Recombinant adeno-associated virus mediated RNA interference inhibits metastasis of nasopharyngeal cancer cells in vivo and in vitro by suppression of Epstein-Barr virus encoded LMP-1.

Nasopharyngeal carcinoma (NPC) is a highly metastatic carcinoma characterized by consistent association with Epstein-Barr virus (EBV). Of the EBV-encoded product, latent membrane protein-1 (LMP-1) is considered to be an onco-protein playing an essential role in cell transformation and metastasis. In this study, we used a recombinant adeno-associated virus type 2 vector (rAAV-2) to deliver small hairpin RNA (shRNA) targeting EBV LMP-1 into the EBV-positive human NPC C666-1 cells and evaluated the effect of long-term suppression of LMP-1 on NPC growth and metastasis in vivo and in vitro. An NPC metastasis nude mouse model with NPC xenograft transplanted in liver was established. The NPC C666-1 cells infected with rAAV-shRNA-LMP-1 or rAAV-EGFP were inoculated in the livers of nude mice. Formation of liver and lung metastasis was evaluated at day 14 after tumor inoculation. Our results demonstrate that rAAV-shRNA-LMP-1 effectively infected C666-1 cells and suppressed LMP-1 expression. Such suppression, in turn, did not significantly inhibit tumor growth, but prevented NPC metastasis in the liver as well as in the lung. Consistent with in vivo data, the in vitro studies in NPC C666-1 cell cultures showed that suppression of LMP-1 by rAAV-shRNA-LMP-1 significantly reduced cell mobility and transmembrane invasion ability. These results demonstrated for the first time that long-term suppression of EBV-encoded LMP-1 in vivo is an effective means for preventing NPC metastasis.

Inhibition of human nasopharyngeal carcinoma growth and metastasis in mice by adenovirus-associated virus-mediated expression of human endostatin.

Nasopharyngeal carcinoma (NPC) is a highly malignant and frequently metastasized tumor. Endostatin has been shown to inhibit NPC growth, but its efficacy against NPC metastasis has not been shown in vivo. Here, we established a NPC metastasis model in mice by transplanting EBV-positive NPC cells, C666-1, in the livers of nude mice and observed lung metastasis. Furthermore, we showed that tail vein injection of recombinant adeno-associated virus encoding human endostatin (rAAV-hEndo) significantly prolonged the median survival rate of NPC metastasis-bearing mice (from 22 to 37 days, P < 0.01). The rAAV-hEndo treatment resulted in a statistically significant reduction in tumor growth and microvessel formation. It also increased the apoptotic index in the primary liver tumor but not in the normal liver tissue. Importantly, no formation of liver or lung metastasis was detected. The potent inhibition of NPC metastasis suggests the feasibility of combining rAAV-hEndo gene therapy with other therapies for the prevention and treatment of NPC metastasis.

Allelic imbalance mapped to 6q14.1 is associated with loss of expression of 5-HT receptor 1B in non-Hodgkin lymphomas.

Previous studies on lymphomas suggested that the long arm of chromosome 6 harbors 1 or more tumor suppressor genes. This study analyzed the status of 25 microsatellite markers in 39 cases, including 9 nodal and 30 extranodal, of non-Hodgkin lymphomas. Thirty of the 39 cases (77%) showed abnormality in at least 1 of the markers. Of the 655 informative results, 135 (20%) were abnormal. These included 5 homozygous deletions, 91 allelic imbalances (AI), and 38 microsatellite instability. The 2 commonest regions of abnormality were mapped to 6q14.1 and 6q27. There was no significant difference in the frequency of these regional losses between nodal and extranodal lymphomas, B-or T-cell lineage, and association with Epstein-Barr virus. The first common deletion region at 6q14.1 is flanked by the HTR1B (5-hydroxytryptamine receptor 1B) gene proximally and a novel unknown gene. AI in the region was found associated with loss of expression HTR1B by RT-PCR. The deletion region at 6q27 was narrowed to approximately 3Mb and maximal at marker D6S386. This locus includes the recently identified SMOC2 (secreted modular calcium-binding protein 2), AF6, and DLL1 (human delta-like 1 protein) genes. RT-PCR analyses of AF6 and DLL1 expression showed poor correlation with the AI results.

Allelic loss of chromosome 6q in gastric carcinoma.

Loss of the long arm of chromosome 6 (6q) has frequently been reported in gastric carcinoma, and most gastric cancer patients have evidence of intestinal metaplasia in the stomach. However, the relationship between loss of chromosome 6q and intestinal metaplasia has not been studied. In the first part of the study, we define the critical deletion region of chromosome 6q using loss of heterozygosity technique (LOH). Seventeen microsatellite markers were used to detect loss of heterozygosity (LOH) in 37 microdissected gastric tumors. We also examined intestinal metaplasia (IM) foci of the stomach in the same cancer patient (17 cases). Losses on chromosome 6q were detected in high frequency (51%) by LOH. Two distinct regions of common allelic loss were identified: one centered on the marker D6S300 (at 6q16.1) and the second on D6S446 (at 6q27), with LOH frequency of 36% and 31.3%, respectively. The deletions fall into 2 discrete regions, suggesting the existence of at least 2 tumor suppressor genes in 6q. The losses at 6q27 were confirmed by fluorescence in situ hybridization study (FISH). In the cases with LOH in the tumor, no LOH were detected in the autologous IM areas, but losses were detected by FISH. In some cases, these genetic changes may be acquired in the transition from normal gastric mucosa to intestinal metaplasia.

Recurrent genomic aberrations in gastric carcinomas associated with Helicobacter pylori and Epstein-Barr virus.

Helicobacter pylori and Epstein-Barr virus (EBV) both have been associated with gastric carcinoma. No specific genomic aberrations have been reported in association with these agents. We studied 20 cases of primary gastric carcinoma (including 11 positive for and 6 for EBV) by comparative genomic hybridization with validation of results by fluorescence in situ hybridization, loss of heterozygosity analysis, and immunohistochemistry. The results were analyzed in respect to presence or absence of and EBV. The tumors were also compared in terms of histologic type, tumor location, and lymph node metastases. The most frequently observed aberrations in the gastric carcinomas were gains of chromosome 19, 17, 1p, 11, 20q, and 22. The more common losses were found in 4q, 6q, 13q, and 15q. Gains in chromosome 19 and losses in 9p23-pter were found more commonly in cases with (P < 0.05). Gains in centromeric region of chromosome 19 were more common in the EBV-negative cases (P < 0.05). Immunohistochemical expression of and correlated with gains in the regions containing these genes. Gains in chromosome 11 and losses in 15q15 were more common in cases with EBV (P < 0.01 and P < 0.001, respectively). There was no significant association between any genomic aberration and histologic type, tumor location, or nodal metastases. and EBV are associated with different genomic imbalances, suggesting that these infectious agents exert different influences in the development of gastric carcinoma.