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Colorectal cancer (CRC) ranks third in terms of incidence and second in terms of mortality, more than 1.8 million new colorectal cancer cases and 881,000 deaths are reported in Global Cancer Statistics 2018. Therefore, there is an urgent need for a new molecular target in considering treatment strategy. In recent years, cancer has been recognized as a stem cell disease. Spheroid colony formation has been demonstrated as an effective method to characterize cancer stem cells (CSCs). Previously, we analyzed the gene expression profile of spheroid colonies from the gastric cancer cell lines by microarray analysis and found that expression of Kinesin Family Member C1 (KIFC1) was upregulated in gastric spheroid colonies. The kinesin superfamily is a group of proteins that share a motor domain and participate in several biological functions. KIFC1 (or HSET) has been reported to play a vital role in centrosome clustering in cancer cells and is related to pathogenesis in several kinds of cancers. However, the expression of KIFC1 in CRC has not been investigated. The aim of this study is to investigate the expression and the biological function of KIFC1 in CRC. We analyzed the expression and distribution of KIFC1 in 129 CRC cases by immunohistochemistry. In contrast to weak or no staining of KIFC1 in non-neoplastic mucosa, CRC tissue showed stronger, more extensive KIFC1 staining. In total, 67 (52%) of 129 CRC cases were positive for KIFC1. KIFC1-positive CRC cases were significantly associated with CD44-positive and aldehyde dehydrogenase 1 (ALDH1)-positive CRC cases. In the CRC cell proliferation analysis, KIFC1 siRNA-transfected cells showed significantly reduced cell growth ability relative to negative control siRNA-transfected cells. The expression level of phosphorylated Akt and Erk were lower in KIFC1 siRNA-transfected cells compared to negative control siRNA-transfected cells. In the sphere formation analysis, the number and the size of spheres from CRC cells were significantly reduced in KIFC1 siRNA transfected cells than in negative control siRNA-transfected cells. These results indicate that KIFC1 plays an important role in CRC pathogenesis and might be involved in cancer stemness. Although further in-depth studies to clarify the biological significance of KIFC1 in colorectal CSCs are needed, KIFC1 may be a promising therapeutic target for the treatment of CRC. Citation Format: Shintaro Akabane, Naohide Oue, Yuto Fujiki, Naohiro Uraoka, Naoya Sakamoto, Kazuhiro Sentani, Hiroyuki Egi, Hideki Ohdan, Wataru Yasui. Clinicopathological significance of KIFC1 in human colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5306.

Objective: Esophageal cancer is one of the deadliest cancers in the world, and the main subtype is esophageal squamous cell carcinoma (ESCC), which comprises 90% of cases. We previously analyzed the gene expression profile of spheroid colonies and parental cells derived from gastric cancer (GC) cell lines by microarray analysis. Among the genes upregulated in spheroid colonies, tryptophan 2,3-dioxygenase (TDO2) is dramatically upregulated in MKN-1 cells (derived from adenosquamous cell carcinoma) compared with other GC cell lines. Expression of TDO2, an enzyme involved in tryptophan catabolism, has been linked with tumor survival and poor prognosis of brain and breast cancer. However, no studies have investigated the potential role of TDO2 in esophageal cancer. Here we explored the expression and biological significance of TDO2 in ESCC. Methods: To explore the expression of TDO2 in cancer and normal samples, we used an online analytical tool, the Broad Institute TCGA Genome Data Analysis Center, http://firebrowse.org/. For quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we used 10 ESCC samples (tumor tissues and the corresponding non-neoplastic tissue). TDO2 protein expression was evaluated in 90 ESCC tissue samples by immunohistochemistry. TDO2 function in ESCC cell lines and spheroid colony formation was evaluated by RNA interference (RNAi). Cell growth was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Modified Boyden chamber assays were performed to examine cell invasiveness. Results: TDO2 expression was upregulated in most cancers except for liver cancer and pancreas cancer. The fold change expression of TDO2 between normal and cancer tissue was the highest in esophageal cancer. TDO2 overexpression was associated with tumor stage, recurrence status and the CD44 cancer stem cell marker in ESCC. TDO2 overexpression was correlated with poor outcome of ESCC patients. Inhibition of TDO2 expression by RNAi in TE-10 and TE-11 cell lines reduced both the number and the size of spheroid colonies as well as cell proliferation and invasive activity. Knockdown of TDO2 expression also induced inactivation of the EGFR signaling pathway. Conclusion: Our results imply that TDO2 could play an important role in the progression of ESCC. Furthermore, TDO2 may be a potential therapeutic target in ESCC. Citation Format: Thang Quoc Pham, Naohide Oue, Yohei Sekino, Yuji Yamamoto, Naoya Sakamoto, Ririno Honma, Kazuhiro Sentani, Wataru Yasui. TDO2 overexpression is associated with cancer stem cells and poor prognosis in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1166.

Genes encoding transmembrane proteins expressed specifically in cancer cell may be ideal biomarkers for diagnosis. Previously, we showed that overexpression of PCDHB9, which was identified as a transmembrane protein, promotes peritoneal metastasis and correlates with poor prognosis in patients with gastric cancer. However, expression of PCDHB9 in esophageal cancer remains unclear. In the present study, we investigated the expression and function of PCDHB9 in esophageal squamous cell carcinoma (ESCC). Immunohistochemical analysis demonstrated that 51 (40%) of 128 GC cases were positive for PCDHB9. PCDHB9 expression was correlated with T classification, N classification and tumor stage. There was no significant correlation between PCDHB9 expression and patient survival. Cell adhesion to fibronectin and invasion activity were significantly reduced by PCDHB9 knockdown. These results suggest that PCDHB9 may be a novel biomarker and therapeutic target for ESCC. Citation Format: Naohide Oue, Yuji Yamamoto, Ryuichi Asai, Naoya Sakamoto, Kazuhiro Sentani, Wataru Yasui. Overexpression of PCDHB9 is correlated with tumor progression in esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 612.

