGF receptor defects may show that such altered expression and amplification is a particular feature of certain human tumours.     

Expression of N-myc in teratocarcinoma stem cells and mouse embryos

The N-myc gene, which is distantly related to the proto-oncogene c-myc, was first detected as an amplified sequence in human neuroblastoma cell lines and tumours. It has since been revealed that there is up to a 300-fold amplification of N-myc DNA in almost 50% of advanced metastatic human neuroblastomas, whereas amplification is not detected in less advanced tumours that have a better prognosis (ref.3 and M.S., unpublished data). Although expression of N-myc is detectable in all neuroblastoma cell lines and tumours examined, its level is greatly enhanced when the N-myc gene is amplified. Recently, it has been shown that on co-transfection with the c-Ha-ras (EJ) gene, N-myc can induce the malignant transformation of rat embryo fibroblasts. Taken together, these data imply a function for N-myc in the development and/or progression of human neuroblastomas. Surveys indicate that N-myc also may be amplified and/or expressed in two other types of human tumours and cell lines derived from them: retinoblastomas and small cell lung cancers. Here, we report that N-myc is expressed at high levels in mouse and human teratocarcinoma stem cells, thus identifying another tumour cell type that expresses the N-myc gene. In addition, we found that N-myc is abundantly expressed in mouse embryos at mid-gestation and that its expression appears to decrease as the embryo approaches term. In the adult mouse, N-myc is expressed at an approximately fivefold lower level in the brain than in teratocarcinoma stem cells and embryos, and at even lower levels in the adult testis and kidney. Our data represent the first demonstration of expression of the N-myc gene in normal cells, and suggest that N-myc may be involved in mammalian embryogenesis.     

Lysyl oxidase is essential for hypoxia-induced metastasis

Metastasis is a multistep process responsible for most cancer deaths, and it can be influenced by both the immediate microenvironment (cell-cell or cell-matrix interactions) and the extended tumour microenvironment (for example vascularization). Hypoxia (low oxygen) is clinically associated with metastasis and poor patient outcome, although the underlying processes remain unclear. Microarray studies have shown the expression of lysyl oxidase (LOX) to be elevated in hypoxic human tumour cells. Paradoxically, LOX expression is associated with both tumour suppression and tumour progression, and its role in tumorigenesis seems dependent on cellular location, cell type and transformation status. Here we show that LOX expression is regulated by hypoxia-inducible factor (HIF) and is associated with hypoxia in human breast and head and neck tumours. Patients with high LOX-expressing tumours have poor distant metastasis-free and overall survivals. Inhibition of LOX eliminates metastasis in mice with orthotopically grown breast cancer tumours. Mechanistically, secreted LOX is responsible for the invasive properties of hypoxic human cancer cells through focal adhesion kinase activity and cell to matrix adhesion. Furthermore, LOX may be required to create a niche permissive for metastatic growth. Our findings indicate that LOX is essential for hypoxia-induced metastasis and is a good therapeutic target for preventing and treating metastases.     

EB病毒及化学致癌物DNP对人胚鼻咽粘膜裸鼠移植的诱癌研究

用EBV,DNP及PMA分别或协同对人胚鼻咽粘膜裸鼠移植物进行诱癌研究,探讨上述因素在人实验性鼻咽癌发生中的作用。免疫荧光法检查证实,经B95-8上清液体外及体内转染的人胚鼻咽粘膜上皮表达EBNA。实验结果显示,实验各组均可见各种增生性改变,对照组仅见一般增生性病变。各诱癌组均可见癌前病变,组间无显著性差异。共出现癌8例(1)EBV组:原位癌1例。 (2)EBV+PMA组:早期浸润癌1例。(3)EBV+DNP组:原位癌及浸润癌各1例。(4)EBV+DNP+PMA组:浸润癌1例。 (5)DNP组:原位癌1例。(6)DNP+PMA组:原位癌及浸润癌各1例。结果提示EBV可能在人鼻咽上皮实验性癌变过程中起病因作用。本研究成功诱发了三例浸润癌,均系DAP与其它因素(PMA、EBV)协同作用,提示人鼻咽癌的发生可能与多种致癌因素的相互作用有关。     

Mitotic EBNA-positive lymphocytes in peripheral blood during infectious mononucleosis

