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Background. Despite recent advances in surgical techniques and treatment options, gastric cancer (GC) remains the third most common cause of cancer-related deaths worldwide. Besides DNA sequence mutations, epigenetic alterations have emerged as significantly major players in cancer development. A-to-I RNA editing is a post-transcriptional modification that converts adenosines to inosines in both coding and noncoding RNA transcripts, and has recently been recognized has a novel epigenetic mechanism in GC pathogenesis. More specifically, A-to-I editing of AZIN1 transcripts was shown to be regulated by an adenosine deaminase acting on RNA-1 (ADAR1), and edited AZIN1 resulted in an aggressive phenotype during disease progression in some human cancers. The aim of this study was to clarify the clinical consequences of RNA editing status of AZIN1 and the RNA editing enzymes (ADAR1 and 2) in GC patients. Methods. Two hundred eighty-eight gastric specimens from one hundred forty-four patients who underwent surgery for GC were evaluated. We analyzed the RNA editing status of AZIN1 by RNA editing site-specific quantitative PCR (RESSqPCR). RESSqPCR allows quantitation of RNA editing levels using wild-type or edited AZIN1-specific primers, and RNA editing levels are calculated by determining the ratios of Ct values between edited vs. wild-type transcript expression levels. Furthermore, expression levels of ADAR1 and ADAR2 were evaluated by qPCR in GC tissues. Results. We observed a higher frequency of the AZIN1 RNA editing in tumors compared with normal mucosa in GC. RNA editing status of AZIN1 was significantly increased in a stage-dependent manner, and high frequency of RNA editing in AZIN1 significantly correlated with advanced T stage and presence of lymph node metastasis in GC patients. Multivariate analysis revealed that high frequency of RNA editing in the AZIN1 gene was an independent prognostic factor for poor disease free survival and overall survival. Furthermore, significant upregulation of ADAR1 and downregulation of ADAR2 was also observed in GC tissues compared with matched normal mucosa, and these alterations also significantly correlated with disease progression factors and poor prognosis in GC patients. Interestingly, ADAR1 expression level was positively correlated with RNA editing status of AZIN1 in GC tissues. Conclusions. Our findings revealed that altered gene-specific A-to-I editing events mediated by ADAR1 promote disease progression in GC. We conclude that assessment of RNA editing status of the AZIN1 gene might offer a novel and superior biomarker for a more accurate diagnosis of disease staging and may help predict clinical outcomes in GC patients. Citation Format: Yoshinaga Okugawa, Yuji Toiyama, Kunitoshi Shigeyasu, Takashi Ichikawa, Satoshi Oki, Koichiro Mori, Yuka Nagano, Hiromi Yasuda, Shigeyuki Yoshiyama, Masaki Ohi, Koji Tanaka, Yasuhiro Inoue, Toshimitsu Araki, Yasuhiko Mohri, Motoyoshi Tanaka, Chikao Miki, Ajay Goel, Masato Kusunoki. Novel findings for the clinical significance of RNA editing status of AZIN1 and ADAR 1 and 2 expression levels in gastric cancer patients [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 4720. doi:10.1158/1538-7445.AM2017-4720

Background Abnormal DNA methylation is an important epigenetic mechanism that has been strongly implicated in the pathogenesis and progression of colorectal neoplasia. In contrast to other potential biomarker species such as mRNA and protein levels, measurement of DNA methylation has been shown to be stable, reproducible and consistent between individuals. Based on these standpoints, detecting aberrant methylation of cancer-related genes helps for the development of diagnostic and prognostic biomarkers in colorectal cancer (CRC). In this study, we comprehensively evaluated whole-genome methylation analysis using colorectal cancer tissues and normal colonic mucosa to discover candidate CRC-specific DNA methylation as potential diagnostic and predictive biomarkers for risk stratification in CRC patients. Patients and Method The study design included an initial discovery phases, and followed by a subsequent clinical validation phase. In discovery phase, we performed whole-genome methylation analysis using CRC tissues and normal colonic mucosae from total fourteen patients to identify CRC-specific methylated CpG cites. Furthermore, we evaluated methylation levels of candidate methylated CpG cites by quantitative bisulfite pyrosequencing using one hundred six colorectal specimens from fifty-three CRC patients to validate the findings from discovery phase. Result In discovery phase, we successfully identified several candidates of CRC-specific hypermethylated- and hypomethylated-CpG cites in CRC tissues compared with normal colonic mucosa. Promoter lesion of PDX1 is one of candidate methylated CpG cites in discovery phase, and we focus a specific CpG cite of PDX1 promoter lesion as a target cite to progress in validation phase. We quantified methylation status of PDX1 using 106 colonic tissues, and demonstrated that PDX1 were significantly hyper-methylated in CRC tissues compared with normal mucosa (p Conclusion Assessment of the PDX1 methylation status could be used as potential of diagnostic and predictive biomarker for risk stratification in CRC patients. Citation Format: Takashi Ichikawa, Yuji Toiyama, Yoshinaga Okugawa, Hiromi Yasuda, Hiroyuki Fujikawa, Yoshiki Okita, Shigeyuki Yoshiyama, Junichiro Hiro, Minako Kobayashi, Toshimitsu Araki, Masaki Ohi, Yasuhiro Inoue, Keiichi Uchida, Yasuhiko Mohri, Masato Kusunoki. Hypermethylation status of PDX1 has potential of diagnostic and predictive biomarker for risk stratification in colorectal cancer patients [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 1795. doi:10.1158/1538-7445.AM2017-1795

Background: MicroRNAs (miRNAs) are dysregulated during colorectal cancer (CRC) development and progression. Profiling of CRC tissue has elicited interest in the potential use of miRNAs as non-invasive biomarkers for early detection of CRC. Exosomes have capacity to envelop specific miRNAs, and maintain the integrity of contents in circulation. Therefore, circulating miRNAs in exosomes are more stable than other forms. Thus miRNAs extracted from exosome in serum may have significant potential as disease specific biomarker; however there is no report to assess whether whole serum or exosome-derived miRNAs from serum is a more accurate diagnostic marker and indicator of tumor progression in CRC. This study aimed to evaluate the diagnostic value of serum and exosomal miRNAs for adenoma patients, respectively. Methods: Following a literature review, we investigated the expression of candidate miRNAs in 20 normal colonic mucosa, 27 adenoma, and 19 CRC tissue samples. Then, we quantified their expression in serum and in exosome from 26 adenoma patients and 47 healthy controls to evaluate their clinical significance. Additionally, we compared the expression levels of serum miRNAs with of exosomal miRNAs to investigate their potential as biomarker. Results: We selected four miRNAs, miR-21, miR-29a, miR-92a, and miR-135b, for further investigation. MiR-21, miR-29a, miR-92a, and miR-135b expression in adenoma were significantly higher than in normal colonic mucosa. Regarding the miRNAs expression in serum, miR-21, miR-29a, miR-92a levels in adenoma patients were significantly higher than in healthy controls, and significantly correlated with adenoma size and total adenoma counts in the colorectum. MiR-21, miR-29a and miR-92a levels in serum could significantly discriminate patients with adenoma, and in the patients with advanced adenoma (>1.0 cm), the capacity to discriminate advanced adenoma improved. On the other hand, exosomal miR-21 and miR-29a levels in serum from adenoma patients were significantly higher than that from healthy volunteer. Only exosomal miR-21 significantly correlated with adenoma size and total adenoma counts in the colorectum, and could differentiate patients with adenomas significantly. In the patients with advanced adenoma, the capacity of exosomal miR-21 for biomarker did not improve, and significance disappeared. Conclusion: Our results revealed that although both serum and exosomal miRNAs could be non-invasive biomarkers for the early detection of CRC, but exosomal miRNAs were unfavorable for the identification of high-risk adenomatous polyps. Serum miR-21, miR-29a and miR-92a are potential diagnostic biomarkers in high-risk adenomatous polyp patients. Citation Format: Ryo Uratani, Yuji Toiyama, Takahito Kitajima, Hiroyuki Fujikawa, Jyunichiro Hiro, Minako Kobayashi, Koji Tanaka, Yasuhiro Inoue, Yasuhiko Mohri, Takao Mori, Toshio Kato, Ajay Goel, Masato Kusunoki. Diagnostic potential of serum and exosomal microRNAs for patients with pre-malignant lesions of colorectal cancer: Detection of adenoma. [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 1952.