Background and Aim: The loss of PTEN (phosphatase and tensin homologue) expression in human colorectal cancer (CRC) is found in approximately 40% of cases, and its functional contribution is not fully understood. To address this question, the colon tumors from the Pten-deficient mice with a disruption of the Adenomatous polyposis coli (Apc) were compared with those from the mice with Apc deficiency alone. Method: To recapitulate human CRC, mouse models carrying transgenes regulated by a 9.5-kb fragment containing sequences from the human CDX2 promoter (CDX2P9.5), which were previously shown to have tightly restricted transgene expression in the colon epithelium, were prepared. CDX2P9.5-NLSCre;Apcflox/+;Ptenflox/+ (referred to as CPC;Apc+Pten mouse) and CDX2P9.5-NLSCre;Apcflox/+(;Pten+/+) mice (referred to as CPC;Apc mouse) were generated, and the overall survival and tumor phenotypes (number, size and severity) at 4, 6, 9, 12 and 15 weeks of age were compared between the two models. In addition, the loss of heterozygosity status of the Apc and Pten wild-type alleles in tumor tissue and normal mucosa in the two mouse models were compared with multiplex polymerase chain reaction (PCR), and Pten transcripts and protein expression were evaluated by quantitative reverse transcription PCR and immunohistochemistry, respectively. Results: CPC;Apc+Pten mice had a significantly shorter life span than CPC;Apc mice (median survival time of CPC;Apc+Pten mice and CPC;Apc mice: 14.4 weeks [n=20] vs. 21.6 weeks [n=20], respectively). The CPC;Apc+Pten mice had more tumors than CPC;Apc mice at all time points measured, but the tumor size in CPC;Apc+Pten mice was significantly larger than that in CPC;Apc mice only at 9 weeks of age. Invasion into the submucosa was more frequently observed in CPC;Apc+Pten mice than in CPC;Apc mice (CPC;Apc+Pten mice and CPC;Apc mice: 36.3%/58.3% and 4.9%/10% at 12/15 weeks of age, respectively). Regarding the findings on multiplex PCR, while the tumors from both models showed biallelic Apc inactivation, the tumors from CPC;Apc+Pten mice showed no loss of the wild-type allele of Pten. Regarding the Pten transcript levels, the levels in the tumors and mucosa from CPC;Apc+Pten mice were almost half of those in CPC;Apc mice. The Pten protein expression level in tumors from CPC;Apc+Pten mice was lower than that in tumors from CPC;Apc mice but not completely suppressed. Conclusion: Pten promotes tumor invasion and carcinogenesis without two hits on the gene. Consistent with the previous finding that two hits on the PTEN gene was a rare event in human CRC with PTEN loss, our data strongly suggest the haploinsufficient tumor-suppressive activity of Pten. These findings show that this CPC;Apc+Pten mouse model recapitulates human CRC with PTEN loss. Citation Format: Haruki Sada, Takao Hinoi, Hiroaki Niitsu, Masatoshi Kochi, Naoya Sakamoto, Kazuhiro Sentani, Naohide Oue, Wataru Yasui, Hideki Ohdan. Pten haploinsufficiency promotes tumor invasion and carcinogenesis in mouse colon epithelium with Apc deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4074.

Background: The transcribed-ultraconserved regions (T-UCRs) are a novel class of long noncoding RNAs transcribed from ultraconserved regions (UCRs) that are located in both intra- and intergenic regions and absolutely conserved among the orthologous regions in most of the vertebrates. There are at least 481 UCRs, and these regions can produce 962 T-UCRs. T-UCRs have distinct signatures in human cancers. However, little has been known about the correlation between T-UCRs and tissue/cancer stem cells so far. Organoid is a novel 3D in vitro stem cell culture technology, which sustains stem cell-driven formation of near-physiologic, self-renewing tissues through using the specific niche factors in a dish. As it can be established from patient-derived tissue samples and recapitulates the genetic diversity of the original tissues, patient-derived tumor organoids could be a reliable model of disease. Because of its biologic features, organoid is assumed to harbor many stem and progenitor cells, so that it is suitable for stem cell analysis. In this study, we aimed to identify T-UCRs that are specifically expressed in cancer stem cell of colorectal cancer (CRC) and validate their functional role in CRC stem cells. Methods: We focused on 61 T-UCRs that were reported as the dysregulated T-UCRs in CRC by microarray analysis. We examined them using RNA samples isolated from 3 pairs of CRC and non-neoplastic organoids by qRT-PCR. Biologic roles of the T-UCRs upregulated in CRC organoids were analyzed using a vector containing the whole sequences of the T-UCRs. Results: Through the validation, we found that Uc.91+, Uc.182+, Uc.249+A and Uc.266+A were specifically upregulated in CRC organoids compared to those in normal organoids. We also validated the T-UCRs expression in several CRC cases using both primary tumors and organoids, which further supported the validity of CRC stem cell specific signature. Each of the expression of the 4 T-UCRs was highly upregulated in 2 CRC cell lines, HCT-116 and SW480, while we did not see any similarities in representative genetic abnormalities between the 2 CRC cell lines. Further in-depth in vitro studies are ongoing to clarify the solid evidences, especially the direct effect of T-UCRs onto the stem cells features such as dormancy and sphere-forming ability. Conclusion: We identified that Uc.91+, Uc.182+, Uc.249+A and Uc.266+A were specifically upregulated in CRC organoids, and the 4 T-UCRs showed similar trend of expression in several CRC cell lines. These 4 T-UCRs are promising candidates that could potentially have an important role in cancer stem cell biology. Citation Format: Ririno Honma, Naoya Sakamoto, Akira Ishikawa, Daiki Taniyama, Kaho Fukada, Yohei Sekino, Shoichiro Mukai, Kazuhiro Sentani, Naohide Oue, Takao Hinoi, Hiroyuki Egi, Hideki Ohdan, Wataru Yasui. Expression analysis of transcribed-ultraconserved regions in cancer stem cell population using colorectal cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2011.