Infectious mononucleosis (IM) is usually a benign lymphoproliferative disease caused by Epstein-Barr virus (EBV). Although EBV induces a state of continuous proliferation in infected B lymphocytes in vitro, the most prominent lymphoproliferation during IM is of activated, or atypical, T lymphocytes presumably responding to the virus or virus-infected cells. However, EBV genome-carrying cells are known to be circulating during IM, as cultured peripheral blood leukocytes from patients with the disease give rise to continuous lymphoblastoid cell lines, each cell of which contains the EBV genome and expresses the EBV determined nuclear antigen (EBNA). The proposal that EBV-infected cells in IM blood are not endowed with enhanced growth potential but are merely latently infected is supported by demonstrations that cells infected in vivo enter a viral replicative cycle when placed in vitro and that most cell lines derived from cultured lymphocytes of IM patients are infected by virus released in vitro. However the cells could also be capable of proliferation in vivo, since virus production and transformation are not mutually exclusive properties of EBV-transformed cells. Recently, EBNA has been detected in a very small fraction of peripheral blood lymphocytes of IM patients after T cells were first removed and this has been interpreted to indicate that cell transformation occurs in vivo during IM. The isolation of colonies of EBNA-positive cells from IM blood leukocytes cultures in soft agar suggests that at least some infected cells are capable of direct outgrowth into transformed cells. We report here direct evidence that circulating EBV-infected cells exhibit increased growth properties during IM.     

Deregulated cyclin E induces chromosome instability.

Cyclin E, a regulatory subunit of cyclin-dependent kinase 2 (Cdk2), is an important regulator of entry into S phase in the mammalian cell cycle. In normal dividing cells, cyclin E accumulates at the G1/S-phase boundary and is degraded as cells progress through S phase. However, in many human tumours cyclin E is overexpressed and the levels of protein and kinase activity are often deregulated relative to the cell cycle. It is not understood how alterations in expression of cyclin E contribute to tumorigenesis. Here we show that constitutive cyclin-E overexpression in both immortalized rat embryo fibroblasts and human breast epithelial cells results in chromosome instability (CIN). In contrast, analogous expression of cyclin D1 or A does not increase the frequency of CIN. Cyclin-E-expressing cells that exhibit CIN have normal centrosome numbers. However, constitutive overexpression of cyclin E impairs S-phase progression, indicating that aberrant regulation of this process may be responsible for the CIN observed. These results indicate that downregulation of cyclin-E/Cdk2 kinase activity following the G1/S-phase transition may be necessary for the maintenance of karyotypic stability.     

A vascular niche and a VEGF-Nrp1 loop regulate the initiation and stemness of skin tumours

Angiogenesis is critical during tumour initiation and malignant progression. Different strategies aimed at blocking vascular endothelial growth factor (VEGF) and its receptors have been developed to inhibit angiogenesis in cancer patients. It has become increasingly clear that in addition to its effect on angiogenesis, other mechanisms including a direct effect of VEGF on tumour cells may account for the efficiency of VEGF-blockade therapies. Cancer stem cells (CSCs) have been described in various cancers including squamous tumours of the skin. Here we use a mouse model of skin tumours to investigate the impact of the vascular niche and VEGF signalling on controlling the stemness (the ability to self renew and differentiate) of squamous skin tumours during the early stages of tumour progression. We show that CSCs of skin papillomas are localized in a perivascular niche, in the immediate vicinity of endothelial cells. Furthermore, blocking VEGFR2 caused tumour regression not only by decreasing the microvascular density, but also by reducing CSC pool size and impairing CSC renewal properties. Conditional deletion of Vegfa in tumour epithelial cells caused tumours to regress, whereas VEGF overexpression by tumour epithelial cells accelerated tumour growth. In addition to its well-known effect on angiogenesis, VEGF affected skin tumour growth by promoting cancer stemness and symmetric CSC division, leading to CSC expansion. Moreover, deletion of neuropilin-1 (Nrp1), a VEGF co-receptor expressed in cutaneous CSCs, blocked VEGF's ability to promote cancer stemness and renewal. Our results identify a dual role for tumour-cell-derived VEGF in promoting cancer stemness: by stimulating angiogenesis in a paracrine manner, VEGF creates a perivascular niche for CSCs, and by directly affecting CSCs through Nrp1 in an autocrine loop, VEGF stimulates cancer stemness and renewal. Finally, deletion of Nrp1 in normal epidermis prevents skin tumour initiation. These results may have important implications for the prevention and treatment of skin cancers.     

Epstein-Barr virus transforms precursor B cells even before immunoglobulin gene rearrangements

The very early stages of the human B-cell differentiation pathway are poorly understood, primarily because of the lack of appropriate permanent cell lines. Epstein-Barr virus (EBV) is a putative human oncogenic virus which transforms human B cells in vitro into continuously proliferating cells. It has been believed that EBV transforms mature B cells, but recently, transformation of immature pre-B-cell lines has been reported, suggesting that EBV might also transform cells much earlier in the B-cell lineage. We report here the establishment of cell lines transformed by EBV at various stages of the B-cell differentiation pathway. Interestingly, two lines showed the complete absence of immunoglobulin synthesis and the lack of immunoglobulin gene rearrangement despite containing EBV genome and surface markers of B cells. Our results indicate that EBV can infect and transform cells of the B lymphocyte lineage even before immunoglobulin gene rearrangement.     