Background & Aims: MicroRNAs (miRNAs) play a crucial role in diverse cellular biological processes such as differentiation, proliferation, migration, invasion and survival. MiRNA expression patterns are found to be frequently dysregulated in human tumors, which is currently being exploited both from a basic science perspective and for its clinical usefulness as disease biomarkers. Circulating miRNAs are attracting major interest as potential noninvasive biomarkers for colorectal cancer (CRC). This study aimed to identify a novel serum miRNA biomarker for the early detection and/or evaluating prognosis of CRC patients. Methods: Comprehensive miRNA array analysis was performed using serum samples from patients with colorectal neoplasia (CRC: n = 3, adenoma: n = 3) and healthy controls (n = 3). Next, to verify whether the candidate miRNA possessed a secretory potential, we screened miRNA expression levels in culture medium from 2 CRC cell lines, followed by serum analysis from 12 stage IV CRC, 12 adenoma and 12 control subjects. Thereafter, we validated expression of candidate miRNAs in 179 primary CRC tissues, as well as serum samples from an independent cohort of 211 CRCs, 56 adenomas, and 57 control subjects. Results: Through microarray analysis, we identified significantly higher levels of miRNA-1290 (miR-1290) in serum from patients with colorectal adenomas and cancers. We verified miR-1290 overexpression in serum of CRC patients in a training cohort. In the validation cohort, serum miR-1290 levels were significantly upregulated in patients with colorectal adenomas (P < 0.0001) and cancers (P < 0.0001). Serum miR-1290 levels could robustly distinguish adenoma (area under the curve [AUC] = 0.718) and CRC patients (AUC = 0.830) from normal subjects. Serum miR-1290 levels significantly diminished after surgical resection of the primary tumor in the same patients’ serum specimens (P < 0.0001). In addition, we observed a statistically significant positive correlation of miR-1290 expression levels between primary CRC tissues and matched serum samples (ρ = 0.482; P < 0.0001). High miR-1290 expression in serum and tissue was significantly associated with tumor aggressiveness and poor prognosis. Moreover, serum miR-1290 levels were an independent prognostic factor in CRC patients (hazard ratio = 4.51; 95% confidence interval = 1.23-23.69; P = 0.0096). Conclusions: Serum miR-1290, which might be secreted from primary CRC, is a novel diagnostic and prognostic biomarker in CRC. Citation Format: Yuji Toiyama, Hiroki Imaoka, Hiroyuki Fujikawa, Junichiro Hiro, Susumu Saigusa, Koji Tanaka, Yasuhiro Inoue, Yasuhiko Mohri, Takao Mori, Toshio Kato, Ajay Goel, Masato Kusunoki. Circulating microRNA-1290 as a novel diagnostic and prognostic biomarker in human colorectal cancer. [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 968.

Purpose. Despite the recent progress in diagnosis and treatment for colorectal cancer (CRC), prognosis of CRC patients still remains poor. Metastatic recurrence is the major causes of cancer-related death in CRC patients, and recent progress in treatment options, especially technical advances in invasive treatment for metastatic lesion, have improved the prognosis of CRC patients with metastasis. Furthermore, almost half of patients that receive chemotherapy derive no clinical benefit, though all are exposed to these severe toxic and expensive therapeutic regimens in patients with metastatic CRC patients. In view of these underlying issues, for improvement of patient's prognosis, there is a keen interest in developing robust biomarkers that can identify the subset of patients who can benefit from intensive post-treatment surveillance protocols for early detection of recurrence, and prompt decision of appropriate treatment protocol. Herein, we systemically and comprehensively evaluated differentially levels of serum cytokines using array-based techniques to identify novel and reliable serum biomarker to predict metastasis and poor outcome in CRC. Methods. The study design included an initial dual screening phases, and followed by a subsequent clinical validation phase in this study.In discovery phase, we examined cytokine profiling using preoperative serum from two different CRC cohorts (n = 30) to identify differentially expressed serum cytokines in CRC patients with metastasis. In validation phase, serum levels of MCP-4 were assessed in 194 CRC patients by enzyme-linked immunosorbent assay, and their relationships with clinicopathological findings, including recurrence and survival, were investigated. Results. In discovery phase, three cytokines (elevated: MCP-4, ENA-78; decreased:Ckb8-1) were overlapped as a differentially expressed cytokines in serum from CRC patients with metastasis compared those without metastasis. High serum MCP-4 was significantly associated with older age (p = 0.033), advanced T stage (p = 0.029), distant metastasis (p = 0.011) and UICC stage classification (p = 0.006). Cox regression analysis showed that elevated MCP-4 was a significant and independent prognostic factor of disease free survival (HR:2.6, 95%CI:1.0-6.7) and overall survival (HR:2.7, 95%CI:1.3-5.9) in all CRC patients. Furthermore, logistic regression analysis revealed that high serum MCP-4 level was an independent marker in predicting distant metastasis in CRC (OR:3.8, 95%CI:1.3-10.9). Conclusion. Our comprehensive study highlights the clinical feasibility of serum MCP-4 as a prognostic and predictive biomarker for distant metastasis and recurrence in CRC patients. Quantification of serum MCP-4 concentration might support the early detection/prediction of recurrence and may contribute to the prediction of clinical outcomes in CRC. Citation Format: Yoshinaga Okugawa, Yuji Toiyama, Koji Tanaka, Mikio Kawamura, Takahito Kitajima, Tadanobu Shimura, Hiroki Imaoka, Hiroyuki Fujikawa, Susumu Saigusa, Yasuhiro Inoue, Motoyoshi Tanaka, Yasuhiko Mohri, Chikao Miki, Ajay Goel, Masato Kusunoki. Elevated serum monocyte chemotactic protein 4 (MCP4) as a novel noninvasive prognostic and predictive biomarker for detection of metastasis in colorectal cancer. [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 5016.

