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Peritoneal metastasis is one of the major patterns of unresectability in colorectal cancer (CRC) and a cause of death in advanced CRC. Identification of distinct gene expressions between primary CRC and peritoneal seeding metastasis is to predict the metastatic potential of primary human CRC. Three pairs of primary CRC (SNU-2335A, SNU-2404A, and SNU-2414A) and corresponding peritoneal seeding (SNU-2335D, SNU-2404B, and SNU-2414B) cell lines were established to determine the different gene expressions and resulting aberrated signaling pathways in peritoneal metastasis tumor using whole exome sequencing and microarray. Whole exome sequencing detected that mutation in CYP2A7 was exclusively shared in peritoneal seeding cell lines. Microarray identified that there were five upregulated genes (CNN3, SORBS1, BST2, EPSTI1, and KLHL5) and two downregulated genes (TRY6 and STYL5) in the peritoneal metastatic cell lines. CNN3 expression was highly augmented in both mRNA and protein levels in peritoneal metastasis cells. Knockdown of Calponin 3 resulted in augmented level of E-cadherin in peritoneal metastasis cells, and migration and invasiveness decreased accordingly. We suggest that CNN3 takes part in cell projection and movement, and the detection and distribution of CNN3 may render prognostic information for predicting peritoneal seeding metastasis from primary colorectal cancer.

Table S1. Primers that were used for RT-PCR. Table S2. STR Profile of radio-resistant rectal cancer cell lines and their parental cell lines. (DOCX 27 kb)

Clinical detection of ovarian clear cell carcinomas is important because of the poor prognosis. To identify microRNA profiles specific for clear cell carcinomas, microRNA expression profiles were compared between clear cell carcinomas and serous carcinomas of the ovary using microRNA microarray. In parallel, clear cell carcinomas were compared with germ cell tumors of the ovary. Six microRNAs differentially expressed between ovarian clear cell and serous carcinomas distinguished uterine clear cell carcinomas from endometrioid carcinomas. MiR-449 was underexpressed in both ovarian and uterine clear cell carcinomas. When germ cell tumors were compared with clear cell carcinomas of the ovary, miR-302d was the most significantly overexpressed microRNA in germ cell tumors. Thus, here we describe microRNA profiles characteristic for clear cell carcinomas of the ovary and uterus.

To elucidate the molecular basis of early gastric cancer (EGC), the genome-wide expression pattern of cancer and normal tissues from 27 patients were analyzed by a microarray-based method. Using an integrative systematic bioinformatics approach, we classified the differentially expressed genes in EGC. Interestingly, the more highly expressed genes in EGC exhibited the most significant correlation with cell migration and metastasis. This implies that, even at the early stage of gastric cancer, the molecular properties usually observed in late-stage cancer are already present. Furthermore, we have found a novel association between the expression pattern and molecular pathways of EGC and estrogen receptor α (ERα)-negative breast cancer through cross-experimental analysis. These results provide new insights into the biological properties of EGC, as well as yielding useful basic data for the study of molecular mechanisms of EGC carcinogenesis.

Objectives: Many efforts have shown multi-oncologic roles of galectin-3 for cell proliferation, angiogenesis, and apoptosis. However, the mechanisms by which galectin-3 is involved in cell proliferation are not yet fully understood, especially in human colon cancer cells. Methods: To cluster genes showing positively or negatively correlated expression with galectin-3, we employed human colon cancer cell lines, SNU-61, SNU-81, SNU-769B, SNU-C4 and SNU-C5 in high-throughput gene expression profiling. Gene and protein expression levels were determined by using real-time quantitative polymerase chain reaction (PCR) and western blot analysis, respectively. The proliferation rate of human colon cancer cells was measured by using a 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Results: Expression of γ-aminobutyric acid B receptor 1 (GABABR1) showed a positive correlation with galectin-3 at both the transcriptional and the translational levels. Downregulation of galectin-3 decreased not only GABABR1 expression but also the proliferation rate of human colon cancer cells. However, Korean herbal extract, HangAmDan-B (HAD-B), decreased expression of GABABR1 without any expressional change of galectin-3, and offset γ-aminobutyric acid (GABA)-enhanced human colon cancer cell proliferation. Conclusions: Our present study confirmed that GABABR1 expression was regulated by galectin-3. HAD-B induced galectin-3-independent down-regulation of GABABR1, which resulted in a decreased proliferation of human colon cancer cells. The therapeutic effect of HAD-B for the treatment of human colon cancer needs to be further validated.

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.

Promoter hypermethylation of the ADAM23 gene, which is normally involved in cell-to-cell and cell-to matrix adhesion, has been reported in pancreatic, breast and brain cancer, and recently the role of this gene was examined in gastric cancer. In this study, we analyzed ADAM23 expression in colorectal cancer cell lines and examined its methylation by methylation-specific PCR (MSP) and bisulfate-modified DNA sequencing analysis. Methylated cells were treated with 5-aza-2'-deoxycytidine to restore the ADAM23 expression. We then examined ADAM23 methylation status in colorectal cancer tissues and their corresponding normal tissues. We found that ADAM23 was aberrantly silenced or expressed at very low levels in 28 of the 32 (88%) colorectal cancer cell lines. MSP analysis showed that ADAM23 was methylated in 29 of 32 (91%) colorectal cancer cell lines and attenuated expression of ADAM23 was found to be related to hypermethylation in its promoter region. Moreover, the CpG dinucleotide methylation threshold of 70-90% was found to be required for complete silencing. In addition, when some cell lines without ADAM23 expression were treated with 5-aza-2'-deoxycytidine, ADAM23 was reexpressed. In colorectal cancer tissues, the promoter region of ADAM23 was hypermethylated in 36 of 76 (47%). These results demonstrated that ADAM23 may be down-regulated by aberrant promoter hypermethylation during the progression of colorectal cancer.

