Regulation of IAPs gene family by interleukin-1 alpha and Epstein-Barr virus in nasopharyngeal carcinoma.

BACKGROUND: Inhibitors of apoptosis proteins (IAPs), which counteract apoptosis by potently inhibiting caspase activation, are promising targets of new anti-tumor therapy. However, their roles in the pathogenesis of nasopharyngeal carcinoma (NPC), an Epstein-Barr virus (EBV)-associated carcinoma, are not fully understood. Herein, we investigated the expression and regulation of IAPs in NPC. METHODS AND RESULTS: Using real-time quantitative polymerase chain reaction (PCR) analysis, we found that among the IAPs family only the transcription of survivin, HIAP-1, and HIAP-2 was consistently up-regulated in NPC and metastatic NPC tissues. Immunohistochemical staining showed that their proteins were more predominantly expressed in tumor cells nests. Noteworthy, these IAPs were upregulated by interleukin-1 alpha stimulation or EBV infection, and subsequently resulted in triggering rapid proliferation of NPC verified by strong Ki-67 staining. CONCLUSION: Survivin, HIAP-1, and HIAP-2 were distinctly upregulated in NPC, suggesting they may play significant roles in NPC tumorigenesis and serve as tumor markers with prognostic and therapeutic implications.

Upregulation of discoidin domain receptor 2 in nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is associated with Epstein-Barr virus (EBV) and has high metastatic potential. Discoidin domain receptors (DDR1, DDR2) are receptor-type tyrosine kinases activated by collagen. Their ability to induce expression of matrix metalloproteinase is related with tumor invasion. Therefore, we aim to investigate DDRs gene expression and its regulation in NPC. METHODS AND RESULTS: By use of real-time quantitative polymerase chain reaction (Q-PCR), DDR2 gene expression but not DDR1 was significantly higher in primary and metastatic NPC. DDR2 was predominantly distributed in NPC tumor cells rather than in infiltrating lymphocytes. EBV Z-transactivator (Zta) transfection may distinctly elevate DDR2 level. Furthermore, data from reporter assay indicate that Zta could transactivate DDR2 promoter activity, suggesting the possible upregulation mechanism. CONCLUSION: DDR2 was differentially upregulated in NPC and modulated by EBV Zta protein. DDR2 may play a role in NPC invasion and serve as a diagnostic and therapeutic target.

IFN-alpha-stimulated genes and Epstein-Barr virus gene expression distinguish WHO type II and III nasopharyngeal carcinomas.

Nonkeratinizing nasopharyngeal carcinoma (NPC) is 100% associated with Epstein-Barr Virus (EBV) and divided into two subtypes (WHO types II and III) based on histology. We tested whether these subtypes can be distinguished at the molecular genetic level using an algorithm that analyzes sets of related genes (gene set enrichment analysis). We found that a class of IFN-stimulated genes (ISG), frequently associated with the antiviral response, was significantly activated in type III versus type II NPC. Consistent with this, replication of the endogenous EBV was suppressed in type III. A strong association was also seen with a subset of ISGs previously identified in systemic lupus erythematosus, another disease in which 'normal' EBV biology is deregulated, suggesting that this pattern of ISG expression may be linked to the increased EBV activity in both diseases. In contrast, unsupervised hierarchical clustering of the complete expression profiles failed to distinguish the two subsets. These results suggest that type II and III NPC have not originated from obviously distinct epithelial precursors; rather, the histologic differences may be a consequence of a differential antiviral response, involving IFNs, to chronic EBV infection.

Epstein-Barr-virus-encoded LMP2A induces primary epithelial cell migration and invasion: possible role in nasopharyngeal carcinoma metastasis.