Background: MicroRNA (miRNA)-1290 is known to activate Wnt signaling pathway and increase the reprogramming-related transcript factors c-Myc and Nanog. Additionally, miR-1290 promotes the epithelial-mesenchymal transition and interferes with cellular differentiation. Previous report demonstrated that overexpressing miR-1290 shows extensive atrophy and intestinal metaplasia of the gastric mucosa, indicating that miR-1290 is involved in the precancerous lesions of the gastric epithelial cells. However, to the best of our knowledge, miR-1290 expression pattern, its clinical significance and molecular mechanism in colorectal cancer (CRC) have not been investigated. Materials and Methods: We screened miR-1290 expression in a subset of 36 tissue samples from normal colonic mucosa (n = 12), adenoma (n = 12) and CRC (n = 12). Next, we further validated miR-1290 expression in a larger, independent cohort, which included normal colonic mucosa (n = 21), adenoma (n = 26) and CRC (n = 179), and investigated the clinical significance of miR-1290 in CRC. In addition, CRC cell lines were transfected with anti-miR against miR-1290 for the assessment of its function. Results: In screening step, miR-1290 expression stepwise increased according to adenoma-carcinoma sequence, and its expression in adenoma and CRC was significantly higher than that in normal colonic mucosa, respectively (adenoma: p40mm), high grade of T stage (T3/4), lymphatic and venous invasion positive, lymph node metastasis and liver metastasis. In addition, Kaplan-Meier survival curves revealed that the CRC patients with high miR-1290 expression were significantly poorer overall survival than that with low expression, respectively (p = 0.0082). Our in vitro study demonstrated that attenuated miR-1290 expression resulted in inhibition of proliferation, invasion and migration capacity of CRC cell lines. Conclusions: We, for the first time, demonstrated that miR-1290 in CRC acts as onco-miRNAs that is supported by several evidences from our in vitro analysis and clinical data. MiR-1290 is significantly associated with malignant potential of CRC and can be the biomarker for predicting poor prognosis in patients with CRC. Citation Format: Yuka Nagano, Yuji Toiyama, Hiroki Imaoka, Hiroyuki Fujikawa, Junichiro Hiro, Susumu Saigusa, Minako Kobayashi, Toshimitsu Araki, Masaki Ohi, Koji Tanaka, Yasuhiro Inoue, Yasuhiko Mohri, Kenichiro Ishii, Masato Kusunoki. MicroRNA-1290 promotes tumor progression and acts as a novel biomarker for poor prognosis in human colorectal cancer. [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 1967.

Background: MicroRNAs (miRNAs) are a class of small non-coding RNAs that inhibit gene expression by binding to 3` untranslated regions of mRNAs and inhibiting their translation. MiRNAs crucially affect cellular processes such as development, proliferation, differentiation and apoptosis, which are deregulated in cancer. MiR-503 is located at Xq26.3 and belongs to an extended miR-16 family of miRNAs. MiR-503 is down-regulated in several types of cancer, including oral, hepatocellular, gastric, and endometrial cancer, suggesting that it plays a tumour-suppressor role in carcinogenesis. In contrast, other recent reports have demonstrated up-regulation of miR-503 in adrenocortical carcinoma, parathyroid and retinoblastoma, compared with adjacent normal tissue. These results suggest that miR-503 shows tissue- or disease-specific expression patterns. However, miR-503 expression patterns in esophageal cancer (EC) and normal esophageal tissue have not been compared and the correlation of miR-503 expression with the clinical features of EC has not been studied. Materials and Methods: A total of 61 formalin-fixed paraffin-embedded (FFPE) tissue samples including 49 EC tissues and 12 adjacent normal esophageal tissues who underwent surgical resection at Mie University Hospital were collected from 2003 to 2013. Tagman qRT-PCR assay was performed to quantify the miR-503 expression in EC and adjacent normal tissues. We analyzed the association between miR-503 expression and clinico-pathological factors in patients with EC. Results: The median age of the patients was 66.2 years (41 - 90). The median follow up time was 24.6 months (3.8 - 80.0). There were 3 patients with stage 0, 16 with stage I, 12 with stage II, 11 with stage III, 3 with stage IVa and 4 with stage IVb by using the criteria of the Japanese Classification of Esophageal Cancer, 10th edition. Forty-four patients had esophageal squamous cell carcinoma (ESCC), five patients had esophageal adenocarcinoma (EAD). MiR-503 was remarkably up-regulated in EC tissues compared to adjacent normal tissue. High miR-503 expression in EC was significantly associated with male sex and large tumor size. Kaplan-Meier analysis showed that patients with high miR-503 expression had significantly poorer disease free and overall survival than those with low expression. Furthermore, high miR-503 expression was an independent predictor for prognosis in EC patients according to multivariate analysis. Conclusions: The results of this study provide novel evidence for associations between miR-503 expression and progression, early recurrence, and poor prognosis in EC, suggesting that it acts as an oncomiR in this type of cancer. MiR-503 may thus be a promising predictive marker for prognosis after surgery in patients with EC. Citation Format: Koichiro Mori, Yuji Toiyama, Shozo Ide, Tomofumi Noguchi, Hiroki Imaoka, Hiromi Yasuda, Susumu Saigusa, Masaki Ohi, Yasuhiro Inoue, Koji Tanaka, Yasuhiko Mohri, Tsutomu Nobori, Masato Kusunoki. MicroRNA-503 promotes tumor progression and acts as a novel biomarker for prognosis in esophageal 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 3401. doi:10.1158/1538-7445.AM2015-3401

Background: Epigenetic changes including the global DNA methylation and the site-specific CpG island promoter methylation have been proposed to play an important role in colorectal carcinogenesis. The long interspersed nucleotide element-1 (LINE-1) methylation is good indicator of global DNA methylation levels, which is known to be hypo methylation in sporadic colorectal cancer (CRC). The promoter region of estrogen receptor 1 (ESR1) has been shown to be highly methylated in CRC compared to normal colorectal mucosa, and to be methylated in normal colorectal mucosa in an age-dependent manner. However, clinico-pathological significance of LINE-1 and ESR1 methylation levels in CRC has not sufficiently been investigated. Aim: The aim of this study was to investigate the association between LINE-1 and ESR1 methylation levels and clinico-pathological findings including with microsatellite instability (MSI), KRAS and BRAF mutations, and tumor-infiltrating lymphocytes (TILs) in CRC. Materials and Methods: We quantified the LINE-1 and ESR1 methylation levels in CRC (n = 182), adjacent normal mucosa (AN) (n = 18), normal colon mucosa (N) (n = 18) using a bisulfite pyrosequencing analysis. MSI status was determined using five mononucleotide-repeat microsatellite markers. KRAS (exons 2 and 3) and BRAF (V600E) mutations were also analyzed by a bisulfite pyrosequencing. TILs were evaluated by counting numbers of intra-tumor Foxp3- and CD8-positive T cell by immunohistochemistry. Results: LINE-1 methylation levels in CRC were significantly lower than in AN or N, respectively. Lower LINE-1 methylation levels were significantly correlated with differentiated cell types, microsatellite stable (MSS) and low TILs in CRC. However, no significant association between LINE-1 methylation levels and tumor progression including with prognostic outcome was recognized. On the other hand, ESR1 methylation levels in CRC were significantly higher than in AN or N, respectively. Higher methylation levels of ESR1 were significantly associated with MSS and KRAS mutation in CRC. Although ESR1 methylation levels were not associated with tumor progression in CRC, survival curves showed that CRC patients with low ESR1 methylation levels had significantly poorer overall survival and disease free survival than those with high methylation levels. Furthermore, ESR1 methylation levels were independent indicators for poor prognosis and early recurrence of CRC patients. Conclusion: We demonstrated novel evidence of ESR1 methylation levels as promising biomarkers for prognosis and predictive recurrence in patients with CRC. Citation Format: Tomofumi Noguchi, Yuji Toiyama, Koichiro Mori, Shozo Ide, Hiroki Imaoka, Hiroyuki Fujikawa, Junichiro Hiro, Susumu Saigusa, Minako Kobayashi, Masaki Ohi, Koji Tanaka, Yasuhiro Inoue, Yasuhiko Mohri, kenichiro ishii, Masato Kusunoki. Clinico-pathological significance of LINE-1 and ESR1 methylation levels in sporadic 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 1061. doi:10.1158/1538-7445.AM2015-1061