Comparative analysis of proteomes using 5-fluorouracil (5-FU)-resistant human colon cancer cell line revealed that decreased galectin-3 expression was significantly associated with retarded proliferation. However, in the presence of 5-FU proliferation rate of cells with suppressed galectin-3 expression did not differ from that of cells with normal galectin-3 expression, even galectin-3 suppression augmented apoptosis. Mechanism by which galectin-3 regulates cancer cell proliferation has been identified in immunoprecipitates of the anti-galectin-3 antibody. Heterogeneous nuclear ribonucleoprotein Q (hnRNP Q) was identified as a protein interacting with galectin-3. Interestingly, while galectin-3 protein was not affected by the hnRNP Q level, its suppression was accompanied by a decrease in hnRNP Q expression. The present study demonstrates that galectin-3 stabilizes hnRNP Q via complex formation, and reduction in the hnRNP Q level leads to slow proliferation and less susceptibility to 5-FU.

The adenomatous polyposis coli (APC), which is the susceptible gene for familial adenomatous polyposis (FAP) and sporadic colorectal cancer, spans 15 exons. The open reading frame of APC is 8529 bp, which encodes 2843 amino acids. Conventional genetic screening involves extensive time as well as high cost and labor. Thus, we developed a novel APC ready-to-use plate for high-throughput mutational analysis by denaturing high performance liquid chromatography (DHPLC). To prepare the ready-to-use APC plate, all 38 primer pairs and PCR mixtures were aliquoted into individual wells of a 96-well plate, and frozen at -20 degrees C until use. All 38 PCR primers were designed to be amplified at the same temperature (52 degrees C). We examined a total of 27 FAP patient samples with APC germline mutations (17 for multiple bp deletions, 1 for 1 bp deletion, 9 for nonsense mutations) and 50 APC-negative noncarriers. All 17 multiple bp deletion mutations were detected during the initial 50 degrees C running analysis and thus ruled out for further analyses. All other mutations were clearly detected under specific optimized conditions. More than 50% of the APC germline mutations were multiple base pair deletions and efficiently selected by omitting time-consuming partial denaturing conditions.

CHFR was recently identified as an early mitotic checkpoint that delays transition to metaphase in response to mitotic stress. Although studies have shown that CHFR is relevant to tumorigenesis, no previous report has investigated whether polymorphisms in the CHFR gene are associated with the risk of cancer development. Here, we genotyped polymorphisms in the CHFR gene and analyzed the possible associations of single polymorphisms and haplotypes with the risk and clinicopathological characteristics of colorectal cancer. Six coding SNPs in the CHFR gene were genotyped in 462 colorectal cancer patients and 245 healthy normal controls, using either the TaqMan assay or direct sequencing. Our results revealed that the V539M polymorphism was significantly associated with a lower risk of colorectal cancer (P=0.03; OR, 0.533; 95% CI, 0.302-0.94), and significantly correlated with no distant metastasis (M0 stage), different TNM stage, and microsatellite instability (MSI) among the colorectal cancer patients. Among the five tested haplotypes, hap 10 (TGACTA) was significantly associated with a lower risk of colorectal cancer (P=0.017; OR, 0.496; 95% CI, 0.279-0.883), and colorectal cancer patients carrying this haplotype showed no distant metastasis, different TNM stage, and microsatellite instability at a significantly higher frequency. These results reveal for the first time that polymorphisms in the CHFR gene are associated with colorectal cancer susceptibility.

Preoperative chemoradiotherapy is widely used to improve local control and sphincter preservation in patients with locally advanced rectal cancer. In the present study, we investigated whether microarray gene expression analysis could predict complete response to preoperative chemoradiotherapy in rectal cancer. Tumor tissues were obtained from 46 patients with rectal cancer (31 for training and 15 for validation testing). All patients underwent preoperative chemoradiotherapy involving 50.4 gray radiotherapy, followed by surgical excision 6 weeks later. Response to chemoradiotherapy was evaluated according to Dworak’s tumor regression grade. Tumor regression Grades 1, 2, and 3 were considered partial responses, and tumor regression Grade 4 was considered a complete response. By using the 31 training samples, genes differentially expressed between partial response and complete response were identified, and clustering analysis was performed. Prediction analysis of response to chemoradiotherapy was performed on the 31 training samples by using a selected set of 95 “predictor” genes. Those findings were validated by independent analysis of the 15 test samples. The 31 training samples comprised 20 partial response and 11 complete response cases. A primary set of 261 genes was identified as differentiating between partial response and complete response. By supervised clustering using these 261 genes, 30 of 31 training samples were clustered correctly according to tumor response. A gene set comprising the top-ranked 95 genes displaying differential expression between partial response and complete response was applied to predict response to chemoradiotherapy. Complete response and partial response were accurately predicted in 84 percent (26/31) of training samples and 87 percent (13/15) of validation samples. Microarray gene expression analysis was successfully used to predict complete responses to preoperative chemoradiotherapy in patients with advanced rectal cancer.

