Akiko Sakai

Genome-Wide Analyses on Loss of Heterozygosity in Head and Neck Squamous Cell Carcinomas

Head and neck squamous cell carcinoma (HNSCC) is a frequent malignancy with a poor survival rate. Identifying the tumor suppressor gene (TSG) loci by genomic studies is an important step to uncover the molecular mechanisms involved in HNSCC pathogenesis. We therefore performed comprehensive analyses on loss of heterozygosity (LOH) using a genome-wide panel of 191 microsatellite markers in 22 HNSCC samples. We found 53 markers with significantly high LOH (>30%) on 21 chromosomal arms; the highest values of those were observed on 3p, 9p, 13q, 15q, and 17p, corresponding to D3S2432 (67%), D9S921-D9S925 (67%) and GATA62F03 (86%), D13S1493 (60%), D15S211 (62%), and D17S1353 (88%), respectively. Fifteen hot spots of LOH were defined in 13 chromosomal arms: 2q22-23, 4p15.2, 4q24-25, 5q31, 8p23, 9p23-24, 9q31.3, 9q34.2, 10q21, 11q21-22.3, 14q11-13, 14q22.3, 17p13, 18q11, and 19q12 as loci reported previously in HNSCCs. Furthermore, we identified five novel hot spots of LOH on three chromosomal arms in HNSCC at 2q33 (D2S1384), 2q37 (D2S125), 8q12-13 (D8S1136), 8q24 (D8S1128), and 15q21 (D15S211). In conclusion, our comprehensive allelotype analyses have unveiled and confirmed a total of 20 possible TSG loci that could be involved in the development of HNSCC. These results provide useful clues for identification of putative TSGs involved in HNSCC by fine mapping of the suspected regions and subsequent analysis for functional genes.

Identification of a candidate tumor suppressor gene RHOBTB1 located at a novel allelic loss region 10q21 in head and neck cancer

Purpose: Aims of the study are to narrow-down the hotspot region on 10q21 defined by previous genome-wide loss of heterozygosity (LOH) analysis in head and neck squamous cell carcinomas (HNSCC) and to define candidate tumor suppressor genes (TSG) concerned with 10q21. Materials and methods: LOH analysis was carried out with ten polymorphic microsatellite markers. Expression analysis was performed by semi-quantitative RT-PCR, and mutation analysis by PCR and direct sequencing. Results: LOH analysis on 10q21 in 52 HNSCC indicated distinctive and frequent allelic loss at D10S589 (42%). Among flanking genes, we found the RHOBTB1 gene as a candidate TSG, since an intragenic marker demonstrated the highest LOH (44%). Expression analysis revealed down-regulation of RHOBTB1 mRNA in 37% of tumors. Interestingly, all the five tumors that showed decreased expression of RHOBTB1 were accompanied with LOH, supporting the haploinsufficiency and class 2 TSG characteristics of RHOBTB1. No pathogenic mutation of RHOBTB1 was found. Furthermore, another gene within the region, EGR2, was also taken under scope. LOH frequencies around the EGR2 gene were relatively low (23 and 33%). Albeit semi-quantitative expression analysis of EGR2 demonstrated downregulation in 45% of tumor samples, no relation was found between the expression levels and LOH status. Conclusion: Frequent allelic loss and decreased expression of RHOBTB1 suggested that this gene has a role in tumorigenesis of a subset of HNSCC.

Genetic risk of hepatocellular carcinoma in patients with hepatitis C virus: A case control study

Backgroud and Aim:  Chronic hepatitis C virus (HCV) infection is a well known risk factor for hepatocellular carcinoma (HCC). The aim of this study is to elucidate the genetic risk of development and recurrence of HCC in patients with HCV.

Isolation and characterization of Escherichia coli hag operator mutants whose hag48 expression has become repressible by a Salmonella H1 repressor

SummaryThe expression of an Escherichia coli K12 flagellin gene, hagA48, is insensitive to the Salmonella H1 repressor (rh1−).By selecting merodiploid cells H2-rh1on-off/F′hag48 for motility in the presence of anti-H48 serum, mutants which had escaped from inhibition by the serum because of repression of their hag48 expression by rh1+ were isolated. Their nucleotide sequences were examined in the region containing the promoter, the position of which was confirmed by S1 nuclease analysis of the transcriptional initiation site. The two independently isolated mutants had the same heptamer insertion AGACGAT at a site overlapping with the promoter sequence, creating a putative operator sequence homologous to Salmonella H1, but not to H2. Other candidates for operator mutants had reduced flagellar synthesis because of mutations between the transcriptional and translational initiation sites or in the structural gene. The sequence analysis also revealed a repetitive extragenic palindrome (REP) consensus sequence and a transcriptional terminator of hag48 in a small, functionally unknown open reading frame (ORF).

