SAA1 polymorphisms are associated with variation in antiangiogenic and tumor-suppressive activities in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a cancer that occurs in high frequency in Southern China. A previous functional complementation approach and the subsequent cDNA microarray analysis have identified that serum amyloid A1 (SAA1) is an NPC candidate tumor suppressor gene. SAA1 belongs to a family of acute-phase proteins that are encoded by five polymorphic coding alleles. The SAA1 genotyping results showed that only three SAA1 isoforms (SAA1.1, 1.3 and 1.5) were observed in both Hong Kong NPC patients and healthy individuals. This study aims to determine the functional role of SAA1 polymorphisms in tumor progression and to investigate the relationship between SAA1 polymorphisms and NPC risk. Indeed, we have shown that restoration of SAA1.1 and 1.3 in the SAA1-deficient NPC cell lines could suppress tumor formation and angiogenesis in vitro and in vivo. The secreted SAA1.1 and SAA1.3 proteins can block cell adhesion and induce apoptosis in the vascular endothelial cells. In contrast, the SAA1.5 cannot induce apoptosis or inhibit angiogenesis because of its weaker binding affinity to αVß3 integrin. This can explain why SAA1.5 has no tumor-suppressive effects. Furthermore, the NPC tumors with this particular SAA1.5/1.5 genotype showed higher levels of SAA1 gene expression, and SAA1.1 and 1.3 alleles were preferentially inactivated in tumor tissues that were examined. These findings further strengthen the conclusion for the defective function of SAA1.5 in suppression of tumor formation and angiogenesis. Interestingly, the frequency of the SAA1.5/1.5 genotype in NPC patients was ~2-fold higher than in the healthy individuals (P=0.00128, odds ratio=2.28), which indicates that this SAA1 genotype is significantly associated with a higher NPC risk. Collectively, this homozygous SAA1.5/1.5 genotype appears to be a recessive susceptibility gene, which has lost the antiangiogenic function, whereas SAA1.1 and SAA1.3 are the dominant alleles of the tumor suppressor phenotype.

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.

Abrogated expression of DEC1 during oesophageal squamous cell carcinoma progression is age- and family history-related and significantly associated with lymph node metastasis.

BACKGROUND: Oesophageal squamous cell carcinoma (SCC) causes the highest number of cancer deaths in some regions of Northern China. Previously, we narrowed down a critical region at 9q33-34, identified to be associated with tumour-suppressive function of deleted in oesophageal cancer 1 (DEC1) in oesophageal SCC. METHODS: We generated DEC1 antibody and constructed tissue microarrays (TMAs) utilising tissue specimens from Henan, a high-risk region for oesophageal SCC, to investigate the importance of DEC1 expression in this cancer. RESULTS: Tissue microarray immunohistochemical staining reveals significant loss of DEC1 from hyperplasia, to tumour, and to lymph node metastasis. In addition, the loss of DEC1 in tumour is age-dependent. Interestingly, there is significant abrogation of DEC1 expression in patients with a family history of oesophageal SCC. Deleted in oesophageal cancer 1 localises to both the cytoplasm and nucleus. The vesicular pattern of DEC1 in the cytoplasm appears to localise at the Golgi and Golgi-endoplasmic reticulum intermediate compartment. CONCLUSION: This is the first TMA study to suggest a clinical association of DEC1 in lymph node metastatic oesophageal SCC, early onset oesophageal SCC and familial oesophageal SCC development. Subcellular localisation of DEC1 and its expression in oesophageal SCC tissues provide important insight for further deciphering the molecular mechanism of DEC1 in oesophageal SCC development.

Identification of a tumor suppressive critical region mapping to 3p14.2 in esophageal squamous cell carcinoma and studies of a candidate tumor suppressor gene, ADAMTS9.

A gene critical to esophageal cancer has been identified. Functional studies using microcell-mediated chromosome transfer of intact and truncated donor chromosomes 3 into an esophageal cancer cell line and nude mouse tumorigenicity assays were used to identify a 1.61 Mb tumor suppressive critical region (CR) mapping to chromosome 3p14.2. This CR is bounded by D3S1600 and D3S1285 microsatellite markers. One candidate tumor suppressor gene, ADAMTS9, maps to this CR. Further studies showed normal expression levels of this gene in tumor-suppressed microcell hybrids, levels that were much higher than observed in the recipient cells. Complete loss or downregulation of ADAMTS9 gene expression was found in 15 out of 16 esophageal carcinoma cell lines. Promoter hypermethylation was detected in the cell lines that do not express this gene. Re-expression of ADAMTS9 was observed after demethylation drug treatment, confirming that hypermethylation is involved in gene downregulation. Downregulation of ADAMTS9 was also found in 43.5 and 47.6% of primary esophageal tumor tissues from Hong Kong and from the high-risk region of Henan, respectively. Thus, this study identifies and provides functional evidence for a CR associated with tumor suppression on 3p14.2 and provides the first evidence that ADAMTS9, mapping to this region, may contribute to esophageal cancer development.

Functional categories of TP53 mutation in colorectal cancer: results of an International Collaborative Study.