Background: Tropomyosin-related receptor kinase B (TrkB) signaling, stimulated by brain-derived neurotrophic factor (BDNF) ligand, promotes tumor progression, and is related to the poor prognosis of various malignancies. Studies in our laboratory revealed: an association between TrkB levels and tumor progression and patient prognosis in gastric cancer; the association of TrkB with chemotherapy resistance in esophageal cancer; and TrkB's involvement in the epithelial-mesenchymal transition in colorectal cancer. We have also demonstrated the involvement of the BDNF/TrkB pathway in tumor progression in gastric cancer. Aim: We sought to examine the clinical relevance of BDNF/TrkB expression in colorectal cancer (CRC) tissues, its prognostic value for CRC patients, and its therapeutic potential in vitro and in vivo. Methods: Two hundred and twenty-three CRC patient specimens were used to determine both BDNF and TrkB mRNA levels. The expression of these proteins in their primary and metastatic tumors was investigated by immunohistochemistry. CRC cell lines and recombinant BDNF and K252a (a Trk inhibitor) were used for in vitro proliferation, migration, invasion, anoikis resistance and in vivo peritoneal metastasis assays. Results: Tissue BDNF mRNA levels were significantly associated with an undifferentiated histology as well as lymph node and liver metastasis. The co-expression of BDNF and TrkB mRNA was significantly associated with lymph node, and liver metastasis and peritoneal carcinomatosis. Patients with high BDNF mRNA had a significantly poor prognosis, and those that co-expressed both BDNF and TrkB mRNA showed a trend toward poor prognosis. BDNF promoted tumor cell proliferation and migration, facilitated invasion and inhibited anoikis in the TrkB-expressing CRC cell lines. These effects were suppressed by K252a. In mice injected with DLD1 co-expressing BDNF and TrkB, and subsequently treated with K252a, peritoneal tumor metastasis was found to be significantly reduced, as compared with control mice. Conclusion: Blockade of BDNF-induced Trk B signaling inhibits peritoneal carcinomatosis arising arising from colorectal cancer. BDNF/TrkB signaling may thus be a potential target for treating the peritoneal carcinomatosis arising from colorectal cancer. Citation Format: Hiroki Imaoka, Koji Tanaka, Yoshinaga Okugawa, Tadanobu Shimura, Takahito Kitajima, Satoru Kondo, Shozo Ide, Susumu Saigusa, Yuji Toiyama, Yasuhiro Inoue, Toshimitsu Araki, Keiichi Uchida, Yasuhiko Mohri, Kenichiro Ishii, Masato Kusunoki. Blockade of BDNF-induced Trk B signaling inhibits peritoneal carcinomatosis arising arising from colorectal cancer. [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 3451. doi:10.1158/1538-7445.AM2014-3451

Background: Inflammation plays a role in the carcinogenesis of sporadic colorectal cancer (CRC), and this can be supported by several evidences showing that effectiveness of nonsteroidal anti-inflammatory drugs as chemopreventive agents for CRC. Angiopoietin-like protein 2 (ANGPTL2) is known to act as a causative mediator of chronic inflammation and inflammatory carcinogenesis. However, the biological role and clinical significance of ANGPTL2 expression remains poorly understood in human cancer. We investigated the functional roles of ANGPTL2 in vitro and evaluated the clinical significance of ANGPTL2 expression in both primary tumor and matched serum in patients with CRC. Methods: ANGPTL2 gene knockdown by siRNA transfection was performed to evaluate ANGPTL2 function in CRC cancer cell line (sw480). Next, we examined ANGPTL2 expression in CRC tissues (n=194) to evaluate the association between its expression and clinicopathological findings by immunohistochemistry (IHC). Finally, we screened serum ANGPTL2 levels from 32 CRC (16 Stage I and 16 Stage IV) and 24 normal controls (NC), and validated its levels using 194 serum samples from CRC and 48 from NC to evaluate its utility as a biomarker by ELISA. Results: Knockdown of ANGPTL2 resulted in significantly inhibition of cell proliferation, migration, invasion, and resistance to anoikis in vitro. ANGPTL2 overexpressed in CRC tissues compared to normal colonic mucosa and high ANGPTL2 expression was associated with advanced T stage (p Conclusion: Our study first reports that overexpression of ANGPTL2 in CRC cells increased malignant potential and metastasis. Serum ANGPTL2, which might be derived from primary CRC tumor, was a novel biomarker for diagnosis and prognosis in patients with CRC. Citation Format: Takahito Kitajima, Yuji Toiyama, Tadanobu Shimura, Shozo Ide, Hiroki Imaoka, Satoru Kondo, Mikio Kawamura, Yoshinaga Okugawa, Aya Kawamoto, Junichiro Hiro, Susumu Saigusa, Masaki Ohi, Koji Tanaka, Yasuhiro Inoue, Yasuhiko Mohri, Sekido Yoshitaka, Masato Kusunoki. Angiopoietin-like protein 2 as a novel serum biomarker for diagnosis and prognosis in patients with colorectal cancer. [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 2858. doi:10.1158/1538-7445.AM2014-2858

Background: Tropomyosin-related receptor kinase B (TrkB) promotes proliferation and invasion related to poor prognoses of various malignancies. TrkB is also involved in resistance to anoikis (a form of detachment-induced apoptosis) and epithelial-to-mesenchymal transition (EMT). Gastric cancer (GC) with mixed histology frequently shows both a well/moderately differentiated (low or intermediate grade) at the surface to the center and a poorly differentiated (high grade) at the invasive front. Dedifferentiation is the progression of cells towards a less differentiated state. Here, we examined the role of TrkB at the invasive front of gastric cancer and its association with tumor cell dedifferentiation. Methods: Immunoreactive TrkB at the invasive front and dedifferentiation, which shows a higher histological grade than that at the surface or center, was evaluated and clarified its clinical and prognostic significance in 320 GC specimens. In vitro study, expression of TrkB, E-cadherin, vimentin, Oct4, and Sox2 at a wound healing edge, which mimics the tumor invasive front, was evaluated in the human GC cell line MKN7. Results: Fifty patients (16%) showed high TrkB expression at the invasive front. Twenty-one patients (6.6%) showed tumor cell dedifferentiation. High TrkB expression at the invasive front and tumor cell dedifferentiation were significantly associated with aggressive phenotypes of GC such as a larger tumor size, depressive macroscopic morphology, lymphovascular invasion, advanced T stage, lymph node involvement, synchronous liver and peritoneal metastasis, and advanced stage. Six patients had high TrkB expression in dedifferentiated GC cells. High TrkB expression at the invasive front was an independent prognostic factor of GC in multivariate analysis (hazard ratio; 1.69, p=0.039). MKN7 cells at the wound healing edge showed increased TrkB, vimentin, Oct4, and Sox2 expression and decreased E-cadherin expression. Tumor cells at the wound edge also showed an increased size and morphological changes in the nucleus or cytoplasm, suggesting that MKN-7 cells at the wound edge may undergo EMT or tumor cell dedifferentiation similar to that of tumor cells at the invasive front of primary tumors. Conclusion: Assessment of immunoreactive TrkB at the tumor invasive front in whole tissue sections provides prognostic information for GC patients. Citation Format: Koji Tanaka, Tadanobu Shimura, Takahito Kitajima, Satoru Kondo, Shozo Ide, Hiroki Imaoka, Yoshinaga Okugawa, Susumu Saigusa, Yuji Toiyama, Yasuhiro Inoue, Tshimitsu Araki, Keiichi Uchida, Yasuhiko Mohri, Masato Kusunoki. Tropomyosin-related receptor kinase B at the invasive front of gastric cancer and tumor cell dedifferentiation. [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 1142. doi:10.1158/1538-7445.AM2014-1142