Nonrandom loss of chromosome 15 in Syrian hamster tumours induced by v-Ha-ras plus v-myc oncogenes

Nonrandom chromosome rearrangements, observed in a variety of human and animal tumours, are associated in some cases with enhanced expression or deregulation of cellular oncogenes. Recently, it was shown that normal, diploid rodent cells are neoplastically transformed following transfection with two cooperating oncogenes, for example myc plus ras. However, the number of steps necessary to convert a normal cell into a malignant cell is unknown. If activation of two oncogenes is sufficient for tumorigenicity, tumours derived from diploid cells transformed by the transfected oncogenes may remain diploid or have only random chromosome alterations. We have performed cytogenetic analyses of tumours formed after transfection of Syrian hamster embryo cells with either v-Ha-ras plus v-myc DNAs or polyoma DNA alone. Whereas polyoma-induced, tumour-derived cells were diploid, tumours induced by v-Ha-ras plus v-myc oncogenes were monoclonal and had a nonrandom chromosome change, monosomy of chromosome 15. Thus, an additional change, loss of chromosome 15, is required for or is advantageous for tumorigenicity induced by v-Ha-ras plus v-myc oncogenes. These results suggest that the neoplastic progression of normal, diploid cells requires more than two steps under certain conditions.     

Selective killing of cancer cells by a small molecule targeting the stress response to ROS

Malignant transformation, driven by gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes, results in cell deregulation that is frequently associated with enhanced cellular stress (for example, oxidative, replicative, metabolic and proteotoxic stress, and DNA damage). Adaptation to this stress phenotype is required for cancer cells to survive, and consequently cancer cells may become dependent upon non-oncogenes that do not ordinarily perform such a vital function in normal cells. Thus, targeting these non-oncogene dependencies in the context of a transformed genotype may result in a synthetic lethal interaction and the selective death of cancer cells. Here we used a cell-based small-molecule screening and quantitative proteomics approach that resulted in the unbiased identification of a small molecule that selectively kills cancer cells but not normal cells. Piperlongumine increases the level of reactive oxygen species (ROS) and apoptotic cell death in both cancer cells and normal cells engineered to have a cancer genotype, irrespective of p53 status, but it has little effect on either rapidly or slowly dividing primary normal cells. Significant antitumour effects are observed in piperlongumine-treated mouse xenograft tumour models, with no apparent toxicity in normal mice. Moreover, piperlongumine potently inhibits the growth of spontaneously formed malignant breast tumours and their associated metastases in mice. Our results demonstrate the ability of a small molecule to induce apoptosis selectively in cells that have a cancer genotype, by targeting a non-oncogene co-dependency acquired through the expression of the cancer genotype in response to transformation-induced oxidative stress.     

Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells

A long-standing hypothesis on tumorigenesis is that cell division failure, generating genetically unstable tetraploid cells, facilitates the development of aneuploid malignancies. Here we test this idea by transiently blocking cytokinesis in p53-null (p53-/-) mouse mammary epithelial cells (MMECs), enabling the isolation of diploid and tetraploid cultures. The tetraploid cells had an increase in the frequency of whole-chromosome mis-segregation and chromosomal rearrangements. Only the tetraploid cells were transformed in vitro after exposure to a carcinogen. Furthermore, in the absence of carcinogen, only the tetraploid cells gave rise to malignant mammary epithelial cancers when transplanted subcutaneously into nude mice. These tumours all contained numerous non-reciprocal translocations and an 8-30-fold amplification of a chromosomal region containing a cluster of matrix metalloproteinase (MMP) genes. MMP overexpression is linked to mammary tumours in humans and animal models. Thus, tetraploidy enhances the frequency of chromosomal alterations and promotes tumour development in p53-/- MMECs.     

Colorectal carcinomas in mice lacking the catalytic subunit of PI(3)Kgamma

Phosphoinositide-3-OH kinases (PI(3)Ks) constitute a family of evolutionarily conserved lipid kinases that regulate a vast array of fundamental cellular responses, including proliferation, transformation, differentiation and protection from apoptosis. PI(3)K-mediated activation of the cell survival kinase PKB/Akt, and negative regulation of PI(3)K signalling by the tumour suppressor PTEN (refs 3, 4) are key regulatory events in tumorigenesis. Thus, a model has arisen that PI(3)Ks promote development of cancers. Here we report that genetic inactivation of the p110gamma catalytic subunit of PI(3)Kgamma (ref. 8) leads to development of invasive colorectal adenocarcinomas in mice. In humans, p110gamma protein expression is lost in primary colorectal adenocarcinomas from patients and in colon cancer cell lines. Overexpression of wild-type or kinase-dead p110gamma in human colon cancer cells with mutations of the tumour suppressors APC and p53, or the oncogenes beta-catenin and Ki-ras, suppressed tumorigenesis. Thus, loss of p110gamma in mice leads to spontaneous, malignant epithelial tumours in the colorectum and p110gamma can block the growth of human colon cancer cells.