Most investigations on MutY human homolog (MYH)-associated polyposis (MAP) have been conducted in Western countries. Limited data on MAP in Asia are currently available. The present study investigated germline mutations of the MYH gene among patients with 10 to 99 adenomatous colorectal polyps and familial adenomatous polyposis (FAP) without adenomatous polyposis coli (APC) germline mutations in Korea.The study population included 46 patients with 10 to 99 adenomatous polyps in the colorectum and 16 FAP patients with no identified APC germline mutations. Subjects were screened for MYH germline mutations, and we additionally screened for MYH mutations in 96 normal control individuals.Two of 46 (4.3%) patients with multiple polyps displayed heterozygous biallelic germline mutations of the MYH gene. A 39-year-old male patient with biallelic MYH mutations (p.G272E and p.A359V) received total proctocolectomy for rectal cancer and 36 colorectal polyps. A 58-year-old female patient with biallelic MYH mutations (p.Q253X and p.Q440P) received right hemicolectomy for ascending colon cancer and 16 colonic polyps. The frequency of biallelic MYH mutation in 14 of 46 multiple-polyp patients, who had 15 to 99 polyps, was 14.3% (2 of 14). No biallelic MYH mutations were detected in the 32 patients with 10 to 14 colorectal polyps, 16 FAP patients, or 96 normal controls.We identified biallelic MYH germline mutations in 2 of 14 (14.3%) Korean patients with 15 to 99 colorectal polyps. In this study, there was no Y165C or G382D hot-spot mutation, which had been reported most frequently in previous studies.

BACKGROUND: Little is known about the genetic risk factors associated with colorectal cancer. Although the Ser326Cys polymorphism of 8-oxoguanine DNA glycosylase (hOGG1) is consistently associated with a range of cancers, there is no consensus regarding this polymorphism and colorectal cancer risk.METHODS: In the present study, conducted in a Korean population, we used the TaqMan assay to investigate whether the hOGG1 Ser326Cys polymorphism was associated with colorectal cancer in 439 colorectal cancer patients and 676 healthy normal controls. We also examined whether the hOGG1 Ser326Cys polymorphism is associated with tumor location, microsatellite instability (MSI) status and tumor-node-metastasis (TNM) stage in colorectal cancer.RESULTS: We found no significant difference between the cancer and control populations in terms of genotype distribution (CC, CG and GG). In addition, we found no association between the hOGG1 Ser326Cys polymorphism and cancer risk, MSI status, TNM stage or tumor location in colorectal cancer patients.CONCLUSIONS: These results suggest that unlike for other cancer types, the hOGG1 Ser326Cys polymorphism is not a major genetic risk factor for colorectal cancer.J Epidemiol 2007; 17: 156-160.

Various types of human cancers harbor BRAF somatic mutations, leading researchers to seek molecular targets for BRAF inhibitors. A mutually exclusive relationship has been observed between the BRAF-V600E mutation and K-ras mutations, suggesting that the BRAF-V600E mutation may differ from the other BRAF mutant types. Here, we used microarray analysis to examine differences between the BRAF and K-ras mutant colorectal samples and within the BRAF group (V600E versus non-V600E), in the hope that the identified gene sets could form the basis for new target development. Eleven colorectal cancers (CRCs) with BRAF mutations and nine with K-ras mutations were examined by high-density microarray analysis. We also tested whether other significant genetic or clinical status involved in CRC development, such as APC and TP53 mutations, MSI and TNM-Duke's staging, were related with the observed BRAF- or K-ras associated expression profiles. Unsupervised two-way hierarchical clustering and multidimensional scaling revealed that the differentially expressed genes clustered according to the mutation status of BRAF and K-ras, and that samples with the BRAF-V600E and non-V600E mutants could be distinguished from each other by gene profiling. Examination of TNM-Duke's staging, MSI and mutations in APC and TP53 revealed that these significant mutations could not account for the hierarchical clustering results observed in our study. We herein identified distinct gene expression patterns and gene sets that may form the basis for identification of BRAF-targeting molecules or provide researchers with a better understanding of the molecular pathogenesis underlying RAS-RAF signaling.