Purpose. The prognosis of advanced gastric cancer patients with peritoneal dissemination remains poor, and a better understanding of this mechanism is critical for development of new treatments that can improve patient survival. Long non-coding RNAs (lncRNAs) are most commonly defined as transcripts of greater than 200 nucleotides in length. Recent evidence suggests that lncRNAs are differentially expressed in cancer cells and have a major role in the development of cancer progression, including metastasis. This study aimed to investigate the clinical and biological significance of metastasis-associated lncRNA in gastric cancer. Experimental Design. We analyzed MALAT1 and HOTAIR expression levels by real-time reverse transcription PCR in 300 gastric tissues (150 gastric cancer tissues and 150 adjacent normal mucosa), and in seven gastric cancer cell lines. Knockdown of the HOTAIR by siRNA transfection was performed to evaluate HOTAIR function in gastric cancer cell lines and animal models. Results. Expression of MALAT1 and HOTAIR were significantly higher in cancerous tissues than in adjacent normal mucosa, and high expression of both lncRNAs significantly correlated with peritoneal metastasis in gastric cancer. In addition, elevated HOTAIR expression was an independent prognostic factor, and was an independent risk factor for peritoneal dissemination. SiRNA knockdown of HOTAIR in MKN45 and KATO-III cell lines resulted in reduced cell proliferation, colony formation, migration and invasion, but simultaneous increase in anoikis rates, compared to the controls. In an in vivo assay, HOTAIR siRNA-transfected MKN45 cells injected into nude mice inhibited the growth of xenograft tumors and peritoneal metastasis compared with scrambled siRNA-transfected cells. Conclusions. Our data highlight that HOTAIR expression may serve as a potentially important disease biomarker for the identification of high-risk gastric cancer patients. Moreover, our findings provide extensive mechanistic evidence for the functional role of HOTAIR over-expression and the ensuing malignant phenotype in gastric cancer cells in both cultured cells and two different animal models (xenograft and peritoneal metastasis). We propose that HOTAIR lncRNA could be used as a clinically useful diagnostic and prognostic biomarker for gastric cancer patients, and might be a novel therapeutic target for developing gastric cancer treatments. Citation Format: Yoshinaga Okugawa, Yuji Toiyama, Keun Hur, Shusuke Toden, Susumu Saigusa, Koji Tanaka, Yasuhiro Inoue, Yasuhiko Mohri, Masato Kusunoki, C. R. Boland, Ajay Goel. The expression of metastasis-associated long non-coding RNA, HOTAIR, is involved in cancer development and peritoneal metastasis in gastric cancer. [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 3553. doi:10.1158/1538-7445.AM2014-3553

Background: c-MET (hepatocyte growth factor receptor) gene is overexpressed in approximately 20% of gastric cancer. Onartuzumab, a MET-selective humanized one-armed monoclonal antibody, has been studied in metastatic Met-positive non-small cell lung cancer and expected to be introduced to MET-positive gastric cancer. Two-photon laser scanning microscopy (TPLSM) has revolutionized in vivo real-time imaging because of the benefits of higher resolution, increased tissue penetration, and reduced photodamage. We established a method of intravital imaging for intra-abdominal organs using TPLSM with an organ stabilizing system (Japanese Patent No.5268282). Aim: To visualize the binding of fluorescence labeled-antibody to its receptor of cancer cells in peritoneal metastatic xenografts of living mice using a TPLSM. Method: Human gastric cancer cell lines (NUGC4) were inoculated into the peritoneal cavity of green fluorescent protein (GFP) expressing nude mice to make macroscopic peritoneal metastases. In vitro study, the binding of Zenon Alexa Fluor 594 labeled anti-c-MET antibody to viable NUGC4 cells was imaged using fluorescence microscopy. In vivo study, the binding of fluorescence labeled-antibody to viable NUGC4 cells of peritoneal metastatic xenografts was imaged using a TPLSM. Result: Zenon Alexa Fluor 594 labeled anti-c-MET antibody (red) was bound to the surface of viable NUGC4 cells, and clearly visualized under a fluorescence microscopy. Peritoneal metastases with label-free NUGC4 cells (black) and murine stromal cells (green) were imaged under TPLSM in vivo real-time. Fluorescence labeled-antibody could be visualized within tumor vessels of peritoneal metastases after its intravenous administration. Thereafter, label-free NUGC4 cells were identified by the binding of fluorescence labeled- anti-c-MET antibody to c-MET of NUGC4 cells. Conclusion: We could visualize fluorescence labeled-antibody in peritoneal metastases of gastric cancer in living mice using a TPLSM. The binding of fluorescence labeled- anti-c-MET antibody to c-MET of viable NUGC4 cells was clearly imaged at high magnification and high resolution in vivo real-time. It is valuable to develop a method of in vivo optical pathology for therapeutic monoclonal antibodies using MPM on metastatic tumor xenografts. Citation Format: Shozo Ide, Koji Tanaka, Tadanobu Shimura, Masato Okigami, Satoru Kondo, Takahito Kitajima, Hiroki Imaoka, Yoshinaga Okugawa, Susumu Saigusa, Yuji Toiyama, Hiromi Yasuda, Masaki Ohi, Yasuhiro Inoue, Toshimitsu Araki, Keiichi Uchida, Yasuhiko Mohri, Masato Kusunoki. In vivo visualization using two-photon laser scanning microscopy of anti-c-MET antibody in peritoneal metastatic gastric cancer. [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 4938. doi:10.1158/1538-7445.AM2014-4938