BACKGROUND: Loss of TP53 function through gene mutation is a critical event in the development and progression of many tumour types including colorectal cancer (CRC). In vitro studies have found considerable heterogeneity amongst different TP53 mutants in terms of their transactivating abilities. The aim of this work was to evaluate whether TP53 mutations classified as functionally inactive (< or=20% of wildtype transactivation ability) had different prognostic and predictive values in CRC compared with mutations that retained significant activity. MATERIALS AND METHODS: TP53 mutations within a large, international database of CRC (n = 3583) were classified according to functional status for transactivation. RESULTS: Inactive TP53 mutations were found in 29% of all CRCs and were more frequent in rectal (32%) than proximal colon (22%) tumours (P < 0.001). Higher frequencies of inactive TP53 mutations were also seen in advanced stage tumours (P = 0.0003) and in tumours with the poor prognostic features of vascular (P = 0.006) and lymphatic invasion (P = 0.002). Inactive TP53 mutations were associated with significantly worse outcome only in patients with Dukes' stage D tumours (RR = 1.71, 95%CI 1.25-2.33, P < 0.001). Patients with Dukes' C stage tumours appeared to gain a survival benefit from 5-fluorouracil-based chemotherapy regardless of TP53 functional status for transactivation ability. CONCLUSIONS: Mutations that inactivate the transactivational ability of TP53 are more frequent in advanced CRC and are associated with worse prognosis in this stage of disease.

Frequent loss of heterozygosity on multiple chromosomes in Chinese esophageal squamous cell carcinomas.

Analysis of the loss of heterozygosity (LOH) detected by polymerase chain reaction techniques using 18 polymorphic markers localized to chromosomes 3p, 5, 17, and 18q in 40 Hong Kong Chinese esophageal squamous cell carcinoma (ESC) patients showed that multiple alterations on several chromosomes are involved in ESC development. The LOH rates detected for markers on chromosome 3 ranged from 44.0 to 85.7%, for chromosome 5 from 40.9 to 61.9%, for chromosome 17 from 40.0 to 100%, and for chromosome 18 from 38.9 to 58.3%. No significant association was observed between LOH and the clinical and histopathological parameters.

Genomic instability and alterations in Apc, Mcc and Dcc in Hong Kong patients with colorectal carcinoma.

Our aim was to reveal the significance of tumor-suppressor genes and genomic instability in 99 Hong Kong Chinese colorectal carcinoma (CRC) patients by PCR-LOH analysis and PCR-PTT assay. The frequencies of allelic loss of Apc, Mcc and Dcc and of APC truncation were 31.3% (15/48), 11.6% (5/43), 44.4% (20/45) and 46/93 (49.5%), respectively. The frequency of Apc LOH was similar to, the Mcc LOH was lower than, and the Dcc LOH was higher than that reported for Caucasians and Japanese. In Hong Kong CRC patients, the replication error-positive (RER(+)) phenotype occurred with a frequency of 10% (10/99), which was similar to other results using microsatellite markers where RER(+) frequencies ranged from 11% to 28%. The rates of genetic alteration in RER(+) tumors were lower in tumors harboring p53, Mcc and Dcc alterations; similar in Apc; and higher in Ki-ras tumors compared with RER(-) tumors, though these differences did not achieve statistical significance. None of the biomarkers examined were predictive of survival independently, but strong trends confirming earlier observations of associations between RER(+) phenotypes with proximal tumor location and poorly differentiated tumor status were noted. The RER(+) phenotype was correlated significantly to the less aggressive Duke's stage B and improved prognosis. Additionally, tumors with RER(+) phenotypes were positively correlated with young age and sex. Our results support the observation that a subset of younger male CRC patients in Hong Kong may develop CRC via the RER pathway and show differences in RER status and sex. A significantly higher percentage of older Hong Kong Chinese CRC patients had APC truncations. Int. J. Cancer (Pred. Oncol.) 84:404-409, 1999.

L-myc genotypes in Hong Kong Chinese colorectal carcinoma patients.

L-myc genotypes have been correlated with prognosis in different human tumors. Its role in colorectal carcinoma (CRC) is still unclear. This study aimed to assess the L-myc genotypes in 99 Hong Kong Chinese CRC patients by PCR-RFLP techniques. The results obtained were correlated with clinical, histological and pathological parameters and genetic alterations. The observed frequency of L-myc genotypes (LL:LS:SS) was 27:46:26. The ratio of S to L alleles was 0.51:0.49. Distribution of L-myc genotypes and alleles in Hong Kong Chinese CRC was similar to that of healthy Chinese and CRC patients of other ethnic origins. The homozygous SS genotype was significantly associated with Dukes' stages C versus B. Other parameters including sex, differentiation status and survival, and genetic alterations such as p53 and Ki-ras mutations and Dcc LOH had no significant association with L-myc SS genotype.

Ki-ras codon 12 point mutational activation in Hong Kong colorectal carcinoma patients.

This study investigated the frequency and importance of Ki-ras codon 12 mutations in 99 Hong Kong Chinese colorectal carcinoma specimens by allele-specific oligonucleotide hybridization. The frequency of mutations detected was 30% and the most common mutation observed resulted in aspartic acid substitutions. Previous studies showed that specific Ki-ras mutations have been significantly associated with prognosis. Ki-ras codon 12 point mutational activation in CRC was significantly associated with the differentiation status of tumors in this study. Ethnic differences in the patterns of Ki-ras codon 12 point mutations were observed.