Nonkeratinizing nasopharyngeal carcinomas (NPC) are >95% associated with the expression of the Epstein-Barr virus (EBV) LMP2A latent protein. However, the role of EBV, in particular, LMP2A, in tumor progression is not well understood. Using Affymetrix chips and a pattern-matching computational technique (neighborhood analysis), we show that the level of LMP2A expression in NPC biopsy samples correlates with that of a cellular protein, integrin-alpha-6 (ITGalpha6), that is associated with cellular migration in vitro and metastasis in vivo. We have recently developed a primary epithelial model from tonsil tissue to study EBV infection in epithelial cells. Here we report that LMP2A expression in primary tonsil epithelial cells causes them to become migratory and invasive, that ITGalpha6 RNA levels are up-regulated in epithelial cells expressing LMP2, and that ITGalpha6 protein levels are increased in the migrating cells. Blocking antibodies against ITGalpha6 abrogated LMP2-induced invasion through Matrigel by primary epithelial cells. Our results provide a link between LMP2A expression, ITGalpha6 expression, epithelial cell migration, and NPC metastasis and suggest that EBV infection may contribute to the high incidence of metastasis in NPC progression.

Differential expression of osteoblast-specific factor 2 and polymeric immunoglobulin receptor genes in nasopharyngeal carcinoma.

BACKGROUND: The molecular mechanisms leading to development of nasopharyngeal carcinoma (NPC) are not well understood. To delineate the features of NPC, we tried to identify unique expression of cellular genes in the tumor biopsy specimens. METHODS AND RESULTS: By use of a combination of differential display and cDNA microarray analysis, we found two genes, 3E5 and 4A5, to show unique expression in the NPC biopsy specimens compared with nontumor nasopharyngeal tissues. Expression of 3E5, the osteoblast-specific factor-2 (OSF-2) gene, was detected at significantly higher levels in NPC biopsy specimens than that in control tissues, a finding confirmed using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). A correlation between expression of OSF-2 and its regulatory cytokine transforming growth factor-beta was observed in nontumor tissues but not in NPC biopsy specimens. On the other hand, expression of 4A5, whose sequences represent the 3' untranslated region of the polymeric immunoglobulin receptor (pIgR) gene, was detected rarely in NPC specimens but frequently in nontumor controls. The expression of pIgR in normal epithelial cells, but not in NPC tumor cells, was verified by RT-PCR and immunohistochemical staining. CONCLUSIONS: NPC shows significant upregulation of OSF-2 and downregulation of pIgR. Expression of OSF-2 is likely to play a role in the pathogenesis of NPC. In addition, expression of OSF-2 and pIgR is disassociated with the expression of their regulatory cytokines in NPC biopsy specimens, suggesting that the tumors may have altered responses to certain cytokines.

Epstein-Barr virus (EBV)-encoded BARF1 gene is expressed in nasopharyngeal carcinoma and EBV-associated gastric carcinoma tissues in the absence of lytic gene expression.

The BARF1 gene is located in the BamHI-A fragment of the Epstein-Barr virus (EBV) genome, encodes 221 amino acids, and has activity as an oncogene. Several reports have demonstrated that BARF1 is expressed in the tissues of various EBV-associated epithelioid malignancies. However,BARF1 is thought to be a lytic gene, since its expression is induced upon induction of the lytic cycle in Burkitt's lymphoma cell lines. Therefore, the possibility cannot be excluded that BARF1 expression in EBV-associated epithelioid malignancies reflects spontaneous induction of the lytic cycle in carcinoma cells. The present study aimed to clarify whether BARF1 was expressed as a latent gene or a lytic gene in epithelioid malignancies. Quantitative real-time RT-PCR assay revealed that BARF1 was highly expressed in nasopharyngeal carcinoma (NPC) and EBV-positive gastric carcinoma tissues in the absence of expression of lytic genes. On the other hand, BARF1 protein was detectable only in two of seven NPC tissue samples by immunoblot analysis. Analysis of BARF1-transfected CNE1 cells revealed that BARF1 was quickly secreted into culture medium and was hardly detectable in the cell lysate, which would account for why some NPC tissues were negative for BARF1 protein expression even though they were strongly positive forBARF1 expression at the transcriptional level. The present findings indicate that BARF1 is expressed in NPC and EBV-positive gastric carcinoma tissues as a latent gene and suggest that BARF1 plays a role in the pathogenesis of these malignancies.