Background: Gastric carcinogenesis is regarded as a multistep process with an intestinal metaplasia-dysplasia-carcinoma sequence, which is initiated by Helicobacter pylori infection that causes a chronic active inflammation in the gastric mucosa. Angiopoietin-like protein 2 (ANGPTL2) is known to act as a causative mediator of chronic inflammation and inflammatory carcinogenesis. However, the biological role and clinical significance of ANGPTL2 expression remains poorly understood in human cancer. We investigated the functional role of ANGPTL2 and evaluated the clinical significance of its expression in both primary tumor and matched serum specimens in patients with gastric cancer (GC). Methods: The function of ANGPTL2 in GC was investigated by siRNA using GC cell lines (MKN1 and KATO III). Next, we examined ANGPTL2 expression in GC tissues (n=192) by immunohistochemistry (IHC) to evaluate associations between its expression and various clinicopathological features. Finally, we determined serum ANGPTL2 levels from 32 GC and 23 normal controls (NC), and validated its expression levels using 194 serum samples from GC and 45 from NC to evaluate its utility as a biomarker by ELISA. Results: Knockdown of ANGPTL2 resulted in significant induction of anoikis (p Conclusion: Our study first reports that overexpression of ANGPTL2 in GC cells results in increased malignant potential and metastasis. Serum ANGPTL2 expression emerged as a novel, non-invasive biomarker for the earlier diagnosis, recurrence and prognosis in patients with gastric cancer. Citation Format: Yuji Toiyama, Takahito Kitajima, Tadanobu Shimura, Hiroki Imaoka, Satoru Kondo, Shozo Ide, Masato Okigami, Hiromi Yasuda, Susumu Saigusa, Masaki Ohi, Koji Tanaka, Yasuhiro Inoue, Yasuhiko Mhori, Ajay Goel, Masato Kusunoki. Angiopoietin-like protein 2, a driver of cancer cell metastasis, is a novel serum biomarker for the diagnosis and prognosis in patients with gastric cancer. [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 883. doi:10.1158/1538-7445.AM2014-883

Background. Vasohibin 1 (VASH1) is an angiogenesis inhibitor which is expressed in the endothelial cells (ECs) of blood vessels of the tumor stroma. VASH1 expression of tumor stromal ECs has been reported to be associated with poor prognosis of human breast cancer and urothelial carcinoma. High VASH1 expression of tumor cells themselves was also reported to be associated with poor prognosis in hepatocellular carcinoma, whereas low VASH1 in tumor cells correlated with unfavorable clinicopathological features in renal cell carcinoma. Aim. This study aims to investigate the expression of VASH1 in tumor cells using immunohistochemistry and to evaluate its clinical and prognostic significance in patients with colorectal cancer (CRC). Materials and methods. Three hundred and nine (n=309) samples of CRC patients who underwent surgery between 2006 and 2011 were investigated for the expression of VASH1 using standard immunohistochemistry (IHC). The IHC score of VASH1 in tumor cells was determined on the basis of both staining intensity and the percentage of positive cells. The IHC score was dichotomized at the cutoff value predictive of overall survival using a non-parametric receiver operating characteristic analysis. The association between the expression of VASH1 in tumor cells and clinicopathological characteristics was analyzed. Impact of VASH1 expression in tumor cells on overall survival (OS) was also analyzed using univariate and multivariate analyses. Results. The number of CRC patients with stage I, II, III, and IV were 65, 91, 82, and 71, respectively. The median follow-up of all patients was 19.0 months (range: 2-77 months). Forty one patients had recurrent disease, and 44 patients had died. In CRC tissues, VASH1 protein was localized mainly in the cytoplasm of tumor cells, and partly in vascular endothelial cells. Imunoreactive VASH1 was detected in tumor cells of 272 CRC patients(88%). High VASH1 expression showed a trend towards an association with degree of differentiation. No significant association between VASH1 expression in tumor cells and the other clinicopathological variables was observed. In univariate analysis, high VASH1 expression was significantly associated with poor prognosis of CRC patients [p=0.02, hazard ratio (HR)=2.21, 95 % CI, 1.13-4.74]. In multivariate analysis, high VASH1 expression retained an independent prognostic factor for CRC patients [p Citation Format: Takahito Kitajima, Koji Tanaka, Susumu Saigusa, Tadanobu Shimura, Satoru Kondo, Yuji Toiyama, Yoshinaga Okugawa, Yasuhiro Inoue, Masaki Ohi, Toshimitsu Araki, Keiichi Uchida, Yasuhiko Mohri, Hiroki Hori, Masato Kusunoki. Prognostic impact of Vasohibin 1 (VASH1) expression in tumor cells of colorectal cancer patients. [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 1149. doi:10.1158/1538-7445.AM2013-1149

Background. Trefoil factor 3 (TFF3) is predominantly expressed by goblet cells of the small intestine and colon and its expression is maintained throughout the colon adenoma-carcinoma sequence. Aberrant TFF3 expression in tumor cells has been reported to be involved in tumor progression and unfavorable prognosis of certain human malignancies. In colorectal cancer (CRC), the association between TFF3 expression and tumor progression remains uncertain. Aim. We aimed to investigate the expression of TFF3 in tumor cells using immunohistochemistry and to evaluate its clinical and prognostic values in patients with colorectal cancer (CRC). Materials and methods. Three hundred (n=300) samples of CRC patients were investigated for the expression of TFF3 in tumor cells using standard immunohistochemistry (IHC). The IHC score of TFF3 in tumor cells was determined on the basis of both staining intensity and the percentage of positive cells. The IHC score was dichotomized at the cutoff value predictive of overall survival using a non-parametric receiver operating characteristic analysis. The association between the expression of TFF3 in tumor cells and clinicopathological features was analyzed. Impact of TFF3 expression in tumor cells on overall survival (OS) was also analyzed using univariate and multivariate analyses Results. TFF3 was expressed in all goblet cells of adjacent normal tissue. In adjacent normal colonic crypt, immunoreactive TFF3 protein was localized mainly in the cytoplasm or supranuclear cytoplasm of epithelial cells. Colorectal cancer cells showed an aberrant cytoplasmic TFF3 staining. By use of our dichotomous scoring system, 110 tumors (66%) were low and 190 (34%) were high for TFF3 immunostaining. Decreased expression of TFF3 was significantly associated with larger tumor size ( Conclusions. Decreased TFF3 expression was associated with colorectal tumor progression. Decreased or lack of TFF3 expression in tumor cells may have a potential prognostic value in CRC patients. Citation Format: Satoru Kondo, Koji Tanaka, Susumu Saigusa, Takahito Kitajima, Tadanobu Shimura, Yuji Toiyama, Yoshinaga Okugawa, Yasuhiro Inoue, Toshimitsu Araki, Keiichi Uchida, Yasuhiko Mohri, Kenichiro Ishii, Masato Kusunoki. Aberrant expression of trefoil factor 3 (TFF3) is associated with tumor progression and an unfavorable prognosis in patients with colorectal 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 1167. doi:10.1158/1538-7445.AM2013-1167

