MHC class I polypeptide-related chain A (MICA) molecule is induced in response to viral infection and various types of stress. We recently reported that a single nucleotide polymorphism (SNP) rs2596542 located in the MICA promoter region was significantly associated with the risk for hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) and also with serum levels of soluble MICA (sMICA). In this study, we focused on the possible involvement of MICA in liver carcinogenesis related to hepatitis B virus (HBV) infection and examined correlation between the MICA polymorphism and the serum sMICA levels in HBV-induced HCC patients. The genetic association analysis revealed a nominal association with an SNP rs2596542; a G allele was considered to increase the risk of HBV-induced HCC (P = 0.029 with odds ratio of 1.19). We also found a significant elevation of sMICA in HBV-induced HCC cases. Moreover, a G allele of SNP rs2596542 was significantly associated with increased sMICA levels (P = 0.009). Interestingly, HCC patients with the high serum level of sMICA (>5 pg/ml) exhibited poorer prognosis than those with the low serum level of sMICA (≤5 pg/ml) (P = 0.008). Thus, our results highlight the importance of MICA genetic variations and the significance of sMICA as a predictive biomarker for HBV-induced HCC.

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Accumulation of iron in tissues increases the risk of cancer, but iron regulatory mechanisms in cancer tissues are largely unknown. Here, we report that p53 regulates iron metabolism through the transcriptional regulation of ISCU (iron-sulfur cluster assembly enzyme), which encodes a scaffold protein that plays a critical role in Fe-S cluster biogenesis. p53 activation induced ISCU expression through binding to an intronic p53-binding site. Knockdown of ISCU enhanced the binding of iron regulatory protein 1 (IRP1), a cytosolic Fe-S protein, to an iron-responsive element in the 5′ UTR of ferritin heavy polypeptide 1 (FTH1) mRNA and subsequently reduced the translation of FTH1, a major iron storage protein. In addition, in response to DNA damage, p53 induced FTH1 and suppressed transferrin receptor, which regulates iron entry into cells. HCT116 p53+/+ cells were resistant to iron accumulation, but HCT116 p53−/− cells accumulated intracellular iron after DNA damage. Moreover, excess dietary iron caused significant elevation of serum iron levels in p53−/− mice. ISCU expression was decreased in the majority of human liver cancer tissues, and its reduced expression was significantly associated with p53 mutation. Our finding revealed a novel role of the p53-ISCU pathway in the maintenance of iron homeostasis in hepatocellular carcinogenesis.

ADAMTS metalloprotease family member ADAMTS9 maps to 3p14.2 and shows significant associations with the aerodigestive tract cancers esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). However, the functional impact of ADAMTS9 on cancer development has not been explored. In this study, we evaluated the hypothesized antiangiogenic and tumor-suppressive functions of ADAMTS9 in ESCC and NPC, in stringent tumorigenicity and Matrigel plug angiogenesis assays. ADAMTS9 activation suppressed tumor formation in nude mice. Conversely, knockdown of ADAMTS9 resulted in clones reverting to the tumorigenic phenotype of parental cells. In vivo angiogenesis assays revealed a reduction in microvessel numbers in gel plugs injected with tumor-suppressive cell transfectants. Similarly, conditioned medium from cell transfectants dramatically reduced the tube-forming capacity of human umbilical vein endothelial cells. These activities were associated with a reduction in expression levels of the proangiogenic factors MMP9 and VEGFA, which were consistently reduced in ADAMTS9 transfectants derived from both cancers. Taken together, our results indicate that ADAMTS9 contributes an important function in the tumor microenvironment that acts to inhibit angiogenesis and tumor growth in both ESCC and NPC. Cancer Res; 70(13); 5567-76. ©2010 AACR.

Hepatitis C virus (HCV) infection is the major cause of hepatocellular carcinoma (HCC) in Japan. We previously identified the association of SNP rs2596542 in the 5' flanking region of the MHC class I polypeptide-related sequence A (MICA) gene with the risk of HCV-induced HCC. In the current study, we performed detailed functional analysis of 12 candidate SNPs in the promoter region and found that a SNP rs2596538 located at 2.8 kb upstream of the MICA gene affected the binding of a nuclear protein(s) to the genomic segment including this SNP. By electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay, we identified that transcription factor Specificity Protein 1 (SP1) can bind to the protective G allele, but not to the risk A allele. In addition, reporter construct containing the G allele was found to exhibit higher transcriptional activity than that containing the A allele. Moreover, SNP rs2596538 showed stronger association with HCV-induced HCC (P = 1.82×10−5 and OR = 1.34) than the previously identified SNP rs2596542. We also found significantly higher serum level of soluble MICA (sMICA) in HCV-induced HCC patients carrying the G allele than those carrying the A allele (P = 0.00616). In summary, we have identified a functional SNP that is associated with the expression of MICA and the risk for HCV-induced HCC.

The key genes involved in the development of esophageal squamous cell carcinoma (ESCC) remain to be elucidated. Previous studies indicate extensive genomic alterations occur on chromosome 9 in ESCC. Using a monochromosome transfer approach, this study provides functional evidence and narrows down the critical region (CR) responsible for chromosome 9 tumor suppressing activity to a 2.4 Mb region mapping to 9q33-q34 between markers D9S1798 and D9S61. Interestingly, a high prevalence of allelic loss in this CR is also observed in primary ESCC tumors by microsatellite typing. Allelic loss is found in 30/ 34 (88%) tumors and the loss of heterozygosity (LOH) frequency ranges from 67 to 86%. Absent to low expression of a 9q32 candidate tumor suppressor gene (TSG), DEC1 (deleted in esophageal cancer 1), is detected in four Asian ESCC cell lines. Stably expressing DEC1 transfectants provide functional evidence for inhibition of tumor growth in nude mice and DEC1 expression is decreased in tumor segregants arising after long-term selection in vivo. There is 74% LOH in the DEC1 region of ESCC primary tumors. This study provides the first functional evidence for the presence of critical tumor suppressive regions on 9q33-q34. DEC1 is a candidate TSG that may be involved in ESCC development.

By using a functional complementation approach, suppression of tumorigenicity was observed after transfer of intact or truncated copies of chromosome 3 into a nasopharyngeal carcinoma (NPC) HONE1 cell line. The extra exogenous chromosome 3 in the microcell hybrids (MCHs) significantly extended the lag period of tumor formation, which may be associated with loss or inactivation of wild type alleles from the normal donor chromosome 3. Representative tumors, which grew in nude mice were reconstituted into culture and expanded as tumor segregants (TSs). In our study, a disintegrin-like and metalloprotease with thrombospondin type 1 motif 9 (ADAMTS9), a gene mapping to 3p14.2, was identified to be critically associated with tumor suppression in NPC. Gene expression analysis showed that ADAMTS9 was either not expressed or was downregulated in HONE1 cells, TSs and NPC cell lines. The mechanism of ADAMTS9 gene inactivation in the NPC cell lines and tissues was attributed to promoter hypermethylation. Using a tissue microarray and immunohistochemical staining, 31 of 66 (47%) of the NPC cases showed downregulated or absence of ADAMTS9 expression. ADAMTS9 expression was downregulated or lost in 17 of 23 (73.9%) lymph node metastatic NPC specimens, which was significantly higher than in 14 of 43 (32.6%) primary tumors. After transfection of the ADAMTS9 gene into 7 NPC cell lines, a dramatic reduction of colony forming ability was observed. These findings support ADAMTS9 as a putative tumor suppressor gene in vivo in NPC that is significantly associated with lymph node metastases.

ASS1 bridges a gap between p53 and Akt.