MYH, OGG1 and MTH1 are members of base excision repair (BER) families, and MYH germline mutations were recently identified in patients with multiple adenomas or familial adenomatous polyposis (FAP). A total of 20 APC-negative Korean FAP patients were analyzed for OGG1, MYH and MTH1 germline mutations. A total of 19 hereditary nonpolyposis colorectal cancer (HNPCC), 86 suspected HNPCC, and 246 sporadic colorectal cancer cases were investigated for OGG1 and MYH mutations. A total of 14 R154H OGG1 polymorphisms were identified in hereditary, sporadic colorectal cancers, and normal controls. For the case-control analysis of OGG1 R154H, a total of 625 hereditary or sporadic colorectal cancer patients and 527 normal controls were screened. R154H was a rare polymorphism associated with sporadic colorectal cancer patents (OR: 3.586, P= 0.053). R154H does not segregate with cancer phenotypes. Upon examining the possibility of recessive inheritance of R154H, we could not identify any complementary mutations in OGG1, MYH or MTH1. Samples with R154H were further screened for mutations of K-ras, beta-catenin, APC, p53, BRAF and the microsatellite instability (MSI) status. Eight somatic mutations were identified in these genes and G:C to T:A transversion mutations were not dominant in samples harboring R154H. This result raises the possibility that OGG1 R154H may function as a low/moderate-penetrance modifier for colorectal cancer development.

Mutations in CDH1, which encodes E-cadherin, have been associated with hereditary diffuse gastric cancer (HDGC) in Western populations but have not been shown to play a major role in Asians. Recently, a patient with familial gastric cancer (FGC) was shown to harbor a germline mutation in the TP53 gene, which encodes p53 and has been previously associated with Li-Fraumeni Syndrome (LFS). To determine whether mutations in TP53 are associated with FGC in Asians, we screened the entire coding region of TP53 in probands from 23 Korean FGC families. We identified a nonsense (E287X) TP53 germline mutation in a family whose history is compatible with both HDGC and LFS. Two members of this family (SNU-G2) were afflicted with brain tumors, seven with gastric cancers, two with sarcomas, and one with both gastric cancer and a sarcoma. The E287X TP53 mutation segregated with the cancer phenotype in the family members from whom DNA samples were available. To our knowledge, this is the first report of a large family with both HDGC and LFS. Our results suggest that TP53 mutational screening in FGC families should be interpreted with caution because additional TP53 mutation-carrying HDGC families may also show LFS-related phenotypes.

A number of genes have been studied in accordance with their metastatic potential. Nevertheless, widely used metastatic colorectal cancer cell lines such as SW-620 or IS3 only show the influence of metastatic genes within limited mutational landscape. Here, three pairs of primary CRC (SNU-2335A, SNU-2335D, SNU-2404A, SNU-2404B, SNU-2414A, and SNU-2414B) and corresponding metastasis cell lines were established and analyzed by whole exome sequencing and microarray. Three pairs of primary CRC (SNU-2335A, SNU-2404A, and SNU-2414A) and corresponding matched metastasis cell lines (SNU-2335D, SNU-2404B, and SNU-2414B) were analyzed for gene expression by microarray and whole exome sequencing. Selected target genes from microarray were analyzed again by real-time PCR in order to confirm the mRNA expression level in the paired CRC cell lines. The genes selected for examination were calponin 3, acidic (CNN3); sorbin and SH3 domain containing 1(SORBS1); epithelial stromal interaction 1 (EPSTI1); bone marrow stromal cell antigen 2(BST2); kelch-like 5 (KLHL5); trypsinogen C (TRY6); and synaptotagmin-like 5 (SYTL5). We calculated the intersection of all three metastatic CRC cell lines. Five genes (BST2, SORBS1, CNN3, EPSTI1, and KLHL5) were up-regulated in metastatic cell lines compared to primary cell lines. Two genes (TRY6 and SYTL5) were down-regulated in metastatic cell lines compared to primary cell lines. Then, we ranked 15 genes that were up-regulated and 15 genes that were down-regulated in omental metastasis regardless of the 2-folds threshold. Of the 30 genes that were differently expressed, we selected three genes (CNN3, SORBS1, and TRY6) that showed the highest fold changes and are found in 2-fold change analysis. We identified 7 genes that were differently expressed in metastatic CRC cell lines compared to primary CRC cell lines. These genes may serve as diagnostic markers and therapeutic targets for patients with metastatic CRC. Note: This abstract was not presented at the meeting. Citation Format: Soon-Chan Kim, Chang-Won Hong, Sang-Geun Jang, Seung-Yong Jeong, Jae-Gahb Park, Ja-Lok Ku. Establishment of six human colorectal cancer cell lines: identification of genes that have metastatic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2439. doi:10.1158/1538-7445.AM2017-2439

The molecular mechanism for aggressive clinical behaviour related to v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2) amplification is not fully-understood. In particular, little is known about microRNAs in the human epidermal growth factor receptor 2 (HER2) signaling network.Using microRNA microarray, the microRNA profiles of 16 HER2-positive breast carcinomas were compared with those of five luminal-type breast carcinomas. Additionally, two frozen, ERBB2-amplified gastric carcinomas were compared with their adjacent normal tissue samples. MicroRNAs that were differentially expressed according to the HER2 status in breast and gastric carcinomas were identified as the HER2 microRNA signature.MiR-337 and miR-302f were commonly overexpressed in HER2-postive breast and gastric cancer. MiR-139 and miR-129 were commonly underexpressed in HER2-positive breast and gastric cancer. A concordant pattern of microRNA expression was noted between discovery sets and the majority of candidate microRNAs (two out of three) in three validation sets.Our study identified novel microRNAs that were differentially expressed according to the HER2 status across different tumor types.

