Anti-angiogenic pathway associations of the 3p21.3 mapped BLU gene in nasopharyngeal carcinoma.

Zinc-finger, MYND-type containing 10 (ZMYND10), or more commonly called BLU, expression is frequently downregulated in nasopharyngeal carcinoma (NPC) and many other tumors due to promoter hypermethylation. Functional evidence shows that the BLU gene inhibits tumor growth in animal assays, but the detailed molecular mechanism responsible for this is still not well understood. In current studies, we find that 93.5% of early-stage primary NPC tumors show downregulated BLU expression. Using a PCR array, overexpression of the BLU gene was correlated to the angiogenesis network in NPC cells. Moreover, expression changes of the MMP family, VEGF and TSP1, were often detected in different stages of NPC, suggesting the possibility that BLU may be directly involved in the microenvironment and anti-angiogenic activity in NPC development. Compared with vector-alone control cells, BLU stable transfectants, derived from poorly-differentiated NPC HONE1 cells, suppress VEGF165, VEGF189 and TSP1 expression at both the RNA and protein levels, and significantly reduce the secreted VEGF protein in these cells, reflecting an unknown regulatory mechanism mediated by the BLU gene in NPC. Cells expressing BLU inhibited cellular invasion, migration and tube formation. These in vitro results were further confirmed by in vivo tumor suppression and a matrigel plug angiogenesis assay in nude mice. Tube-forming ability was clearly inhibited, when the BLU gene is expressed in these cells. Up to 70-90% of injected tumor cells expressing increased exogenous BLU underwent cell death in animal assays. Overexpressed BLU only inhibited VEGF165 expression in differentiated squamous NPC HK1 cells, but also showed an anti-angiogenic effect in the animal assay, revealing a complicated mechanism regulating angiogenesis and the microenvironment in different NPC cell lines. Results of these studies indicate that alteration of BLU gene expression influences anti-angiogenesis pathways and is important for the development of NPC.

HBx mutants differentially affect the activation of hypoxia-inducible factor-1α in hepatocellular carcinoma.

BACKGROUND: Mutations in HBx gene are frequently found in HBV-associated hepatocellular carcinoma (HCC). Activation of hypoxia-inducible factor-1α (HIF-1α) contributes to HCC development and progression. Wild-type HBx has been demonstrated to activate HIF-1α, but the effect of HBx mutations on HIF-1α has not been elucidated. METHODS: HBx mutations were identified by gene sequencing in 101 HCC tissues. Representative HBx mutants were cloned and transfected into HCC cells. Expression and activation of HIF-1α were analysed by western blot and luciferase assays, respectively. The relationship between HBx mutants and HIF-1α expression in HCC tissues was also evaluated. RESULTS: The dual mutations K130M/V131I enhanced the functionality of HBx as they upregulated the expression and transcriptional activity of HIF-1α. The C-terminal truncations and deletion mutations, however, weakened the ability of HBx to upregulate HIF-1α. Meanwhile, the C-terminus was further found to be essential for the stability and transactivation of HBx. In the HCC tissues, there was a positive association between the HBx mutants and HIF-1α expression. CONCLUSION: Different mutations of HBx exert differentiated effects on the functionality of HIF-1α, however, the overall activity of HBx mutants appears to increase the expression and transcriptional activity of HIF-1α.

Single nucleotide polymorphism in the promoter region of human alpha-fetoprotein (AFP) gene and its significance in hepatocellular carcinoma (HCC).

BACKGROUND: Variations of serum AFP levels in HCC patients and cell lines are likely due to the differential activity of enhancer/silencer elements that control AFP. To understand the potential mechanism underlying the differential expression of AFP, we have examined the sequence of the AFP promoter in HCC. METHODS: Direct DNA sequencing was carried out to sequence 980 bp of AFP promoter of DNA samples isolated from 83 HCC patients. RESULTS: Three novel SNPs in the promoter region of the AFP gene, which have not been previously reported, were found at positions -330, -401 and -692. The level of serum AFP was significantly higher in HCC patients with the CT genotype of 330 SNP or the AG genotype of the 401 SNP. The genotype of CG in 692 SNP was also associated with a significant elevated level of serum AFP, and further this genotype appeared to be associated with the high risk of HCC development. 401 SNP and 692 SNP were located at the positions of known binding sites for transcription factors that have a role in the production of AFP and the growth of tumors. CONCLUSIONS: The novel polymorphisms identified in the promoter region of the AFP gene may be pathologically significant in HCC.

Identification of hepatitis B virus X gene mutation in Hong Kong patients with hepatocellular carcinoma.

BACKGROUND: Chronic infection by hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) in man. The viral transactivator HBV X (HBx) gene plays a critical role in the molecular pathogenesis of HBV-related HCC. OBJECTIVES: The aim of this study was to investigate whether there were particular HBx mutations associated with the Chinese Hong Kong patients with HCC. STUDY DESIGN: We have examined HBx in 113 tumor tissue samples from patients with HCC and 48 serum samples from the same group. In addition, we also examined the expression of HBx protein and the index of apoptotic cell death in tumor tissues of HCC. The entire coding region of HBx gene from the sample was sequenced and aligned with the published HBx gene sequence. RESULTS AND CONCLUSIONS: We have identified total 54 different types of mutations in HBx gene. HBx mutations occurred in a very high percentage of samples tested. Mutation of HBx was found in 95.2% and 95.3% of the tumor tissue and serum samples, respectively. Most of samples contained more than one type of the mutation. Relative risk analysis indicated that the mutations in 12 sites of tissue HBx and nine sites of serum HBx were highly associated with HCC, suggesting a potential role of these mutants in carcinogenesis. An insert mutation at position 204: Insert 204AGGCCC, was always found to co-exist with point mutations at 260 (G-->A) and 264 (G/C/T-->A). Furthermore, this particular pattern of HBx mutation was most frequently detected. Immunochemical staining of HBx protein revealed that the nuclear localization of HBx protein in hepatocytes of tumor tissues was highly associated with this particular pattern of HBx mutation. In conclusion, HBx mutation occurs frequently in HCC samples tested and a sample usually has multiple types of mutations. A special pattern of insert at 204 and point mutations at 260 and 264 was identified, and it appears to be associated with the nuclear localization of HBx protein. The development of multiple types of mutations in a given sample may contribute to the process of multiple steps in hepatocarcinogenesis.