Colorectal cancer (CRC) is a leading cause of cancer mortality and the third most common cancer worldwide. Annually, there are more than 900,000 cases occur worldwide, nearly 500,000 of which are fatal. CRC have accumulated defects in oncogenes and tumor suppressor genes. Many CRCs likely arise through clonal outgrowth and evolution of an epithelial cell or cells in an adenomatous precursor lesion via so-called adenoma-carcinoma sequence. Intensive studies have uncovered that the spectra of these genetic modifications are highly variable across the tumors. While genetic changes in tumors of individual patients can be assessed in several stratified methods, these data still need to be properly interpreted for necessitating model systems for analysis of chemosensitivity of CRC patients, which could enable us to organically coordinate among genotype-to-phenotype correlations, clinicopathological features and clinical outcomes. One of the most plausible models for implementing this magnificent scheme is most likely to be “organoid”, which is a novel 3D intestinal stem cell culture system and has successfully reconstitute normal and cancerous colonic crypts in in vitro through using the specific niche factors in a dish, such as EGF, Wnt3a, Rspondin-1 and Noggin, together with a TGF-b inhibitor and a p38 inhibitor. Two groups, Van der Wetering et.al and Fujii et.al, have previously reported the establishment of organoid biobank of colorectal cancer patients, and both of them proved the usefulness of this platform for underpinning the significance of genotype-phenotype analysis in every single patient and providing insights into tailor-made therapy in CRC. In this present study, we generated a pairs of cancerous and normal adjacent organoids from a number of CRC patients and aimed to figure out sustainability and reproducibility of CRC organoids especially in terms of phenotypic and genotypic features even after several times of passage. We compared growth activity and histopathological features of the CRC organoids among several time points after the passage by evaluating ki-67 labeling index and morphology of the organoids. In order to confirm the stability of genetic status of representative cancer-related genes, we focused on TP53, KRAS, BRAF, and MSI status. We then tried to elucidate the difference of chemosensitivity between MSI-high and MSI-low/MSS CRC cases with similar genetic background in most of the representative cancer-related genes. Although further in-depth studies on CRC organoids are needed, our approach would be instrumental in accurately predicting the drug sensitivity of CRC patients, which could possibly lead to the establishment of personalized medicine in CRC. Citation Format: Naoya Sakamoto, Akira Ishikawa, Ririno Honma, Takao Hinoi, Naohide Oue, Kazuhiro Sentani, Shoichiro Mukai, Tomohiro Adachi, Hiroyuki Egi, Hideki Ohdan, Wataru Yasui. A colorectal cancer organoid library provides a promising screening tool for assessment of chemosensitivity [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 4836. doi:10.1158/1538-7445.AM2017-4836

Prostate cancer (PCa) is the most prevalent cancer among men and the second leading cause of cancer related death in western world. Currently, there is a lack of effective treatment options for advanced PCa. Understanding the molecular mechanisms in prostate cancer development and progression would allow the identification of novel prognostic markers or therapeutic targets. The transcribed-ultraconserved regions (T-UCRs) are novel class of noncoding RNAs that are absolutely conserved (100%) between orthologous regions of the human, rat and mouse genomes. Some studies showed that T-UCRs exhibit distinct profiles in various human cancers. We examined the expression profile of representative 26 T-UCRs using qRT-PCR in 12 PCa tissues and 10 non-neoplastic prostate tissues, and found that the expression of Uc.63+ was higher in PCa tissues than that in non-neoplastic tissues. Further analysis revealed that the expression of Uc.63+ significantly correlated with higher PSA levels (p = 0.007) and higher Gleason score (p < 0.001). In order to verify biological function of Uc.63+ in PCa, we firstly transfected Uc.63+ expression vector into LNCaP cells expressing low level of Uc.63+. MTT assay and wound healing assay revealed that overexpression of Uc.63+ significantly increased the cell growth and migration. We also confirmed that downregulation of Uc.63+ using siRNA in DU145 and PC3 expressing high level of Uc.63+. Knockdown of Uc.63+ decreased the cell growth and migration. A recent report indicates that T-UCRs may act as endogenous competing RNA. miR-130b was found to have binding sites within the Uc.63+ using online software. We examined the expression of miR-130b by qRT-PCR in PCa tissues and non-neoplastic prostate tissues. We found a significant downregulation of miR-130b in PCa tissues compared with non-neoplastic prostate tissues, which figured out that there was a significant inverse correlation between Uc.63+ and miR-130b in PCa (p = 0.002). The expression of miR-130b in LNCaP was higher than that in DU145 and PC3. It has been shown that MMP2 is a direct target of miR-130b. Western blot analysis and qRT-PCR revealed that MMP2 expression was higher in LNCaP cells transfected with Uc.63+ expression than that in LNCaP cells transfected with empty vector. These results suggest that Uc.63+ may contribute to the progression of PC through miR-130b interaction. Citation Format: Yohei Sekino, Keisuke Goto, Ririno Honma, Yoshinori Shigematsu, Naoya Sakamoto, Kazuhiro Sentani, Naohide Oue, Jun Teishima, Akio Matsubara, Wataru Yasui. Expression of the transcribed ultraconserved region Uc.63+ in prostate cancer [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 3478. doi:10.1158/1538-7445.AM2017-3478

Urothelial carcinoma of the bladder is a common malignancy that causes approximately 150,000 deaths per year worldwide. So far, no molecularly targeted agents have been approved for treatment of the disease. In the present study, we built an in-house oligonucleotide array, on which 394 genes were selected based on our SAGE data and previously reported array data, in order to identify the genes of most relevance to gastric carcinogenesis. Among these genes, we focused on SEC11A, because it is frequently overexpressed in gastric cancer (GC). SEC11A encodes the SPC18 protein, which is one of the subunits of the signal peptidase complex (SPC). Most secretory proteins contain amino terminal- or internal signal peptides that direct their sorting to the endoplasmic reticulum (ER). From the ER, proteins are transported to either the extracellular space or the plasma membrane through the ER-Golgi secretory pathway. The ER signal peptides are then cleaved by the SPC. In GC, SPC18 contributes to progression via EGFR pathway associated with TGF-a secretion. However, the expression and function of SPC18 have not been investigated in bladder cancer (BC). In this study, we analyzed the expression and distribution of SPC18 in human BC. Expression of SPC18 was observed in 44 (54.3%) out of 81 BC cases that was associated with T category (p=0.0002), Grade (p=0.0425), vascular invasion (p=0.0112) and CK5/6 positive/ CK20 negative basal type BC (p=0.0004). The univariate analysis indicated that expression of SPC18 (HR, 3.14; 95% CI, 1.47-7.47; P=0.0027) was associated with overall survival. In the multivariate model, SPC18 expression was an independent prognostic indicator (HR, 3.46; 95% CI, 1.35-10.00; P=0.0087). BC cells KMBC2 transfected with SPC18 expression vector significantly induced the cell growth (p Citation Format: Yoshinori Shigematsu, Yohei Sekino, Naoya Sakamoto, Kazuhiro Sentani, Naohide Oue, Tetsutaro Hayashi, Jun Teishima, Akio Matsubara, Wataru Yasui. Expression and functional analysis of signal peptidase complex 18 in bladder cancer [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 522. doi:10.1158/1538-7445.AM2017-522