Distinguishing between primary and metastatic adenocarcinomas in the lung may sometimes be difficult by conventional histopathological methods. In addition, novel biomarkers are needed for the more accurate subtyping of primary lung carcinomas.MicroRNA microarrays were performed on 26 primary lung adenocarcinomas, 3 squamous cell carcinomas, 6 small cell lung cancers (SCLCs), and 2 colorectal cancer metastases in the lung.Forty-four microRNAs differentially expressed between three histological subtypes at p10(-6) predicted histology with 100% accuracy in 100 randomly drawn datasets. Prominent among differentially expressed genes were miR-375, miR-217 and miR-216a, which were found overexpressed in SCLC compared to lung adenocarcinomas. Lung adenocarcinomas overexpressed miR-29b-1, miR-375, miR-2110, miR-29c-star, 199b-5p, and 146b-3p and underexpressed miR-617, miR-205-star, and miR-1246 compared to squamous cell carcinomas. In primary vs. metastatic lung adenocarcinomas, miR-552 and miR-592 were differentially expressed at p10(-6); the level of expression of miR-552 in colorectal cancer metastases was 39-times higher and that of miR-592 was six-times higher. Furthermore, microRNA profiles of primary colorectal cancer in our database indicated that these two microRNAs were overexpressed in primary colorectal cancer relative to primary lung adenocarcinomas.MicroRNA profiles predict the histology of primary lung carcinomas, and differentiate between primary lung adenocarcinomas and colorectal cancer metastases.

(a) An introductory sentence indicating the purposes of the study Distant metastasis is the major cause of cancer-related death in colorectal cancer (CRC) patients. Although the Calponin family has emerged as a distinguishing feature in epithelial-to-mesenchymal transition (EMT) in human cancer, the role of CNN3 member in the metastatic CRC has not been investigated. (b) A brief description of pertinent experimental procedures. Three pairs of primary CRC and corresponding matched metastasis cell lines were analyzed for gene expression by microarray and western blotting. CNN3 was transduced to CRC cell line with lower CNN3 expression level (SW-480), and CNN3 with higher CNN3 expression CRC cell lines (SW-620, HCT-116) was suppressed by lentiviral-induced shRNA. Functional analysis of CNN3 overexpression and suppression was investigated in these CRC cell lines for proliferation and invasion. Expression of several potential CNN3 target genes (ERK1/2) and EMT markers (E-cadherin and vimentin) in primary CRC and matching metastasis CRC cell lines was validated. (c) A summary of the new, unpublished data. Global CNN3 expression levels were significantly higher in metastasis cell lines compared with the matched primary cell lines in both mRNA (FC = 2.3, p = 0.017) and protein levels. CRC cell lines with suppressed CNN3 showed decreased cell proliferation and reduced invasive behavior in CRC cell lines. Suppression of CNN3 in CRC cell lines with higher CNN3 expression resulted in increased E-cadherin and decreased vimentin. pERK1/2 were decreased in accordance with suppression of CNN3 in both SW-620 and HCT-116. Overexpression of CNN3 in CRC cell lines caused reduced E-cadherin in SW-480 and HT-29. (d) A statement of the conclusions CNN3 is involved in mediating EMT and metastatic behavior in the colon. Its expression is accordantly related to EMT markers, and CNN3 may serve as a possible diagnostic marker and therapeutic target for patients with CRC. Citation Format: Soon-Chan Kim, Chang-Won Hong, Sang-Geun Jang, Yeh-Ah Kim, Seung-Yong Jung, Jea-Gab Park, Ja-Lok Ku. Calponin-3, acidic (CNN3) mediates EMT-related genes in human colorectal cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1642.

Background Non-Hodgkin lymphoma (NHL) is a hematologic malignancy for which good diagnostic markers are lacking. Despite continued improvement in our understanding of NHL, efforts to identify diagnostic markers have yielded dismal results. Here, we translated low-mass-ion information in urine samples from patients with NHL into a diagnostic marker. Methods To minimize experimental error, we tested variable parameters before MALDI-TOF analysis of low-mass ions in urine. Urine from 30 controls and 30 NHL patients was analyzed as a training set for NHL prediction. All individual peak areas were normalized to total area up to 1000 m/z. The training set analysis was repeated four times. Low-mass peaks that were not affected by changes in experimental conditions were collected using MarkerView™ software. Human Metabolome Database (HMDB) searches and ESI LC-MS/MS analyses were used to identify low-mass ions that exhibited differential patterns in control and NHL urines. Identified low-mass ions were validated in a blinded fashion in 95 controls and 66 NHL urines to determine their ability to discriminate NHL patients from controls. Results The 30 highest-ranking low-mass-ion peaks were selected from the 60-urine training set, and three low-mass-ion peaks with high intensity were selected for identification. Of these, a 137.08-m/z ion showed lower mass-peak intensity in urines of NHL patients, a result that was validated in a 161-urine blind validation set (95 controls and 66 NHL urines). The 130.08-m/z ion was identified from HMDB searches and ESI LC-MS/MS analyses as hypoxanthine (HX). The HX concentration in urines of NHL patients was significantly decreased (P < 0.001) and was correlated with the mass-peak area of the 137.08-m/z ion. At an HX concentration cutoff of 17.4 μM, sensitivity and specificity were 79.2% and 78.4%, respectively. Conclusions The present study represents a good example of low-mass-ion profiling in the setting of disease screening using urine. This technique can be a powerful non-invasive diagnostic tool with high sensitivity and specificity for NHL screening. Furthermore, HX identified in the study may be a useful single urine marker for NHL screening.