Background. Previously, we have reported the association between the expression of tropomyosin-related kinase B (TrkB) in tumor cells and gastric tumor progression, chemoresistance of esophageal cancer, and epithelial-mesenchymal transition in colorectal cancer. In gastric cancer, high tumoral TrkB mRNA level demonstrated to be an independent prognostic factor. Aim. In this study, we investigated the TrkB protein expression in tumor cells using immunohistochemistry, and evaluated its clinical and prognostic significance in patients with human gastric cancer (GC). Materials and methods. Three hundred and twenty (n=320) samples of GC patients were investigated for the TrkB protein expression in tumor cells using a standard immunohistochemistry (IHC). The IHC score of TrkB in tumor cells was determined on the basis of both staining intensity and the percentage of positive cells. The IHC score was dichotomized at the median value as a cutoff value. The association between the TrkB protein expression in tumor cells and clinicopathological features was analyzed. Impact of TrkB protein expression in tumor cells on overall survival (OS) was also analyzed using univariate and multivariate analyses. Results. Immunoreactive TrkB protein was localized in both cytoplasm and nucleus of gastric cancer cells. High cytoplasmic TrkB expression was significantly associated with larger tumor size (p Conclusions. Tumoral TrkB expression was associated with tumor progression in human gastric cancer. Increased TrkB at the tumor invasive front was an independent prognostic factor in patients with gastric cancer. Citation Format: Koji Tanaka, Tadanobu Shimura, Susumu Saigusa, Satoru Kondo, Takahito Kitajima, Yuji Toiyama, Yoshinaga Okugawa, Yasuhiro Inoue, Masaki Ohi, Toshimitsu Araki, Keiichi Uchida, Yasuhiko Mohri, Masato Kusunoki. Expression of tropomyosin-related kinase B (TrkB) at the tumor invasive front as an independent prognostic factor 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 1144. doi:10.1158/1538-7445.AM2013-1144

Background. Angiopoietin-like protein 2 (ANGPTL2) is a secreted protein belonging to the angiopoietin family.It has been reported to act as a causative mediator of chronic inflammation andmetabolic abnormalities. In ovarian and lung cancer, ANGPTL2 expression in tumor cells was reported to be associated with unfavorable prognosis. So far,clinical and prognostic significance of ANGPTL2 in human gastric cancer remains unknown. Aim. This study investigated the expression of ANGPTL2 in tumor cells using immunohistochemistry and to evaluate its clinical and prognostic significance in patients with human gastric cancer (GC). Materials and methods. Two hundred and eighty two (n=282) samples of GC patients were investigated for the expression of ANGPTL2 in tumor cells using standard immunohistochemistry (IHC). The IHC score of ANGPTL2 in tumor cells was determined on the basis of both staining intensity and the percentage of positive cells. The IHC score was dichotomized at the cutoff value predictive of overall survival using a non-parametric receiver operating characteristic analysis. The association between the expression of ANGPTL2 intumor cells and clinicopathological features was analyzed. Impact of ANGPTL2 expression in tumor cells on overall survival (OS) was also analyzed usingunivariate and multivariate analyses. Results. ANGPTL2 protein was localized mainly in the cytoplasm of tumor cells, and partly in the nucleus. High cytoplasmic ANGPTL2 was significantly associated with higher age (p=0.04), larger tumor size (p In univariate analysis, high cytoplasmic ANGPTL2 and higher cytoplasmic ANGPTL2 at the invasive margin were significantly associated with poor prognosis (p Conclusions. Tumoral ANGPTL2 expression was associated with gastric tumor progression. Increased ANGPTL2 at both tumor center and invasive margin were independent prognostic indicators in patients with gastric cancer. Citation Format: Tadanobu Shimura, Koji Tanaka, Susumu Saigusa, Satoru Kondo, Takahito Kitajima, Yuji Toiyama, Yoshinaga Okugawa, Yasuhiro Inoue, Toshimitsu Araki, Keiichi Uchida, Yasuhiko Mohri, Tsutomu Nobori, Masato Kusunoki. Prognostic value of angiopoietin-like protein 2 (ANGPTL2) at the tumor margins in patients with 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 1139. doi:10.1158/1538-7445.AM2013-1139

Background: Growing evidence indicates that the expression of several miRNAs can be epigenetically regulated through methylation of their promoter CpG sequences. MiR-124 is one such tumor suppressive miRNA, which is expressed at lower levels in various human cancers due to methylation of its promoter region. The present study aimed to determine miR-124 methylation status and determine its clinical significance in sporadic and ulcerative colitis-associated colorectal cancer (CRC). Materials & Methods: We analyzed 579 colorectal tissues, which included 176 sporadic CRC and the corresponding adjacent normal colonic mucosa (NC), 57 colorectal adenomas and 20 normal colonic mucosae from non-cancer patients (NN). In addition, we also examined 135 normal mucosae (N-UC), 12 dysplasia(D-UC) and 12 colitic cancers (C-UC) from UC patients. We performed quantitative bisulfite pyrosequencing analysis to determine miR-124 promoter methylation levels. Results: MiR-124 methylation rate was significantly higher in CRCs and adenomas compared to NC or NN (P Citation Format: Yuji Toiyama, Keun Hur, Koji Tanaka, Yasuhiro Inoue, Takeshi Nagasaka, Masato Kusunoki, C. Richard Boland, Ajay Goel. The clinical significance of tissue miR-124 methylation in sporadic and ulcerative colitis-associated colorectal 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 651. doi:10.1158/1538-7445.AM2013-651

Background: MicroRNAs (miRNAs) are rapidly emerging as potential diagnostic and prognostic markers of human cancer. Recent studies indicate that tumor-derived miRNAs can also be present in human serum and plasma in remarkably stable form. Therefore, specific miRNAs that are frequently over-expressed in cancer tissues present with logical targets for the development of noninvasive miRNA biomarkers in blood. The aim of this study was to investigate whether serum miR-21 can be used as a biomarker for the earlier diagnosis and prognosis of colorectal cancer (CRC). Materials & Methods This study was divided into two steps. The first step included determination of miR-21 expression using quantitative TaqMan assays in a ‘training set’ comprised of pre-operative serum samples from 12 CRC patients and 12 healthy subjects. To enhance assay specificity, we analyzed miR-21 expression in matched CRC and normal tissues from 8 of the 12 patients. The second step included an independent confirmation of these results in a ‘validation set’ of serum samples from 184 CRC patients, 43 patients with advanced adenomas and 53 healthy controls. We then examined miR-21 expression in matched normal and cancer tissue samples from 167 CRC patients. In addition, we analyzed post-operative serum samples from 60 CRC patients. Results: In the training set, miR-21 expression was significantly up-regulated in both serum (p 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 4145. doi:1538-7445.AM2012-4145

Purpose. Smad interacting protein 1 (SIP1) encodes a transcription factor and is one of the epithelial-mesenchymal transition (EMT)-inducible genes that play a key role in tumor progression in various cancers. The aim of this study is to clarify the clinical significance of SIP1 expression in gastric cancer patients, and to clarify its biological function especially in intestinal type gastric cancer. Methods. We studied one hundred thirty-four patients undergoing surgery for gastric cancer. We analyzed SIP1 mRNA levels by real-time reverse transcription PCR in gastric cancer tissue and adjacent normal mucosa, and in gastric cancer cell lines. SIP1 protein expression in primary cancer and in peritoneal dissemination samples was measured using immunohistochemical analysis. Knockdown of the SIP1 gene by siRNA transfection was performed to evaluate SIP1 function in gastric cancer cell. Results. Expression of the SIP1 gene was significantly higher in cancerous tissue than in adjacent normal mucosa (p 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 450. doi:1538-7445.AM2012-450