Epstein-Barr virus latent membrane protein 1 induces the matrix metalloproteinase-1 promoter via an Ets binding site formed by a single nucleotide polymorphism: enhanced susceptibility to nasopharyngeal carcinoma.

The Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1) has a significant role in several malignancies, including nasopharyngeal carcinoma (NPC). LMP1 is the principal oncoprotein, and we have shown that it also induces a set of factors that mediates invasion, angiogenesis and metastasis. Matrix metalloproteinase-1 (MMP1) is also involved in several malignancies. A single guanine insertion polymorphism (2G) in the MMP1 promoter creates an Ets binding site that causes high levels of transcription and correlates with risk for some malignancies. Here, we evaluate the impact of this 2G insertion type on NPC. We genotyped 44 Japanese and 39 Taiwanese NPC patients, as well as 58 Japanese and 23 Taiwanese healthy controls. The proportion of 2G homozygotes was higher in the NPC groups than in controls (Japanese: p = 0.02, odds ratio (OR) = 2.49; Taiwanese: p = 0.02, OR = 3.66). An analysis of overall survival rates in the patients with NPC, and the 1G/1G genotype disclosed a favorable prognosis (5-year survival rate = 100%, p = 0.04). Multivariate analysis showed that 1G/1G has independent prognostic significance. We also examined whether LMP1 enhances MMP1 expression in epithelial cells in culture. LMP1-transfected cells with 2G/2G genotype expressed MMP1, which was abolished by activator protein-1 (AP1) dominant-negative (DN) and Ets-DN. LMP1 also induced active MMP3, which can cleave latent MMP1, and AP1-DN and Ets-DN suppressed the MMP3 expression. These results suggest that LMP1-induced MMP1 and MMP3 are closely linked and show that LMP1 activates MMP1 via an Ets binding site formed by 2G, which is a candidate marker for both risk and prognosis of NPC.

Epstein-Barr virus-encoded small RNA induces insulin-like growth factor 1 and supports growth of nasopharyngeal carcinoma-derived cell lines.

To assess the role of insulin-like growth factor 1 (IGF-1) in the growth of nasopharyngeal carcinoma (NPC), three NPC-derived cell lines, C666-1, CNE1 and HONE1, were examined. C666-1 cells maintained NPC phenotype of Epstein-Barr virus (EBV) expression and were positive for IGF-1 secretion, and their growth was strikingly inhibited by treatment with an anti-IGF-1 antibody under low serum condition. On the other hand, CNE1 and HONE1 cells were EBV-negative and did not secrete IGF-1. Although they could not grow under low serum condition, addition of recombinant IGF-1 made them grow. EBV conversion of CNE1 and HONE1 cells reproduced NPC phenotype of EBV expression and accompanied IGF-1 expression. Although they could grow under low serum condition, their growth was strikingly inhibited by treatment with the anti-IGF-1 antibody. These results suggest that EBV infection induces IGF-1 in NPC cell lines, and that the secreted IGF-1 acts as an autocrine growth factor. These findings seem to be operative in vivo, as NPC biopsies consistently express IGF-1. Further studies demonstrated that increased IGF-1 expression reflected transcriptional activation, and EBV-encoded small RNA (EBER) was responsible for IGF-1 induction. EBER is invariably expressed in EBV-associated malignancies, including NPC. The present findings strongly suggest that EBER directly affects the pathogenesis of NPC.

Lymphoepithelial carcinoma versus large cell undifferentiated carcinoma of the major salivary glands.