Background Elucidating the molecular mechanisms by which tumors become resistant to Herceptin is critical for the treatment of Her2-overexpressed metastatic breast cancer. Methods To further understand Herceptin resistance mechanisms at the molecular level, we used comparative proteome approaches to analyze two human breast cancer cell lines; Her2-positive SK-BR-3 cells and its Herceptin-resistant SK-BR-3 (SK-BR-3 HR) cells. Results Heat-shock protein 27 (HSP27) expression was shown to be upregulated in SK-BR-3 HR cells. Suppression of HSP27 by specific siRNA transfection increased the susceptibility of SK-BR-3 HR cells to Herceptin. In the presence of Herceptin, Her2 was downregulated in both cell lines. However, Her2 expression was reduced by a greater amount in SK-BR-3 parent cells than in SK-BR-3 HR cells. Interestingly, co-immunoprecipitation analysis showed that HSP27 can bind to Her2. In the absence of Herceptin, HSP27 expression is suppressed and Her2 expression is reduced, indicating that downregulation of Her2 by Herceptin can be obstructed by the formation of a Her2-HSP27 complex. Conclusion Our present study demonstrates that upregulated HSP27 in human breast cancer cells can reduce Herceptin susceptibility by increasing Her2 protein stability.

We herein describe the development of a sensitive microarray hybridization method called competitive DNA hybridization (CDH) and its use for analysis of BRAF somatic mutations. These mutations have been identified in many human cancers, and fast, reliable BRAF mutation detection may one day facilitate directed therapy of BRAF-mutated tumors. Our fast, reliable mutation detection by CDH is based on the principle that competition among multiple fluorescent-labeled samples for binding to shared wild-type sequences should reduce nonspecific results and increase the positive signals of unshared mutated sequences. The positive signals can then be discriminated based on the labeling of each sample (ie, with Cy3, Cy5, or Alexa-594). For testing of this method, we developed a BRAF oligonucleotide microarray containing 65 mutation types (more than 95% of the known BRAF mutations) and validated this microarray with 20 colorectal cancer tissues/cancer cell lines with BRAF mutations and 60 BRAF-negative samples. In sum, we were able to screen up to nine cancer samples on a single BRAF microarray (three per CDH on three regions per slide), indicating that this method may dramatically decrease the experimental time, cost, and effort of mutation detection in BRAF and other genes amenable to microarray analysis.

Background Glutathione S-transferases are a group of enzymes that participate in detoxification and defense mechanisms against toxic carcinogens and other compounds. These enzymes play an important role in human carcinogenesis. In the present study, we sought to determine whether GSTT2 promoter single nucleotide polymorphisms (SNPs) are associated with colorectal cancer risk. Methods A total of 436 colorectal cancer patients and 568 healthy controls were genotyped for three GSTT2 promoter SNPs (-537G>A, -277T>C and -158G>A), using real-time TaqMan assay and direct sequencing. An electrophoretic mobility shift assay (EMSA) was performed to determine the effects of polymorphisms on protein binding to the GSTT2 promoter. Results The -537A allele (-537G/A or A/A) was significantly associated with colorectal cancer risk (OR = 1.373, p = 0.025), while the -158A allele (-158G/A or A/A) was involved in protection against colorectal cancer (OR = 0.539, p = 0.032). Haplotype 2 (-537A, -277T, -158G) was significantly associated with colorectal cancer risk (OR = 1.386, p = 0.021), while haplotype 4 (-537G, -277C, -158A) protected against colorectal cancer (OR = 0.539, p = 0.032). EMSA data revealed lower promoter binding activity in the -537A allele than its -537G counterpart. Conclusion Our results collectively suggest that SNPs and haplotypes of the GSTT2 promoter region are associated with colorectal cancer risk in the Korean population.

AIM: To evaluate the usefulness of denaturing high performance liquid chromatography (DHPLC) for analyzing microsatellite instability (MSI) status in stool DNA of patients with colorectal cancer. METHODS: A total of 80 cancer tissues from patients with primary sporadic colorectal tumor (proximal cancer: 27, distal cancer: 53) and matched stool (which were employed for comparison with the tissues) were analyzed for MSI status in BAT 26. DNA samples extracted from stool were evaluated by nested polymerase chain reaction (PCR) and DHPLC for MSI analysis. RESULTS: Six cases (7.5%) of MSI were identified in BAT 26 from 80 cancer tissues. All the stool DNA samples from patients whose cancer tissue showed MSI also displayed MSI in BAT 26. CONCLUSION: As MSI is one of the established fecal DNA markers to screen colorectal cancer, we propose to use DHPLC for the MSI analysis in fecal DNA.