Objective: Standard treatment for stage II or III gastric cancer is curative resection followed by adjuvant chemotherapy. Treatment outcomes are expected to be further improved by individualized therapy based on biomarker analysis. We extracted mRNA and protein from frozen specimens of gastric cancer and analyzed markers for stratifying the risk of recurrence after curative resection of stage II or III gastric cancer. We report our currently available findings. Methods: Candidate markers for stratification of recurrence risk were identified by quantitative real-time polymerase chain reaction (Q-PCR) at the gene level and liquid chromatography-tandem mass spectrometry (LC-MS/MS) at the protein level. The study group for quantitative real-time PCR comprised 255 patients with stage II or III gastric cancer (training set, 145 patients; validation set, 110 patients). The relative expression levels of 127 genes were measured in gastric cancer tissue. Genes identified be independent predictors of poor outcomes in the training set were verified in the validation set. The study group for exploratory proteomics analysis LC-MS/MS comprised 24 patients with T4a Nx M0, Stage III gastric cancer (recurrence and death, 12 patients; relapse-free survival >5 years, 12 patients). Proteins with max fold change >2 and Anova (P) Results: On Q-PCR, 5 genes; SPARC, HER2, CXCR4, INHBA, and VSNL1 were identified as candidate markers. On exploratory proteomics analysis by LC-MS-MS, 4 proteins were identified as candidate markers for recurrence risk stratification. Conclusions: Promising candidate markers for stratification of the risk of recurrence after curative resection of stage II or III gastric cancer were identified at the gene and protein levels. These candidate markers are now being analyzed by tissue microarray, using specimens from about 500 patients each at two high-volume centers, with the goal of developing clinical applications. Citation Format: Takashi Oshima, Yayoi Kimura, Kentaro Sakamaki, Yohei Miyagi, Takaki Yoshikawa, Yasushi Rino, Kazuhiro Sentani, Naohide Oue, Wataru Yasui, Toshio Imada, Munetaka Munetaka. Stratification markers for the risk of recurrence after curative resection of stage II or III gastric cancer and potential clinical applications. [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 4932.

Purpose: Oncogenic KRAS mutations are found in 40-50% of human colorectal cancers. However, specific therapeutics has not been yet available. We aimed to search new therapeutic molecular targets or biomarkers in KRAS mutated colorectal cancers. Experimental design: We generated Apcflox/flox; CDX2P9.5-G22Cre and LSL-KrasG12D; Apcflox/flox; CDX2P9.5-G22Cre mice, that had been considered as sporadic colon cancer mouse model with Kras wild and mutant, respectively. We observed the carcinogenesis at the age of 3-4 weeks by necropsy, and harvested the tumors for gene expression profiling. We compared the gene expression by Microarray (GeneChip, Affymetrix) between each 3 tumors from these mice strains. Genes with >5 fold changes between the two groups were selected as candidate genes, and subsequently narrow these down by bibliographic search for further analyses in human colorectal cancers. Result: In macroscopic findings, extensive polyps were generated in cecum and proximal colon, but not in other site of gastrointestinal tract. Tumors occurred in the both strains were considered as well differentiated adenocarcinomas in hematoxylin-eosin staining. It was confirmed that tumors generated in Apcflox/flox; CDX2P9.5-G22Cre mice had Cre-targeted Apc 580D alleles and wild-type Kras, and that tumors generated in LSL-KrasG12D; Apcflox/flox; CDX2P9.5-G22Cre mice had both Cre-targeted Apc 580D alleles and activated oncogenic KrasG12D. In the subsequent gene expression profiling, we identified 31 genes with >5 fold change in Microarray analyses. Of these, we focused on some genes that showed lower expression in Kras mutant tumor than in Kras wild type tumor. We confirmed the lower expressions of these genes in murine tumors by quantitative RT-PCR, and the lower expression in early-staged human colorectal cancers with oncogenic KRAS mutant by immunohistochemistry staining. To analyze the functions, the retroviral vectors that lead overexpression of these genes were constructed and infected to SW480 and RKO CRC cell lines. Proliferation and migration is being analyzed using these cell lines. Conclusion: We identified novel genes associated with oncogenic KRAS mutation, using the analyses of gene expression in colon cancer mouse model. Citation Format: Hiroaki Niitsu, Takao Hinoi, Yasuo Kawaguchi, Kazuhiro Sentani, Naohide Oue, Yusuke Sotomaru, Tomohiro Adachi, Yasufumi Saito, Masashi Miguchi, Masatoshi Kochi, Manabu Shimomura, Wataru Yasui, Hideki Ohdan. Gene expression profiling for oncogenic Kras mutation in mice and human colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 823. doi:10.1158/1538-7445.AM2015-823

Background & Aims: The transforming growth factor-beta (TGF-beta) signal is a tumor-suppressor pathway that is commonly inactivated in about 90% of microsatellite instability (MSI) colorectal cancer (CRC). However, there was little evidence what gene is regulated by TGF-beta signal in the multistep progression sequence of CRC. The first aim of the present study was to generate a mouse model that is null for Tgfbr2 and Apc in the colon epithelium and forms tumors in the colon. The second aim was to analyze the tumors that arose in the mice model for the purpose to identify the gene regulated by TGF-beta signal. Method & Result: Previously we have described the generation of the ‘CDX2P-G19Cre;Apcflox/flox mice’ (called Apc KO mice) which is randomly null for Apc in the colonic epithelium. By mating Tgfbr2flox/flox mice with Apcflox/flox and CDX2P9.5-G19Cre mice, we have finally generated a mouse model ‘CDX2P-G19Cre;Apcflox/flox;Tgfbr2flox/flox mice’ (called Apc+Tgfbr2 KO mice) which is null for Apc and Tgfbr2. In these model, the tumors with well differentiated adenocarcinoma arose mainly in proximal colon and most of mice died at 4 weeks age due to tumor bleeding. Therefore the mice were harvested at 3 weeks age to evaluate the development of colon tumors. Total RNAs of only cancerous tissue areas were extracted from frozen samples by the laser capture microdissection method. We compared gene expression profiles of these mice's tumors (n = 3, respectively) with Mouse Exon 1.0 ST Array (Affymetrix). Gene X expression of Apc+Tgfbr2 KO mice tumors was most highly upregurated by 9.25-fold compared with Apc KO mice (p = 0.045). The array data was validated by quantitative PCR. For human CRC samples, mutations of repetitive mononucleotide tracts in the coding regions of TGFBR2 were identified by direct sequencing. By immunohistochemical analysis, the expression of X was classified according to the percentage of stained cancer cells. The expression was considered to be ‘positive’ if ≥30% of cancer cells were stained. An analysis demonstrated that 11 (100%) of 11 mutated TGFBR2 cases were positive for X, whereas 10 (66.7%) of 15 wild type TGFBR2 cases were positive (P = 0.033), indicating that high expression of X was correlated with TGFBR2 mutation in human CRCs samples. Additionally, the cell proliferation assay revealed that silencing of X led to a significant reduction in CRC cell proliferation. Conversely, forced expression of X enhanced CRC cell proliferation in vitro. Conclusion: We have generated an in vivo model system that Apc and Tgfbr2 were inactivated only in the colonic epithelium and tumors with well differentiated adenocarcinoma arose mainly in proximal colon. The analysis of this model revealed that Gene X is regulated by a TGF-beta signal and likely promotes cell proliferation in CRC. Citation Format: Masashi Miguchi, Takao Hinoi, Manabu Shimomura, Tomohiro Adachi, Yasufumi Saito, Hiroaki Niitsu, Masatoshi Kochi, Yusuke Sotomaru, Hideaki Ijichi, Tsuneo Ikenoue, Kunitoshi Shigeyasu, Kohji Tanakaya, Kazuhiro Sentani, Naohide Oue, Wataru Yasui, Hideki Ohdan. The generation of colorectal cancer mouse model based on microsatellite instability and the identification of transforming growth factor-beta signal target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2301. doi:10.1158/1538-7445.AM2015-2301

