Ja-Lok Ku

Identification of Genes with Differential Expression in Acquired Drug-Resistant Gastric Cancer Cells Using High-Density Oligonucleotide Microarrays

Purpose: A major obstacle in chemotherapy is treatment failure due to anticancer drug resistance. The emergence of acquired resistance results from host factors and genetic or epigenetic changes in the cancer cells. The purpose of this study was to identify differentially expressed genes associated with acquisition of resistance in human gastric cancer cells. Experimental Design: We performed global gene expression analysis in the acquired drug-resistant gastric cancer cell lines to the commonly used drugs 5-fluorouracil, doxorubicin, and cisplatin using Affymetrix HG-U133A microarray. The gene expression patterns of 10 chemoresistant gastric cancer cell lines were compared with those of four parent cell lines using fold-change and Wilcoxon’s test for data analysis. Results: We identified over 250 genes differentially expressed in 5-fluorouracil-, cisplatin-, or doxorubicin-resistant gastric cancer cell lines. Our expression analysis also identified eight multidrug resistance candidate genes that were associated with resistance to two or more of the tested chemotherapeutic agents. Among these, midkine (MDK), a heparin-binding growth factor, was overexpressed in all drug-resistant cell lines, strongly suggesting that MDK might contribute to multidrug resistance in gastric cancer cells. Conclusions: Our investigation provides comprehensive gene information associated with acquired resistance to anticancer drugs in gastric cancer cells and a basis for additional functional studies.

Common variant in 6q26-q27 is associated with distal colon cancer in an Asian population

Background and aim Colorectal cancer (CRC) is a multifactorial disease with both environmental and genetic factors contributing to its development. The incidence of CRC is increasing year by year in Japan. Patients with CRC in advanced stages have a poor prognosis, but detection of CRC at earlier stages can improve clinical outcome. Therefore, identification of epidemiologial factors that influence development of CRC would facilitate the prevention or early detection of disease. Methods To identify loci associated with CRC risk, we performed a genome-wide association study (GWAS) for CRC and sub-analyses by tumour location using 1583 Japanese CRC cases and 1898 controls. Subsequently, we conducted replication analyses using a total of 4809 CRC cases and 2973 controls including 225 Korean subjects with distal colon cancer and 377 controls. Results We identified a novel locus on 6q26-q27 region (rs7758229 in SLC22A3, p=7.92×10−9, OR of 1.28) that was significantly associated with distal colon cancer. We also replicated the association between CRC and SNPs on 8q24 (rs6983267 and rs7837328, p=1.51×10−8 and 7.44×10−8, ORs of 1.18 and 1.17, respectively). Moreover, we found cumulative effects of three genetic factors (rs7758229, rs6983267, and rs4939827 in SMAD7) and one environmental factor (alcohol drinking) which appear to increase CRC risk approximately twofold. Conclusions We found a novel susceptible locus in SLC22A3 that contributes to the risk of distal colon cancer in an Asian population. These findings would further extend our understanding of the role of common genetic variants in the aetiology of CRC.

Germline mutations of E-cadherin gene in Korean familial gastric cancer patients.

AbstractGastric cancer is the most common cancer in Korea. Germline mutations of the E-cadherin gene have recently been identified in familial gastric cancer patients. We screened five Korean familial gastric cancer patients to investigate germline mutations of the E-cadherin gene. These patients fulfilled the following criteria: presence of at least two gastric cancer patients within first-degree relatives and one patient diagnosed before the age of 50 years. Abnormal band patterns were found in exons 6 and 10 in two familial gastric cancer patients by polymerase chain reaction-single strand conformation polymorphism analysis (probands from the SNU-G2 and SNU-G1001 families, respectively). DNA sequencing analysis of the E-cadherin gene of these two patients revealed missense mutations in each exon. The SNU-G2 proband harbored a missense mutation from aspartic acid (GAT) to glycine (GGT) at codon 244 in exon 6 of the E-cadherin gene, and the SNU-G1001 proband had a missense mutation from valine (GTG) to alanine (GCG) at codon 487 in exon 10. The SNU-G2 proband was diagnosed with gastric cancer at the age of 38; three brothers and two sisters had died of gastric cancer under the age of 50, and their mother had died of gastric cancer at the age of 63. The SNU-G1001 proband was diagnosed with gastric cancer at the age of 42 and one brother had died of gastric cancer at the age of 49. In summary, we found germline mutations of the E-cadherin gene in two of five Korean familial gastric cancer patients screened.

Promoter hypermethylation downregulates RUNX3 gene expression in colorectal cancer cell lines.