Recently, RASSF2A was identified as a potential tumor suppressor epigenetically inactivated in human cancers. Here, we evaluated the methylation status of RASSF2A in colorectal cancer (CRC) and analyzed its correlation with K-ras/BRAF mutations, microsatellite instability status and other clinicopathological features. Using methylation-specific PCR and bisulfite sequencing, we analyzed the methylation status in primary CRC, adenomas and corresponding normal tissues and then compared it with the presence of K-ras and BRAF mutations. We also examined the expression and methylation status of RASSF2A in CRC cell lines. We found that aberrant methylation of RASSF2A promoter regions is associated with gene silencing in CRC cell lines. In primary CRC, the frequency of RASSF2A methylation was 72.6%, and it was found in 16 of 16 (100%) adenomas. In addition, there was a positive correlation between K-ras/BRAF mutations and RASSF2A methylation in primary CRC. Furthermore, a significant positive correlation between K-ras/BRAF mutations and RASSF2A methylation was also observed in microsatellite-stable (p = 0.033) and distal CRC (p = 0.025). These results show that RASSF2A methylation is a frequent event in colorectal tumorigenesis and positively correlates with K-ras/BRAF mutation in microsatellite-stable or distal CRC.

MDK is a heparin-binding growth factor associated with cancer development. Here, we sought to examine the association of MDK expression with resistance and sensitivity to different chemotherapeutic agents. We established stable HeLa cell transfectants (HeLa-MDK) and tested for decreased sensitivity to chemotherapeutic agents (5-FU, doxorubicin, and cisplatin). In addition, we used siRNA to block MDK expression in SNU-638 human gastric cancer cells and examined the chemosensitizing effect. HeLa-MDK cells treated with 5-FU, doxorubicin, and cisplatin showed a fold increase in the average IC(50) and an increased cell survival. siRNA-based knockdown of MDK expression in SNU-638 cells decreased the average IC(50) by 18-44% in cells treated with three drugs. Further investigations on the molecular mechanism should be clarified, but these results indicate that MDK up- and down-regulation appears to be capable of changing the chemosensitivities of cancer cells and MDK may have possible importance as a candidate therapeutic molecule.

Von Hippel-Lindau (VHL) disease is an autosomal dominant inherited tumor syndrome associated with germline mutations in the VHL gene. VHL disease, as well as several other cancer syndromes, has been associated with an increased risk of pheochromocytomas, which are catecholamine-secreting tumors of the adrenal gland. VHL disease genotype-phenotype correlations have been well established based on the type of mutations in the VHL gene. However, although many groups have reported VHL germline mutations in different countries, no previous report has described VHL gene mutations in VHL disease and/or pheochromocytoma patients in Korea. In this study, we used direct sequencing to investigate VHL germline mutations in Korean patients with VHL disease or pheochromocytomas (11 VHL patients and 3 additional members from 7 families, 2 patients from 1 family with familial pheochromocytoma, and 2 cases of isolated pheochromocytoma). We found a total of 7 VHL germline mutations (6 missense and 1 frameshift), 3 of which were novel (323_324delGC, 355T>C and 361G>A). No VHL germline mutation was found in the 2 patients with isolated pheochromocytomas and paragangliomas. This study provides informative data for VHL germline mutations and VHL-related phenotypes in Korea.

Familial adenomatous polyposis (FAP) is a clinically well-defined hereditary disease caused by germline mutations in the adenomatous polyposis coli (APC) gene. FAP is characterized by polyposis in the large bowel and variable extracolonic manifestations. With an increase of reported APC germline mutations, many reports have investigated genotype-phenotype correlations in FAP patients. Here, we analyzed the APC gene for germline mutations in 83 unrelated Korean FAP patients and investigated genotype-phenotype correlations. We identified germline APC mutations in 59 (71%) of the cases, including 34 frameshift mutations, 19 nonsense mutations, and six splice site mutations. Among 59 patients with the identified germline mutation of the APC gene, 37 had been reported previously and were included in the genotype-phenotype analysis. In the other 22 patients, we identified seven novel mutations: c.1438C>T, c.2232_2233dupCT, c.3426delT, c.3739_3769del31, c.3931_3935delATTGG, c.4332dupA, and c.4722_4725delACTA. Desmoid tumors were identified in six of the examined FAP patients, five of whom had APC germline mutations; these mutations involved codons 849, 864, 1309, 1444 and 1464, respectively (c.2547_2548delTA, c.2592_2593insCT, c.3927_3931delAAAGA, c.4332dupA and c.4391-4394delAGAG). Four of the included FAP patients had papillary thyroid cancers; all were female and had germline APC mutations (c.1863_1865delTTAincCT, c.2805C>A, c.3183_3187delACAAA and c.3927_3931delAAAGA).