Spheroid colony formation is a typically method to identify cancer stem cells (CSC). The growth of spherical colonies is considered indicative of self-renewal ability, and is consistent with a CSC phenotype. However, mechanism of spheroid formation remains unclear in gastric cancer (GC). In the present study, we analyzed spheroid formation associated genes in GC. Comprehensive gene expression profile of MKN-45 and MKN-74 GC cell lines that were grown as monolayers or spheroids was obtained by Affymetrix GeneChip, and we found several kinds of genes whose expression was up-regulated or down-regulated in cells that were grown as spheroids. Among these, 10 kinds of kinesin genes were up-regulated in both MKN-45 and MKN-74 cells that were grown as spheroids. Kinesins are categorized into 14 subfamilies and classified as mitotic kinesins, which are involved in cell division, and non-mitotic kinesins, which are principally involved in intracellular transport. In spheroids, mitotic kinesins were up-regulated. In the present study, we focused on KIFC1, because antibody against KIFC1 is available. qRT-PCR analysis revealed that up-regulation of KIFC1 in both MKN-45 and MKN-74 cells that were grown as spheroids. Immunohistochemical analysis of KIFC1 was performed in 20 GC cases. Expression of KIFC1 was up-regulated in GC tissues in comparison with corresponding non-neoplastic mucosa. These results suggest that KIFC1 plays an important role in gastric carcinogenesis and may be a therapeutic target of GC. Citation Format: Naohide Oue, Shoichiro Mukai, Trang Pham T.B., Kazuhiro Sentani, Reiko Ito, Wataru Yasui. Significance of KIFC1 in spheroid formation of gastric cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1413. doi:10.1158/1538-7445.AM2015-1413

Introduction: Previous study showed that Synbiotics might have beneficial effect in human and animal colitis like ulcerative colitis. However, it is not well known that the influence of Synbiotics for tumorigenesis in colon cancer. The aim of present study was to investigate the influence of Synbiotics in colon cancer mouse model and colitis associated cancer mouse model. Methods: Mouse stains carrying colon epithelium-preferential Apc mutation (CPC;Apc mice), that show sporadic colorectal adenomas and carcinomas are used in this study. We also used CPC;Apc mice consumed 1%Dextran Sulfate Sodium (DSS) as colitis associated colon cancer model, developing apploximately fourfold more adenomas and adenocarcinomas than CPC;Apc not consumed. At first, 9 of CPC;Apc mice were administerated symbiotics (Probiotics (Lactobacillus) 0.1g + Prebiotics (Oligosaccharide) 10mg/0.3ml/body) between 7 and 20 age in weeks and 10 was not administerated. Secondaly, using 9 of CPC;Apc consumed 1%DSS was administrated Symbiotics, 8 of not administrated, 7 of only Probiotics, and 7 of only Prebiotics. Body weight was measured weekly. Fecal microbial analysis was performed. At sacrifice in 21 age in weeks, the small and large bowel were histologically assessed. Results: Synbiotics administration group has no differences of tumorigenesis comparing to control group (tumor incidence rate(88.9%(8/9) vs. 62.5% (5 / 8); control vs. Synbiotics; p = 0.21)average tumor number(3.2 vs. 2.1; p = 0.38), average tumor greatest diameter 5.8 vs. 5.1 mm; p = 0.51). Using CPC;Apc consumed 1%DSS, Symbiotics administration group showed inhibiting morbidity (50% (8 / 16) vs. 80% (8 / 10); control vs. Synbiotics; p = 0.04) and weight loss, and less tumor incidence than control (18.8 vs. 9.6; control vs. Synbiotics; p = 0.01). On the other hands, administration of probiotics or prebiotics alone had no significant differences of tumorigeneis. Conclusion: This data suggest that Synbiotics administration may have influence of inhibiting tumorigenesis in the colitis associated colon cancer. Citation Format: Yasufumi Saito, Takao Hinoi, Tomohiro Adachi, Manabu Shimomura, Masashi Miguchi, Hiroaki Niitsu, Masatoshi Kochi, Hideki Ohdan, Kazuhiro Sentani, Naohide Oue, Wataru Yasui. Impact of synbiotics administration on tumorigenesis of colon cancer mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2303. doi:10.1158/1538-7445.AM2015-2303