BACKGROUND: Undifferentiated carcinomas of the major salivary glands are rare malignant neoplasms of the head and neck region, and patients with these lesions have a poor prognosis. Patients with lymphoepithelial carcinoma (LEC), a specific subtype of undifferentiated carcinoma, however, have a better prognosis, and LEC seems to differ from large cell undifferentiated carcinoma (LCUC) clinically. METHODS: Sixteen patients with LEC and 12 patients with LCUC were retrieved from the records of 295 patients who had malignancies of the major salivary glands. A retrospective study on clinical manifestations, treatments, long-term outcomes, and an association with Epstein-Barr virus (EBV) by EBV-encoded small RNA-1 in situ hybridization was conducted to identify their differences. RESULTS: The median patient age was 44.5 years in the LEC group and 56 years in the LCUC group. At the time of presentation, patients with LCUC had a history of rapid-growing tumor and more advanced locoregional disease (Stage IV in 75% of patients with LCUC compared with 13% of patients with LEC). All 16 patients with LEC underwent curative surgery and radiotherapy, and their 5-year survival rate was 85.6%. In the LCUC group, only 7 patients were eligible to undergo radical surgery and receive radiotherapy, and their 2-year survival rate was only 36%. Age > 50 years was associated with a significantly worse prognosis for patients with LCUC. Neck metastasis and tumor size > 6 cm tended to be poor prognostic factors. Tumors were positive for harboring the EBV genome in all 16 LEC samples but in none of the LCUC samples. CONCLUSIONS: The clinicopathologic features of LEC and LCUC of the major salivary glands were different. LEC was associated with EBV, and patients with LEC had a much better prognosis compared with the prognosis for patients with LCUC. Therefore, LEC should be put in an independent group and should not be included in the same category as undifferentiated carcinomas of the salivary gland.

Immune responses to Epstein-Barr virus lytic proteins in patients with nasopharyngeal carcinoma.

Immune responses to three Epstein-Barr virus (EBV) lytic proteins, DNase, thymidine kinase (TK), and BMRF-1 gene products (50/52 kDa diffused early antigen, EA-D complex) were determined in EBV-infected control individuals and patients with nasopharyngeal carcinoma (NPC). Immunofluorescence assays (IFA) were used to detect their humoral immune responses using recombinant EBV lytic proteins expressed in a baculovirus system as antigens. Cell proliferation assays were performed to evaluate their cellular immune responses by monitoring 3H-thymidine incorporation. Seventy patients with NPC and 32 non-cancer controls were analyzed. The results of IFA showed antibody titers to all three EBV lytic proteins to be higher in the patients with NPC especially for the IgA class. Positivity rates of the three IgA antibodies also were higher in the patients with NPC population. Furthermore, the profiles of the IgA antibodies correlated with those to total early antigens (EA) expressed in the early phase and viral capsid antigen (VCA) expressed in the late phase, of EBV replication. The most interesting finding was that antibody titers to the three EBV lytic proteins were associated significantly with metastases of cervical lymph nodes in patients with NPC. As for cellular immunity to the EA-D complex and DNase, weak responses were observed in the cell proliferation assays. Peripheral blood cells from most individuals could not be stimulated to proliferate, except for a few patients with NPC whose antibody titers against the EA-D complex and DNase also were very high.

Diagnostic value of serum EBV-DNA quantification and antibody to viral capsid antigen in nasopharyngeal carcinoma patients.

We compared the amount of serum Epstein-Barr virus DNA (EBV-DNA) detected in patients with nasopharyngeal carcinoma (NPC) in a high-incidence area, represented by Taiwan, and a low-incidence area, represented by Japan, using real-time quantitative PCR. The median serum EBV-DNA value in 41 Japanese NPC cases was 5450 copies/ml, and that in in 23 Taiwanese cases was 2125 copies/ml. The median serum EBV-DNA value in all 64 NPC cases was significantly higher than in control groups. Using receiver-operating-characteristic (ROC) curves, the sensitivity and specificity of EBV-DNA quantification were determined (cut-off point, 6.87 copies/ml; sensitivity, 0.855; specificity, 0.885) and compared with those of EBV-viral-capsid-antigen (VCA) titers; the results showed that EBV-DNA was a more sensitive and specific parameter than EBV-VCA titer. Then, we analyzed 19 NPC patients in whom recurrence developed (11 Japanese and 8 Taiwanese), and 26 NPC patients in continuous remission. Although there was no significant difference in EBV-DNA values between Japanese and Taiwanese patients, the value was significantly higher in the 19 patients with recurrence than in those in remission. ROC analysis again revealed a higher diagnostic value of EBV-DNA than EBV-VCA. These results suggest EBV-DNA is a more reliable tumor marker than EBV-VCA in both high-incidence and low-incidence areas of NPC.