It was recently reported that RUNX3 gene expression is significantly downregulated in human gastric cancer cells due to hypermethylation of its promoter region or hemizygous deletion (Cell, 109, 2002). To verify the genetic alterations and methylation status of the RUNX3 gene in colorectal carcinogenesis, we analysed for mutations, loss of heterozygosity (LOH), and RUNX3 gene promoter hypermethylation, in 32 colorectal cancer cell lines. RT–PCR analysis showed undetectable or low RUNX3 expression in 16 cell lines, and no mutations were found in the RUNX3 gene by PCR-SSCP analysis. Of these 16 cell lines, hypermethylation of the RUNX3 promoter was confirmed in 12. The following observations were made: (i) RUNX3 was re-expressed after 5-aza-2′-deoxycytidine treatment, (ii) the RUNX3 promoter was found to be methylated by MS-PCR, and (iii) hypermethylation of the RUNX3 promoter was confirmed by direct sequencing analysis after sodium bisulfite modification in the above 12 cell lines. RUNX3 was neither methylated nor expressed in four cell lines. Of these four, microsatellite instability (MSI) at the RUNX3 locus was found in three, SNU-61 (D1S246), SNU-769A, and SNU-769B (D1S199). This study suggests that transcriptional repression of RUNX3 is caused by promoter hypermethylation of the RUNX3 CpG island in colorectal cancer cell lines, and the results of these experiments may contribute to an understanding of the role of RUNX3 inactivation in the pathogenesis of colorectal cancers.

Heterogeneity of Genetic Changes Associated with Acquired Crizotinib Resistance in ALK-Rearranged Lung Cancer

Background: Anaplastic lymphoma kinase (ALK)-rearranged non–small-cell lung cancer (NSCLC) is markedly sensitive to the ALK inhibitor crizotinib. However, acquired resistance to crizotinib is inevitable through several mechanisms. Therefore, this study was conducted to identify genetic alterations associated with crizotinib resistance. Methods: Tumor samples were derived from seven ALK-positive NSCLC patients who showed acquired resistance to crizotinib, and these patients were analyzed for ALK, EGFR, and KRAS mutations and ALK and EGFR gene amplifications. In vitro cytotoxicity of crizotinib and ALK downstream signals were compared between crizotinib-naive and -resistant NSCLC cells. Results: After a median duration of 6 months (range, 4–12 months), seven ALK-positive NSCLC patients developed acquired resistance to crizotinib. Three patients harbored secondary ALK mutations, including one patient with both mutations: L1196M (n = 2) and G1269A (n = 2). Of note, one patient displayed ALK gene copy number gain (4.1-fold increase compared with the pre-crizotinib specimen) and EGFR L858R mutation with high polysomy. The amphiregulin concentration was high in the supernatant fluid from five patients with malignant pleural effusion (116.4–18934.0 pg/ml). SNU-2535 cells derived from a patient who harbored the G1269 mutation were resistant to crizotinib treatment similar to H3122 CR1 cells. L1196M and G1269A mutant clones were less sensitive to crizotinib and ALK downstream signals were ineffectively suppressed in these clones. Conclusions: Genetic changes associated with crizotinib resistance are heterogeneous in ALK-rearranged NSCLC patients who respond to crizotinib and subsequently develop resistance.

Decreased pyruvate kinase M2 activity linked to cisplatin resistance in human gastric carcinoma cell lines

Resistance to anticancer drugs is a major obstacle preventing effective treatment of disseminated cancers. Understanding the molecular basis to chemoresistance is likely to provide better treatment. Cell lines resistant to cisplatin or 5‐fluorouracil (5‐FU) were established from human gastric carcinoma cell lines SNU‐638 and SNU‐620. Comparative proteomics involving 2‐dimensional gel electrophoresis (2‐DE) and matrix‐associated laser desorption ionization‐mass spectroscopy (MALDI‐MS) was performed on protein extracts from these parental and drug‐resistant derivative lines to screen drug resistance‐related proteins. Pyruvate kinase M2 (PK‐M2) was identified as a protein showing lower expression in cisplatin‐resistant cells compared to parental cells. Consistent with this finding, PK‐M2 activity was also lower in cisplatin‐resistant cells. Suppression of PK‐M2 expression by antisense oligonucleotide resulted in acquired cisplatin resistance in SNU‐638 cells. Furthermore, PK‐M2 activity in 11 individual human gastric carcinoma cell lines positively correlated with cisplatin sensitivity. Taken together, PK‐M2 protein and activity levels were lower in cisplatin‐resistant human gastric carcinoma cell lines compared to their parental cell lines. Furthermore, suppression of PK‐M2 expression using antisense oligonucleotides increased cisplatin resistance. These data clearly link PK‐M2 and cisplatin resistance mechanisms.

Mutational analysis of BRAF and K-ras in gastric cancers: absence of BRAF mutations in gastric cancers

Recently, BRAF mutations were found in a variety of human cancers. Interestingly, the most common of BRAF mutation (V599E) has not been identified in tumors with K-ras mutations. Whereas the majority of human cancer types has been screened for BRAF mutations, no detailed studies on gastric cancers have been investigated. Thus, we decided to investigate the incidence of BRAF mutations in gastric cancers, and the relationship between BRAF and K-ras mutations in such cancers. Three non-pathogenic BRAF polymorphisms and seven K-ras missense mutations were found in 66 gastric cancers and 16 gastric cancer cell lines. Although only 9% of our gastric cancer panels had K-ras mutations, the incidence of BRAF mutations was not high. Thus, BRAF mutations, which are present in a variety of other human cancers, do not seem to be involved in gastric cancer development.