Background: Cervical cancer is the second-most common cancer in women worldwide. In Japan, it was the 11th leading cause of death from cancer for women in 2008 but there has been a unique increase of cervical cancer incidence among women in the 20-39-year age group in spite of the decrease of it among women over age 40. High-risk (hr) HPV infection initiates an early event through which an accumulation of genetic and epigenetic aberrations follows. Aberrant DNA methylation (abMet) is shown to provide disease biomarkers with great potential applicable to clinical specimens. The authors undertook a prospective cohort study, as the first trial in the world up to our knowledge, to examine the relationship between the abMet of DLX4 and SIM1 genes and progression of LSIL. Methods: A total of 113 patients were selected from the Comparative Study on Conventional Pap smear and Liquid-based Cytology (ThinPrep) for Uterine Cervix (CCLBC), in which 11,039 samples were enrolled between October 2007 and March 2010 to compare the cytological features of conventional Pap smears and liquid-based cytology specimens using the ThinPrep method. They were classified into 4 groups according to their cervical cytology, hrHPV infection and follow up. Cytology samples were examined for abMet of DLX4 and SIM1 genes and their protein expressions. CaSki cells were treated with 5-Aza-2´-deoxycytidine (5-aza-dC). Results: 40 samples in group 1 were negative for intraepithelial lesion or malignancy. 21 LSILs in group 2 showed a continuance of LSIL for longer than 365 days, and 12 LSILs in group 3 showed an up-grading to high-grade (H) SIL+ within 365 days after the diagnosis of LSIL. 40 in group 4 were squamous cell carcinoma. All but group 1 were infected with hrHPV. Significant difference existed in frequency of abMet between groups 2 and 3 (p=0.044), between groups 3 and 4 (p=0.020) for DLX4, and between groups 1 and 3 (p=0.0003), as well as between groups 2 and 3 (p=0.005) for SIM1 gene. DLX4 protein expression was significantly reduced in the DLX4 abMet positive tissues, as compared to the negative tissues (p=0.008), and 5-aza-dC treatment extracted DLX4 protein expression of CaSki cells in a dose-dependent manner (p Conclusion: AbMet of DLX4 and SIM1 genes should be a useful and novel progression marker of uterine cervical LSIL with hrHPV infection. Citation Format: Junichi Sakane, Kiyomi Taniyama, Kazuaki Miyamoto, Akihisa Saitou, Kazuya Kuraoka, Kazuhiro Sentani, Naohide Ooue, Wataru Yasui. Aberrant DNA methylation of DLX4 and SIM1 is a predictive marker for disease progression of uterine cervical low-grade squamous intraepithelial lesion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 289. doi:10.1158/1538-7445.AM2014-289

Gastric cancer (GC) is one of the most common malignancies worldwide and is one of the leading causes of cancer related mortality. Among gastric cancer subtypes, scirrhous type GC is highly aggressive and has a worse prognosis because of its diffuse infiltration pattern. Because of such pathological features, early clinical diagnosis of scirrhous gastric carcinoma remains difficult. Moreover, peritoneal dissemination occurs frequently even after radical surgery and is the cause of death in many patients. A potential diagnostic or therapeutic target to identify or to inhibit the mechanism of peritoneal dissemination would be an advantageous in this field and need to be elucidated. Escherichia coli ampicillin secretion trap (CAST) method is a powerful technique to identify novel genes that encode secreted or transmembrane proteins. We previously reported DSC2 (desmocollin 2), identified by this method, as a key regulator of GC with intestinal phenotype, using MKN-1 and MKN-28 GC cell lines. In this study, mRNAs were extracted from HSC-44PE and 44As3 scirrhous GC cell lines to analyze the gene expression profile. HSC-44PE cell line was established from pleural fluid of a patient with scirrhous GC. 44As3, derived from HSC-44PE cells, by isolating a highly disseminated clones with a strong capability of inducing ascites in mouse model. Randomly primed cDNAs ligated with pCAST vector were picked up and sequenced thereafter. Among the candidate genes, ZDHHC14 (zinc finger, DHHC-type containing 14) showed higher expression in 44As3 CAST library than in HSC-44PE library. ZDHHC14 is one of the palmitoyl transferase family members and is supposed to be associated with substrates linked to dissemination cascade in scirrhous type GC. Indirect immunofluorescence microscopic analysis also revealed that ZDHHC14 expression was found mostly in large cells of both cell lines, however, ZDHHC14 staining was more frequently observed in 44As3 than in HSC-44PE. High level of ZDHHC14 expression was observed in 27% of GC samples by qRT-PCR. Cell invasion assay and MTT assay revealed that ZDHHC14 influenced the invasion and growth capacity of GC cells. Identification of the palmitoylated proteins, the substrates of ZDHHC14, remain under investigation. ZDHHC14 identified by CAST method, might contribute in the process of peritoneal dissemination of the gastric cancer cells and it might be a potential therapeutic target for scirrhous type GC. Citation Format: Htoo Zarni Oo, Naoya Sakamoto, Naohiro Uraoka, Katsuhiro Anami, Kazuhiro Sentani, Naohide Oue, Kazuyoshi Yanagihara, Wataru Yasui. ZDHHC14, a potential regulator in peritoneal dissemination of scirrhous type gastric cancer, identified by CAST method. [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 1925. doi:10.1158/1538-7445.AM2013-1925

Gastric cancer (GC) is one of the most common malignancies worldwide. Scirrhous type GC is highly metastatic and characterized clinically by rapid disease progression and poor prognosis. MicroRNAs (miRNAs), small non-coding RNA of 19-25 nucleotides, play a part in crucial biological processes and also have an important role in cancer development and progression. In the present study, we show that microRNA-143 (miR-143) is expressed at higher level in diffuse type GC than in intestinal type GC by microRNA microarray analysis. We investigated whether miR-143 is useful as a diagnostic marker of scirrhous type GC. Quantitative RT-PCR analysis revealed that miR-143 expression was low in GC in comparison with adjacent non-tumorous mucosa. Expression levels of miR-143 were higher in scirrhous type GC than those in other types of GC. Treatment of 5-aza-2′-deoxycytidine retrieved miR-143 expression, suggesting that miR-143 might be repressed by DNA methylation. Quantitative RT-PCR and in situ hybridization analysis showed that miR-143 is expressed at higher level in normal fibroblast and cancer associated fibroblast cell lines than in GC cell lines, indicating that major source of miR-143 in cancer tissue is stromal fibroblasts rather than cancer cells. miR-143 knock down by RNAi repressed collagen type III expression in normal fibroblast and cancer associated fibroblast cell lines. Furthermore, TGF-β treatment induced expression of miR-143 and collagen type III mRNA, and this induction of collagen type III mRNA was repressed by miR-143 knock down. We also studied the relation of miR-143 expression with GC patient prognosis (n=68). High miR-143 expression was associated with worse prognosis (P=0.0141). These data suggest that miR-143 regulates fibrosis of scirrhous type GC through induction of collagen expression in stromal fibroblasts and miR-143 expression is a prognostic marker of GC. Citation Format: Yutaka Naito, Naoya Sakamoto, Naohiro Uraoka, Kazuhiro Sentani, Naohide Oue, Masakazu Yashiro, Kosei Hirakawa, Wataru Yasui. Function of microRNA-143 and its potential of prognostic factor in scirrhous type gastric cancer. [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 4197. doi:10.1158/1538-7445.AM2013-4197