Significant association between the Axin2 rs2240308 single nucleotide polymorphism and the incidence of prostate cancer

The Wnt signaling pathway plays a crucial role in human cancer development, and axis inhibition protein 2 (Axin2) is a master scaffold protein involved in Wnt signaling. Axin2 negatively regulates Wnt signaling and acts as a tumor suppressor protein. The present study evaluated the association between the Axin2 single nucleotide polymorphism (SNP) rs2240308 [guanine (G)/adenine (A)] and the incidence of prostate cancer. In total, 103 patients with prostate cancer and 100 cancer-free control males were included in this case-control study, and were genotyped using the genomic DNA extracted from peripheral blood samples. The results revealed a higher incidence of prostate cancer in the subjects with the homozygous GG genotype and a reduced cancer incidence in the patients with the GA genotype of the rs2240308 SNP (G/A) in the Axin2 gene. The adjusted odds ratio for carriers with the GA genotype was 0.377 (95% CI, 0.206–0.688; P=0.001) and that for the AA genotype was 0.830 (95% CI, 0.309–2.232; P=0.712) compared with the GG genotype. Therefore, the GA genotype was found to exhibit a protective effect that decreased the risk of prostate cancer. To the best of our knowledge, this is the first study to demonstrate the significant association between this SNP (rs2240308, G/A) and the risk of prostate cancer. This association indicates the possibility that the variations in the Axin2 gene in this position may play a significant role in promoting the development of cancer in the prostate. We believe that the Axin2 SNP (rs2240308) could be a useful biomarker for the predisposition and early diagnosis of the disease.

TP53 codon 72 polymorphism is associated with pancreatic cancer risk in males, smokers and drinkers

Tumor protein p53 (TP53) is the best-known tumor suppressor gene and plays a crucial role in carcinogenesis. The TP53 Arg 72 Pro polymorphism has been reported to be a risk factor for several types of cancer, but its association with pancreatic cancer has not been fully evaluated. Therefore, we investigated the effects of this polymorphism on pancreatic cancer in relation to smoking and drinking habits by examining the distribution of the SNP genotypes in 226 pancreatic cancer patients and 448 healthy controls. The frequencies of Arg/Arg, Arg/Pro and Pro/Pro were found to be 37, 49 and 15% in the pancreatic cancer cases and 44, 46 and 10% in the controls, respectively. Compared to the controls with the Arg/Arg genotype, cases with Pro/Pro homozygosity exhibited a significantly increased risk [adjusted odds ratio (OR)=1.70; 95% confidence interval (CI) 1.01-2.88]. In stratified studies, the association was particularly strong in males (OR=2.62; 95% CI 1.32-5.23), particularly in those smoking in excess of 20 pack-years and drinking in excess of 23 g ethanol/day (OR=5.02; 95% CI 1.12-22.51). We found that the TP53 Pro/Pro genotype compared to the Arg/Arg genotype had a profound effect on pancreatic cancer risk among males, particularly among heavy smokers and excessive alcohol drinkers.

A subtype of human papillomavirus 5 (HPV-5b) and its subgenomic segment amplified in a carcinoma: Nucleotide sequences and genomic organizations☆

A subtype of human papillomavirus 5 (HPV-5b) is closely associated with carcinomas in the disease epidermodysplasia verruciformis (EV). The complete genome was cloned from virus particles in benign lesions of a patient with EV and sequenced: it was 7779 nucleotides long and consisted of six open reading frames (ORFs) (E6, E7, E1, E2, E4, and E5) in the early region, three ORFs (L2, L3, and L1) in the late region, and a noncoding region, all existing on one DNA strand. The 40% segment of the HPV-5b genome specifically amplified in carcinomas was cloned from a primary carcinoma of the same EV patient and sequenced: it was 3143 nucleotides long and corresponded to a segment of the original HPV-5b genome containing the entire sequences of E6, E7, and the noncoding region and portions of E1 and L1. Compared to the whole genomic DNA, no mutations were detected in this probable malignancy-associated viral subgenomic segment cloned from carcinoma. These results suggest that amplification of the viral segment containing E6, E7, and the noncoding region may play a role in the malignant conversion of HPV-5b-infected benign lesions and that mutations in these genes or regions are not necessarily required.