Prognostic significance of microsatellite instability in sporadic colorectal cancer

Background and aimsColorectal cancers exhibiting microsatellite instability (MSI) appear to have unique biological behavior. The influence of MSI on the prognosis of sporadic colorectal cancers is controversial and requires further investigation. The aim of this study was to analyze the association between MSI status and clinicopathological features and prognosis in sporadic colorectal cancer patients.Patients and methodsOf the 322 consecutive colorectal cancer patients operated upon at the Seoul National University Hospital between January and December 1998, we examined the clinicopathological features and prognosis of 248 patients with sporadic primary colorectal cancer. The MSI status of these 248 patients has been reported in a previous study. Of the 248 patients, 23 (9.3%) had MSI+ tumors. The patients’ clinicopathological parameters were obtained from their medical records, and follow-up and survival data were obtained from medical records and phone calls.ResultsMSI+ sporadic colorectal cancers were found predominantly in the proximal colon (p<0.001) and were associated with poor differentiation (p=0.030), a lower preoperative serum carcinoembryonic antigen (CEA) level (p=0.012), and less frequent systemic metastasis (p=0.034) than MSI− tumors. Low tumor grade (p=0.022), low tumor T-stage (p=0.002), no lymph node metastasis (p<0.001), no systemic metastasis (p<0.001), adjuvant chemotherapy (p<0.001) and MSI+ status (p=0.038) were independent favorable prognostic factors for survival in sporadic colorectal cancer patients.ConclusionMSI status was an independent favorable prognostic factor for survival in sporadic primary colorectal cancer patients.

Role of reactive oxygen species in the induction of apoptosis by α-tocopheryl succinate

α‐Tocopheryl succinate (TOS), a vitamin E analog, is a promising anticancer agent due to its abilities to inhibit proliferation and to induce apoptosis in a variety of human malignant cell lines, while being relatively less active toward normal cells. However, the molecular mechanisms underlying the apoptotic effects of TOS are not precisely understood. Reports that TOS can generate reactive oxygen species (ROS) prompted us to investigate the role of ROS in TOS‐induced apoptosis in cancer cells. We found that the human lung cancer A549 and H460 cell lines were much more sensitive to TOS‐induced apoptosis than the human glioblastoma T98G and U87MG cell lines. Our data suggested that the differential TOS sensitivity was not caused by differences in the uptake and retention of TOS between TOS‐sensitive and –resistant cancer cells. The differential ability of cancer cells to generate ROS in response to TOS appears to be an important factor in determining the susceptibility of cells to TOS‐induced apoptosis. Our results further suggest that TOS‐induced generation of ROS is involved in caspase‐independent apoptosis. Taken together, our findings suggest an important role of ROS generation in TOS‐induced, caspase‐independent apoptosis of cancer cells.

Establishment and characterisation of six human biliary tract cancer cell lines.

Human cell lines established from biliary tract cancers are rare, and only five have been reported previously. We report the characterisation of six new six biliary tract cancer cell lines (designated SNU-245, SNU-308, SNU-478, SNU-869, SNU-1079 and SNU-1196) established from primary tumour samples of Korean patients. The cell lines were isolated from two extrahepatic bile duct cancers (one adenocarcinoma of common bile duct, one hilar bile duct cancer), two adenocarcinomas of ampulla of Vater, one intrahepatic bile duct cancer (cholangiocarcinoma), and one adenocarcinoma of the gall bladder. The cell phenotypes, including the histopathology of the primary tumours and in vitro growth characteristics, were determined. We also performed molecular characterisation, including DNA fingerprinting analysis and abnormalities of K-ras, p15, p16, p53, hMLH1, hMSH2, DPC4, β-catenin, E-cadherin, hOGG1, STK11, and TGF-βRII genes by PCR–SSCP and sequencing analysis. In addition, we compared the genetic alterations in tumour cell lines and their corresponding tumour tissues. All lines grew as adherent cells. Population doubling times varied from 48–72 h. The culture success rate was 20% (six out of 30 attempts). All cell lines showed (i) relatively high viability; (ii) absence of mycoplasma or bacteria contamination; and (iii) genetic heterogeneity by DNA fingerprinting analysis. Among the lines, three lines had p53 mutations; and homozygous deletions in both p16 and p15 genes were found three and three lines, respectively; one line had a heterozygous missense mutation in hMLH1; E-cadherin gene was hypermethylated in two lines. Since the establishment of biliary tract cancer cell lines has been rarely reported in the literature, these newly established and well characterised biliary tract cancer cell lines would be very useful for studying the biology of biliary tract cancers, particularly those related to hypermethylation of E-cadherin gene in biliary tract cancer.