Gastric cancer (GC) is one of the most common human cancers. We previously performed Serial Analysis of Gene Expression (SAGE) on four primary GCs and identified several GC-specific genes. In the present study, we have built an in-house oligonucleotide array on which 394 genes selected based on our SAGE data and previously reported array data to allow for the study of the genes that are most relevant to gastric carcinogenesis. We focused on the SEC11A gene, which encodes SPC18 protein. SPC18 is one of the member of signal peptidase complex. SPC18 mRNA expression was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in GC tissue samples. Expression and distribution of SPC18 protein was investigated by immunohistochemistry in two independent GC cohorts (Hiroshima cohort, n = 99 and Chiba cohort, n = 989). To determine the effect of SPC18 on cell growth and invasiveness in vitro, MTT and Boyden chamber invasion assays were performed. To evaluate the influence of SPC18 on cell growth in vivo, GC cells were injected into SCID mice. The levels of TGF-α and EGF in media from the GC cells were measured by ELISA. Overexpression of SEC11A mRNA was observed in 40% of GC cases by qRT-PCR. Immunohistochemical analysis of SPC18 revealed that 26% of GC cases were positive for SPC18 in Hiroshima cohort and 20% of GC cases were positive for SPC18 in Chiba cohort. SPC18-positive GC cases showed poorer survival than SPC18-negative GC cases in both Hiroshima cohort and Chiba cohort by Kaplan-Meier analysis. Forced expression of SPC18 activates GC cell growth in vitro and in vivo. The levels of TGF-α and EGF in culture media from GC cells were reduced by knockdown of SPC18. These results indicate that SPC18 contributes to malignant progression through promotion of EGF and TGF-α secretion in GC. Citation Format: Naohide Oue, Tetsutaro Hayashi, Reiko Ito, Akiko Nagatsuma, Kazuhiro Sentani, Naoya Sakamoto, Katsuhiro Anami, Naohiro Uraoka, Yutaka Naito, Kazuyoshi Yanagihara, Atsushi Ochiai, Hiroki Sasaki, Wataru Yasui. SEC11A contributes to malignant progression through promotion of EGF and TGF-α secretion in gastric cancer. [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 412. doi:10.1158/1538-7445.AM2013-412

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide. Because most ESCC is diagnosed at advanced stages, the overall 5-year survival rate of ESCC still remains low in spite of the use of various treatment modalities. Therefore, identification of new diagnostic markers and therapeutic targets for ESCC is important. Serial Analysis of Gene Expression (SAGE) is one of the common comprehensive methods to analyze transcript expression levels, and enables the quantitative and simultaneous analysis of a large number of transcripts. To identify potential diagnostic markers and therapeutic targets for ESCC, the SAGE library was generated from one ESCC sample. A total of 14,430 tags were generated, including 5,765 that were unique. By comparing SAGE tags from the ESCC sample with those from normal human squamous esophagus, several genes that is upregulated in ESCC compared with normal squamous esophagus in ESCC were identified. Among these, we focused on the nardilysin (N-arginine dibasic convertase, NRD1) gene. NRD1 has been reported to be associated with ectodomein shedding of TNF-α through activation of ADAM proteases. There is only one report in the literature mentioning the association of NRD1 with cancer (breast cancer), however, there is no report that links expression of NRD1 and ESCC. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed a high level of NRD1 expression in three of five esophageal cancer cell lines and seven of 14 ESCC samples, but not in 14 normal systemic tissues. Western blot analysis also showed upregulation of NRD1 protein in total five esophageal cancer cell lines. Immunohistochemical analysis revealed expression of NRD1 in cytoplasm of 43 out of 109 ESCC cases (39%), as well as heart, skeletal muscle, and testis in agreement with previous reports. In clinicopathological analysis, expression of NRD1 correlated with the advanced T grade, N grade and tumor stage, and was an independent predictor of survival in patients with ESCC. Knockdown of NRD1 in TE5 cells inhibited cell invasion ability. Furthermore, qRT-PCR analysis showed a reduction of matrix metalloproteinase-2 (MMP-2) expression in NRD1 knocked down cells. In conclusion, NRD1 is an independent predictor of survival, and contribute to cell invasion through regulation of MMP-2 in ESCC. NRD1 could be a novel diagnostic and therapeutic target for ESCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-91. doi:1538-7445.AM2012-LB-91

Regenerating gene family, member 4 (REG4, encodes Reg IV), was originally identified from cDNA library of inflammatory bowel disease tissues, and is specifically expressed in gastrointestinal tract. We have identified REG4 as an overexpressed gene in gastric cancer from SAGE analysis in gastric cancer tissues and noticed as a novel serum marker of cancer. The expression of Reg IV is associated with intestinal mucin differentiation, and RegIV is expressed in CDX2-positive cells. CDX2, the intestine-specific caudal-related homeobox transcription factor, plays a key role in intestinal development and differentiation. In the present study, we investigated whether CDX2 induce RegIV expression. Western blot analysis indicated that forced expression of CDX2 in HT29 colon cancer cell line induced Reg IV expression. In silico analysis of 2-kilobase upstream region of REG4 revealed at least four putative CDX2 consensus sequences. To assess the role of these putative CDX2 consensus sequences in regulating REG4 transcription, we performed luciferase assay into HSC39 gastric cancer cell line, in which CDX2 is highly expressed. Reporter gene constructs containing 2-kilobase of 5’-flanking region from the REG4 gene showed strong reporter activity. Significant change of reporter activity was not observed between 2.0 and 0.65-kilobase upstream region of REG4 constructs, apart from that significant lower expression level at 0.1-kilobase. These results suggest that expression of REG4 is regulated by CDX2, and upstream region of REG4 has enhancer sequences at proximal region at 0.1-kilobase upstream region. Based on the analysis of single mutation in the putative CDX2 consensus sequences in the 5’-flanking region of the REG4 gene, two putative CDX2 consensus sequences were found to activate REG4 expressions. Therefore, RegIV may be a key factor mediating CDX2 function in intestinal determination. To identify the CDX2-binding site in detail, ChIP assay and mutagenesis with multiple mutations in the putative CDX2 consensus sequences must be carried out. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 272. doi:10.1158/1538-7445.AM2011-272