In cancer research, gene expression and mutations are increasingly investigated by use of oligonucleotide microarrays, which use immobilized oligonucleotides and sequence-specific DNA probe hybridization to investigate differences between nondiseased and cancer tissues (1). In our previous works, we used oligonucleotide microarray-based mutation analysis to detect germline or somatic mutations (2)(3). Activating mutations of the K- ras gene occur in ∼20–50% of colorectal cancers, with ∼85% of the mutations restricted to codons 12 and 13 (4). K- ras gene mutations have been widely studied as markers for cancer prognosis, and population-based studies have suggested that mutated K- ras might be associated with some tumor phenotypes (4)(5)(6). Studies of associations between K- ras mutations and specific clinical features generally require the analysis of large numbers of samples (5). Thus, researchers need a high-throughput technique for assessing K- ras mutations. Oligonucleotide microarrays may provide a valid option because they allow scientists to accurately and rapidly process large numbers of samples. Because the K- ras gene is known to have two mutational hot spots (codons 12 and 13), it has been used as a target gene for testing newly developed techniques for mutation detection, including various applications of the DNA chip (7)(8). Here we describe a new method for K- ras oligonucleotide microarray analysis called competitive DNA hybridization (CDH). CDH is a novel, efficient, high-capacity hybridization technique in which various fluorescently labeled samples are mixed to compete with each other in a hybridization reaction. A total of 204 Korean patients with colorectal cancer from Seoul National University Hospital and the National Cancer Center of Korea were screened for somatic K- ras mutations in this study. Written informed consent was obtained from all patients; the cancers included 103 cancers originating from the proximal colon and 101 …

E-cadherin, the main adhesion molecule of epithelial cells, has been implicated in carcinogenesis because its expression is frequently lost in human epithelial cancers. E-cadherin protein expression is significantly reduced in sporadic colorectal cancers (CRC), but apparently not as a consequence of allele loss or somatic mutation. We reported recently that a single nucleotide polymorphism (-347 G-->GA) in the E-cadherin promoter suppressed E-cadherin expression and was associated with familial gastric cancer. Here we sought to investigate whether the functional polymorphisms of E-cadherin might affect CRC. We genotyped 407 individuals (260 CRC patients and 147 normal controls) for the -347 G-->GA promoter polymorphism of E-cadherin using denaturing high-performance liquid chromatography and direct sequencing. We also measured the activity of promoters harboring the polymorphism by dual luciferase reporter assay in several CRC cell lines. We found that the E-cadherin GA genotype (G/GA heterozygous and GA homozygous) was more common in CRC patients than in normal controls (P = 0.011). Subjects with the E-cadherin GA genotype had an overall 1.75-fold increased risk of CRC. We also observed an increased risk association between the E-cadherin GA genotype and both proximal colon (P = 0.019) and distal CRC (P = 0.036). Interestingly, the GA allele decreased transcriptional efficiency by 12-, 9- and 10-fold compared with the G allele in SNU-C4, SNU-C5 and SNU-1033 cell lines, respectively. Additionally, we examined whether there was a correlation between the E-cadherin promoter polymorphism and microsatellite instability status, and found no such correlation. Taken together, our results suggest that the E-cadherin -347 G-->GA polymorphism may be associated with CRC.

Colorectal cancer (CRC) is the third most common type of cancer in developed countries. Proper radiotherapy with chemotherapy is the worldwide treatment for CRC patients. It has been reported that chemoradiotherapy is related to chemical and radiation resistance and may cause local recurrence. To investigate features in resistant tumors, various cancer cell lines that have resistance to chemo and radiotherapy have been established. In this study, three radio-resistant human colorectal cancer cell lines (SNU-61R80GY, SNU-283R80GY, and SNU-503R80GY) were induced by exposing them to a total of 80 greys of Cs-137 to define their characteristics compared to their parental cell lines (SNU-61, SNU-283, and SNU-503). In order to explain cellular properties of parental and induced radio-resistant cell lines, cell proliferation assay, soft agar colony forming assay, cell cycle analysis, and apoptotic assay were performed. Interestingly, remarkable increases of cell proliferation and colony forming abilities on the induced radio-resistant cell line (SNU-503R80GY) were observed compared to the parental cell line (SNU-503) without irradiation although there was no significant change with cell cycle and apoptotic assay using FACS and western blot analysis. Gene expression microarray analysis was then conducted to identify genetic differences between the induced radio-resistant cell lines and the parental cell lines and thirty-three genes with more than 2-fold up-regulated upon induced radio-resistant cell lines were analyzed. According to the gene ontology analysis of these genes, most of them were associated with responses to hypoxia and oxygen levels, oxidation reduction, regulation of epithelial cell differentiation, and cell-cell adhesion. Five genes with more than 3-fold up-regulated upon all three induced radio-resistant cell lines were also sorted out as candidate marker genes for radio-resistance. Furthermore, various genes that are related with cell migration, growth, differentiation, adhesion, and ERBB signaling pathways were screened by RT-PCR. In conclusion, induced radio-resistant human colorectal cancer cell lines have different features such as differential gene expression patterns, and a great ability of cell proliferation, and colony forming compared to the parental cell lines. These results suggest that the induced radio-resistant cell lines are more tumorigenic than the parental cell lines. These cellular and genetic characteristics on the induced radio-resistant cell lines could be considered markers for radio-resistance. The genes that were 3-fold up-regulated on the induced radio-resistant cell lines could be also regarded as candidate markers for radio-resistance. In order to verify our hypothesis, we are in the process of performing the same experiments using other human colorectal cancer cell lines. Citation Format: Ye-Ah Kim, Sang-Geun Jang, Young-Kyoung Shin, Ja-Lok Ku. Identification of genes with differential expression in induced radio-resistant human colorectal cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 441. doi:10.1158/1538-7445.AM2013-441