Postirradiation vertigo in nasopharyngeal carcinoma survivors.

OBJECTIVE: Vertigo rarely manifested as an initial symptom of nasopharyngeal carcinoma or an early symptom after irradiation; however, increasing numbers of long-term nasopharyngeal carcinoma survivors experienced it. The purpose of this study was to investigate the causes of vertigo in irradiated nasopharyngeal carcinoma survivors. SETTING: University hospital. PATIENTS: From January 1992 to December 2001, a total of 113 nasopharyngeal carcinoma patients (67 men and 46 women) with postirradiation vertigo consecutively visited our vertigo clinic. The mean interval from completion of irradiation to the occurrence of vertigo was 10 years. Each patient underwent otoscopic examination and a battery of audiovestibular function tests. Then, correlation between the vertigo and the radiation effect was explored. RESULTS: Postirradiation vertigo was mainly attributable to peripheral labyrinthine disorder (69%), followed by central vestibular lesions (31%). The vertiginous and associated symptoms including severity, nausea/vomiting, oscillopsia, or imbalance in cases of peripheral labyrinthine disorder were milder than those in central vestibular lesion. Meanwhile, the former had less life impact and better response to therapy compared with the latter. The mean radiation dosage in both groups was 73 +/- 6 Gy and 74 +/- 5 Gy, respectively, without a significant difference. Of these 113 patients, 85 patients (75%) had radiation otitis media in one or both ears, and 28 patients disclosed bilateral intact eardrums. The prevalence of radiation otitis media in patients with postirradiated vertigo was 53% versus 73% for those with radiation dosage less than or more than 71 Gy, respectively, exhibiting a significant difference. However, the prevalence of radiation otitis media is unrelated to radiation interval. In comparison with absent caloric responses in radiation otitis media ears, 32% versus 57% in those of less than or more than 71 Gy, respectively, indicates a significant difference. CONCLUSIONS: Vertigo is a late complication in irradiated nasopharyngeal carcinoma survivors, which is mainly caused by the sequela of radiation otitis media. Because the latter is correlated with the radiation dosage, 70 Gy is recommended as the maximum dosage for nasopharyngeal carcinoma. Eradicating radiation otitis media in nasopharyngeal carcinoma survivors may subsequently prevent the postirradiation vertigo.

Vascular leiomyoma of the head and neck.

OBJECTIVES/HYPOTHESIS: Vascular leiomyoma, a benign tumor composed of smooth muscle cell and vascular endothelium, is rare in the head and neck region. The authors report their experience with 21 patients. STUDY DESIGN: Retrospective review. METHODS: From 1988 to 2001, the clinical records of 21 patients with vascular leiomyoma of the head and neck were reviewed. The pathological material of each tumor was reviewed again for confirmation of the diagnosis and histological classification proposed by Morimoto. RESULTS: Twelve male and 9 female patients were studied. The mean age was 48 years. The locations and numbers of cases of the tumors were as follows: auricle, five; nasal cavity, three; external nose, 3; neck, 3; lip, 3; inner canthus, 2; forehead, 1; and hard palate, 1. All tumors were painless, and most were less than 2 cm in diameter. Three vascular leiomyomas of the neck were larger than 2 cm. Two of the three tumors originating in the nasal cavity presented with nasal obstruction or epistaxis. Regarding histological subtype, 14 of 21 (67%) tumors were solid type; 6 (28%) were cavernous type, and only one (5%) was venous type. Only one tumor (5%) recurred after excision. CONCLUSION: Vascular leiomyoma usually presents as a small, painless mass. Auricle, nose, lip, and neck are the most common sites of occurrence. Unusually large vascular leiomyomas are developed in the deep space of the neck. Imaging study or cytological examination is not helpful for diagnosis. Histological classification is not necessary. Simple excision yields excellent results.