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide. To identify potential targets for diagnosis and therapeutics of ESCC, we performed Serial Analysis of Gene Expression (SAGE) on the ESCC sample. We generated the SAGE library from one ESCC sample and obtained a total of 14,430 tags, including 5,765 that were unique. By comparing SAGE tags from ESCC samples with those from normal human squamous esophagus (Gene Expression Omnibus accession number, GSM52501), we found several genes and tags that were differentially expressed between ESCC and normal squamous esophagus. Among these, we focused on the ADAM metallopeptidase with thrombospondin type 1 motif, 16 (ADAMTS16) gene. ADAMTS has been described as part of a family of zinc dependent proteases (metzincin family), which play important roles in a variety of normal and pathological conditions including arthritis and cancer. Although ADAMTS16 is expressed in some organs, the relationship with cancers, including ESCC, has not been studied. Quantitative reverse transcription-polymerase chain reaction analysis showed a high level of ADAMTS16 expression in 8 out of 20 ESCC samples (40%) but not in 15 kinds of normal tissues. We also investigated the mRNA expression levels of SCCA1, which encodes SCC antigen. High levels of SCCA1 mRNA expression were found in 4 of 20 cases (20%). We then investigated the relation of ADAMTS16 expression to clinicopathologic characters and found that ADAMTS16 overexpression correlated with the advanced T grade (p = 0.0215). Western blot analysis revealed that ADAMTS16 protein expression was detected in all 4 ESCC cases, whereas in 1 of the 4 corresponding non-neoplastic mucosa. Furthermore, ADAMTS16 protein was detected in culture media from the TE5 ESCC cell line which expressed significant level of ADAMTS16 mRNA, indicated that ADAMTS16 is surely secreted. To evaluate the biological significance of ADAMTS16 in ESCC, we performed MTT assay and transwell invasion assay. These examination revealed that knockdown of ADAMTS16 by siRNA in TE5 cells inhibited both cell growth and invasion ability. These results suggest that ADAMTS16 has a high potential as a serum tumor marker and therapeutic target for ESCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2178.

Prostate cancer (PCa) is one of the most common human cancers. Identification of novel genes that encode membrane-spanning or secreted proteins expressed specifically in PCa has a great advantage to develop of ideal biomarkers for cancer diagnosis and therapeutics. Escherichia coli Ampicillin Secretion Trap (CAST) is a signal sequence trap method to identify genes encoding cell surface and secreted proteins. In this study, to identify novel diagnostic and therapeutic targets of PCa, CAST method was used. We generated cDNA libraries from normal prostate and two PCa cell lines, LNCaP and DU145. These CAST libraries were ligated into the pCAST which was plasmid with a mutant beta-lactamase lacking the endogenous signal peptide. Survival on ampicillin was observed only when various cDNA fragments encoding a signal sequence were inserted. We sequenced 960 ampicillin-resistant colonies for normal prostate CAST library, 1344 colonies for LNCaP CAST library and 960 colonies for DU145 CAST library. By comparing these sequences to those deposited in the public databases using BLAST (NCBI), 223 genes from normal prostate CAST library, 234 genes from LNCaP and 228 genes from DU145 were identified. To extract highly expressed genes in PCa, the genes expressed in normal prostate were excluded. Quantitative RT-PCR analyses of 64 candidate genes were performed and compared mRNA levels in 9 PCa tissues, 2 normal prostate tissues and 14 kinds of normal tissues. Quantitative RT-PCR analyses revealed that CDON was highly expressed in PCa. Overexpression of CDON (tumor/normal ratio > 2) was observed in 8 (89%) of 9 PCa tissues. CDON is a cell surface receptor of the immunoglobulin /fibronectin type III repeat family. By western blot analysis, low to moderate CDON expression was noted in LNCaP cell extracts and moderate to high CDON expression was noted in DU145 cell extracts. The expression of CDON in LNCaP and DU145 was suppressed by treatment with CDON specific siRNA. Cell invasion assay and MTT assay revealed that knockdown of CDON in LNCaP cells did not inhibit both cell growth and invasion ability, but knockdown of CDON in DU145 cells inhibited both cell growth and invasion ability. In conculusion, CAST is a robust and rapid method to identify cell surface and secreted proteins. CDON was up-regulated in PCa and participated in cell growth and invasion. These finding suggest that CDON may be a good therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 242.

Gastric cancer (GC) is one of the most common human cancers. Identification of novel genes encoding secreted or transmembrane proteins expressed specifically in GC is important for the development of ideal biomarkers for cancer diagnosis and therapeutics. In the present study, we tried to identify genes encoding cell surface and secretory proteins in GC using Escherichia coli ampicillin secretion trap (CAST) method. CAST libraries were generated from two GC cell lines, MKN-1 and MKN-28. Ampicillin-resistant 1,440 colonies from each library were sequenced and these sequences were compared to the public databases using BLAST (NCBI). Many genes encoding secreted and transmembrane proteins including some known genes were identified. Quantitative RT-PCR was performed to confirm the overexpression of the candidate genes in GC, and the expressions of several genes are narrowly restricted in GC. Among these genes, we focused on the desmosomal cadherin desmocollin 2 (DSC2). DSC2 is ubiquitously expressed in several normal tissues where desmosomes exist. In cancers, the reduction of DSC2 in sporadic colorectal cancer is reported as desmocollin switching. It is also known that the homeodomain transcription factors Cdx1 and Cdx2 regulate DSC2 expression in colon cancer cells. Quantitative RT-PCR analyses with 14 kinds of normal tissues and 9 GC cases showed that DSC2 is highly expressed in GCs. In normal tissues, the expression was seen in the heart and pancreas at the lower levels, but not in the stomach. Overexpression of DSC2 was observed in 13 (31%) of 41 GC cases. Immunohistochemical analyses revealed that DSC2 was preferentially expressed in the cases with mucosal or submucosal invasion. DSC2 expression was significantly associated with the well differentiated cases. There were no correlations between DSC2 expression and lymph node metastasis, pathological stage, or overall survival. Additionally, DSC2 was significantly associated in GCs positive for MUC2 and CDX2. Cell invasion assay and MTT assay revealed that knockdown for DSC2 of GC cell lines by siRNA did not inhibit either cell growth or invasion ability. These findings suggest that DSC2 is involved in GC with intestinal differentiation. DSC2 is a good biomarker of intestinal type GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 235.