Background and aim: Metastatic spread is the major cause of mortality in patients with colorectal cancer (CRC). Liver metastasis is the most frequent and thus the main therapeutic target in metastatic CRC patients. The mechanism of colorectal liver metastasis (CLM) is still poorly understood. In this study, we presented high-resolution images of dynamics of tumor-host interaction in liver metastasis model of the living mice using two photon laser scanning microscopy (TPLSM). We also visualized the formation of CLM from single cancer cells to micrometastatic colonies in the same mice by intravital TPLSM at several times. Materials and methods: Red fluorescent protein (RFP) expressing SL4 murine colorectal cancer cell line (RFP-SL4) and green fluorescent protein (GFP)-transgenic mice (GFP mouse) were used as syngeneic tumor model. RFP expressing HT29 human colorectal cancer cell line (RFP-HT29) and GFP nude mice (C57BL/6-BALB/c-nu/nu-EGFP) were used as xenogeneic tumor model. 1×106 RFP expressing cancer cells were injected into the spleen of GFP (nude) mice for an experimental liver metastasis model. Intravital TPLSM was performed by exteriorizing the liver lobe of living mice and fixing it with a solder lug terminal and an adhesive agent (organ stabilizing system, patent number: 2007-129723). Interval TPLSM was also performed for intravital TPLSM at several times in the same mice. Results: Viable cancer cells in the vital liver of living mice were successfully imaged at high-resolution in vivo real-time by TPLSM. Single cancer cells were arrested within hepatic sinusoids 2 hours after injection at a magnification of X600∼X1200. Platelet aggregation surrounding a single cancer cell was observed 2h after injection, indicating a phenomenon of tumor-cell induced platelet aggregation (TCIPA). Cancer cells were extravasated from hepatic sinusoid to Disse space 24h after injection. Protrusions of a Kupffer cell surrounding a cancer cell were observed, indicating that a Kupffer cell appears to be phagocytosing a cancer cell. RFP-SL4 were formed micrometastatic colonies with extensive stromal reaction. Liver metastasis of RFP-HT29 was observed as a clustering of micrometastatic nodules. Conclusions: Our advanced technique using both intravital and interval TLPSM enables a dynamic high-resolution imaging of not only tumor-host interaction in cancer metastasis, but also metastatic process from a single cancer cell to micrometastasis formation in the living animals. 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 5278. doi:10.1158/1538-7445.AM2011-5278

Purpose. Interleukin-6 (IL-6) binds not only to membrane form but also to soluble IL-6 receptor (sIL-6R), derived from the extracellular part of the membrane receptor. Although most soluble receptors act as functional antagonists to their cytokine, sIL-6R plays a role in agonistic activity. The aim of this study was to clarify the relationship between concentration of intra-tumoral soluble interleukin-6 receptor levels and cancer progression in colorectal cancer patients, and to clarify its kinetics with clinical outcome. Methods. We studied 161 patients undergoing surgery for colorectal cancer. We measured concentrations of sIL-6R in tumors and normal mucosa, and in supernatants from colonic cancer cell lines. Expressions of IL-6, membranous IL-6R and gp130 were evaluated by immunohistochemically Results. The net balance between the concentration of sIL-6R in cancer tissue and normal mucosa (sIL-6R Ca/N expression ratio: the cancer tissue sIL-6R concentration divided by normal mucosa sIL-6Rntration) was 1.262 ± 1.156. The decreased sIL-6R Ca/N expression ratio was significantly associated with T classification (p=0.0076), distant metastasis (p=0.0102), UICC stage (p=0.0251) and poor prognosis (p=0.0003). In Cox multivariate analysis, distant metastasis and decreased sIL-6R Ca/N expression ratio were independent risk factors for poor prognosis. Colon cancer cell lines produced sIL-6R, and the production was exaggerated by IL-1beta stimulation, and was suppressed by the addition of IL-1 receptor antagonist. Immunohistochemically, IL-6, membranous IL-6R and gp130 were intensely expressed in cancer cell specifically, and IL-6 expression in cancer cytoplasm was associated with poor prognosis (p=0.0266). The decreased levels of sIL-6R Ca/N expression ratio in cancer tissue was inversely correlated with the intense IL-6 immunoreactivity in cancer cytoplasm (p=0.0088). Conclusion: Relative decrease in sIL-6R in the tumor stroma which reflects increased IL-6/sIL-6R affinity may play a key role in the progression of colorectal carcinoma via IL-6 tran-signaling. 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 1334.

Purpose: Slug is a member of the snail family of transcription factors, plays a crucial role in the regulation of epithelisal-mesenchymal transition(EMT) by suppressing several epithelial markers. In this study, we investigated the clinical significance of Slug expression in colorectal cancer and identified the function of Slug gene in vitro. Methods: Slug gene expression in surgically resected colorectal carcinoma of 184 patients were quantified by real-time quantative reverse transcription-PCR to assess the association between Slug expression and clinicopathologic factors and survival. To identify the function of Slug RNA interference (RNAi) was used to inactivate this gene in the colon cancer cell line SW480. Immunohistochemical analysis was performed to characterize the pattern of Slug expression in colorectal cancer. Results: Slug expression in cancer tissue was significantly higher than that in normal tissue and significantly higher in patients with invasive tumor stage, lymphatic invasion, lymph node involvement, liver metastasis and advanced TNM stage. Increased Slug expression demonstrated a significantly associated with poor prognosis and independent prognostic factor not only in all colorectal cancer patients but also in patients with curative intent. Furthermore, increased Slug expression was an independent marker for predicting lymph node metastasis. Function of Slug gene investigated by RNAi have ability of cell proliferation and invasive ability to basal membrane in cancer cell line and this protein was mainly expressed in the invasive front of the tumor. Conclusions: Slug is up-regulated in human colon cancer and seems to play a role in cancer invasion from primary lesion. Our present data might be suggested that quantification of slug gene in colorectal cancer could be useful marker to identify the patients with lymph node positive or with poor prognosis in curative intent. 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 2249.

Background: The pathogenesis of colorectal cancer in familial adenomatous polyposis (FAP) has been studied genetically. Recently, intestinal stem cell (ISC) or cancer stem cell (CSC) is being focused on colorectal carcinogenesis. The aim of this study is to investigate the immunohistochemical features of stem cell transcription factors on colorectal adenocarcinomas, adenomataous polyps, and normal crypts in patients with FAP. Methods: Lgr5 as an ISC marker, CD133 and CD44 as CSC markers, and NANOG, OCT4, and SOX2 as embryonic stem cell transcription factors were evaluated immunohistochemically. Matched specimens of colorectal adenocarcinoma, adenomatous polyps, and adjacent normal crypts were obtained from individual FAP patients (n=4), to clarify the association between stepwise progression from normal crypt to adenomatous polyp to adenocarcinoma and expression of stem cell transcription factors. Results: Lgr5, NANOG, OCT4, and SOX2 were mainly located in cytoplasm of cancerous and adenomatous cells. CD133 showed cytoplasmic or intraluminal membranous staining in cancerous and adenomatous cells. Immunoreactive CD44 protein was present in cell membrane or cytoplasm of cancerous and adenomatous cells. Immunoreactive positivity and intensity was greater in colorectal adenocarcinomas than in adenomatous polyps, except for NANOG. Adjacent normal crypts showed negative or weak staining of all markers. CD133, CD44, Lgr5, OCT4, and SOX2 were strongly expressed on cancerous cells at the advancing front of colorectal adenocarcinoma. Conclusions: ISC or CSC may be associated with colorectal carcinogenesis of FAP. Stem cell transcription factors may be involved in tumor invasion of colorectal adenocarcinoma in FAP patients. 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 3358.