Expression of an Escherichia coli flagellin gene, hag48, in the presence of a Salmonella H1-repressor.

SummaryAn Escherichia coli K12 flagellin gene, hag48, was found to be expressed in the presence of the Salmonella rh1 gene product. The strains which had hag48 on a chromosome or on an F′ factor were constructed from strains H2-e,n,xon-offrh1+ and fixed H2-e,n,xonrh1+ in which rh1+ is contranscribed with H2 in its “on” state. Motility of these strains in semisolid medium was inhibited by anti-H48 serum and motile clones (swarms) that escaped from it were hag mutants in case of the hag48 e,n,xon-off strain tested. H48 flagellin was detected by electrophoresis, though its amount was less than e,n,x flagellin, from all the strains that were nonmotile in the presence of anti-H48 serum.

The allelic distribution of a single nucleotide polymorphism in the PDCD5 gene locus of Japanese non-small cell lung cancer patients

It has been reported that the rs1862214 single nucleotide polymorphism (SNP) in the programmed cell death 5 gene (PDCD5) is associated with smoking-related lung cancer risk and prognosis in a European population with a history of smoking. The aim of this study was to investigate the status and impact of SNPs in the PDCD5 locus of a Japanese population. We developed an assay based on real-time PCR with melting curve analysis for determining the rs1862214 SNP, and examined this SNP in 165 lung cancer patients and 180 healthy volunteers. Of the 165 lung cancer patients (107 smokers), 25 (17), 72 (47) and 68 (43) had the CC, CG and GG genotypes of rs1862214, respectively. Of the 180 volunteers (117 smokers), 31 (24), 81 (52) and 68 (41) had the CC, CG and GG genotypes of rs1862214, respectively. No significant difference in allelic distribution was found between Japanese patients and healthy controls, even among smokers. Based on the published data, the distribution of this SNP appears to be significantly different in Japanese and European populations. No significant difference in prognosis according to the SNP was observed, either in patients with a history of smoking or in the total number of patients. This too differs from the results from a European population. In conclusion, we developed a convenient real-time PCR-based assay for the genotyping of rs1862214 in the PDCD5 locus. The distribution of the rs1862214 SNP in our Japanese population differs from its distribution in a European population, and is not related to the risk of cancer or to poor prognosis in lung cancer. This suggests the presence of an ethnicity-related difference in the role of PDCD5 in the pathogenesis of lung cancer.

Unique circulating microRNAs in relation to EGFR mutation status in Japanese smoker male with lung adenocarcinoma

The incidence of lung adenocarcinoma has been increasing recently in smokers. The molecular target therapy has been developed for lung adenocarcinoma patients harboring EGFR gene mutation. However, the treatment modalities for patients without mutation are currently limited. Thus, analysis of EGFR gene mutation status at early stage is important strategy to classify the patients for improving treatments and prognosis efficiently. This study aimed to identify microRNA (miRNA) signature in relation to mutation status in EGFR gene in early stage of lung adenocarcinoma male patients with smoking history. MiRNA profiles were assessed by microarray in paired plasma and tissue pooled from 10 EGFR wild type (EGFR-wt) and 10 EGFR mutated (EGFR-mut) patients. Expressions of selected miRNAs were verified further by real-time qRT-PCR in 83 plasma samples consisting of 55 EGFR-wt patients and 28 EGFR-mut patients and their correlation with clinicopathological parameters and EGFR gene mutation status were evaluated. We found that seven miRNAs (miR-16-5p, miR-23a-3p, miR-103a-3p, miR122-5p, miR-223-3p, miR-346 and miR-451a) were differentially expressed in stage I and stage I+II. Especially, miR-23a-3p was only miRNA shown higher expression in EGFR-wt patients than EGFR-mut patients. Thus, our findings could be useful non-invasive biomarkers to differentiate mutation status in EGFR gene in smoker lung adenocarcinoma male patients.