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Cancer-associated fibroblasts (CAFs) are reportedly involved in invasion and metastasis in several types of cancer, including gastric cancer (GC), through the stimulation of CXCL12/CXCR4 signaling. However, the mechanisms underlying these tumor-promoting effects are not well understood, which limits the potential to develop therapeutic targets against CAF-mediated CXCL12/CXCR4 signaling. CXCL12 expression was analyzed in resected GC tissues from 110 patients by immunohistochemistry (IHC). We established primary cultures of normal fibroblasts (NFs) and CAFs from the GC tissues and examined the functional differences between these primary fibroblasts using co-culture assays with GC cell lines. We evaluated the efficacy of a CXCR4 antagonist (AMD3100) and a FAK inhibitor (PF-573,228) on the invasive ability of GC cells. High CXCL12 expression levels were significantly associated with larger tumor size, increased tumor depth, lymphatic invasion and poor prognosis in GC. CXCL12/CXCR4 activation by CAFs mediated integrin β1 clustering at the cell surface and promoted the invasive ability of GC cells. Notably, AMD3100 was more efficient than PF-573,228 at inhibiting GC cell invasion through the suppression of integrin β1/FAK signaling. These results suggest that CXCL12 derived from CAFs promotes GC cell invasion by enhancing the clustering of integrin β1 in GC cells, resulting in GC progression. Taken together, the inhibition of CXCL12/CXCR4 signaling in GC cells may be a promising therapeutic strategy against GC cell invasion.

Purpose: Cancer cells exhibit altered glucose metabolism, termed glycolysis which is described by the increased uptake of glucose and the conversion of glucose to lactate in cancer cells under adequate oxygen tension. Alternating glucose transporters (GLUTs) expression was detected in the cancer specific metabolism to adapt to the rapid growth and tumor microenvironment in diverse malignant tumors. In this study, GLUTs profiling of ESCC patients, the relation between proliferation and GLUT1 expression using siRNA for GLUT1 were analyzed. Furthermore, the prognostic features according to GLUT1 expression was evaluated on esophageal squamous cell carcinoma (ESCC) patients. Methods: The ratio of the 11 GLUTs (GLUT1- GLUT14) expression between tumor and normal tissue (T/N) were analyzed in silico (GDS3838). GLUT1 expression was down-regulated using 3 siRNAs in vitro. Immunohistochemical staining of GLUT1 was performed using paraffin sections of tissues obtained from 145 resectable ESCC patients without preoperative treatment. Results: Expression of GLUT1 (T/N 2.44; 95% confidence interval (CI): 1.78-3.34, P Conclusions: GLUTs expression was altered in ESCC patients and GLUT1 expression was most up-regulated among the 11 GLUTs. Growth assay with siRNAs for GLUT1 revealed that GLUT1 played a curial role of ESCC cells proliferation. High expression of GLUT1 was associated with depth of invasion and poor prognosis of ESCC patients. These findings provide evidence of the significance of GLUT1 expression as a biomarker and GLUT1 may be therapeutic target in ESCC patient. Citation Format: Hiroshi Sawayama, Masaaki Iwatsuki, Naoya Yoshida, Yoshifumi Baba, Yasuo Sakamoto, Koichi Kinoshita, Kenichi Nakamura, Daisuke Kuroda, Eto Tsugio, Tasuku Toihata, Tomoyuki Uchihara, Eri Oda, Taisuke Yagi, Mayuko Ohuchi, Takatugu Ishimoto, Hideo Baba. Glucose transporter 1 is associated with proliferation and prognosis on esophageal squamous cell carcinoma [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 4407. doi:10.1158/1538-7445.AM2017-4407

Background: Peritoneal metastasis is one of the most frequent causes of death in patients with advanced gastric carcinoma (GC). However, molecular mechanisms driving peritoneal dissemination still remain poorly understood. Here, we aimed to give new insights to the molecular mechanisms that drive the peritoneal dissemination of GC. This study identified candidates of potential regulators of peritoneal dissemination and focused on the significance of one of them in vitro experiments and clinical samples. Methods: We utilized previously reported combined expression analysis with two cell lines and samples and 200 GC patients to identify driver genes of peritoneal dissemination. The cell lines consist of HSC58, an established cell lines from scirrhous type primary GC and its progeny cell line, 58As9, with in vivo-selected highly peritoneal metastatic potential. In vitro experiments, we utilized the two gastric cancer cell lines, 58As9 and NUGC4 and evaluated the effect of SERPINE2 knockdown on proliferation, spheroid formation, invasion, and motility. As for evaluation of clinical samples, 176 primary gastric carcinomas were included, which were obtained from patients who underwent curative gastrectomy in Kumamoto university between 2005 and 2015. We evaluated expression of SERPINE2 in immunohistochemistry and divided the cohort into two groups by SERPINE2 expression and analyzed relationship with clinicopathological factors and survival. Results: Enrichment analyses of the profile of the peritoneal dissemination-associated expression signature revealed that poor prognosis and peritoneal metastasis was related with the two pathways, focal adhesion (FDR q=0.00195) and extracellular matrix (FDR q=0.00195). Among the genes related with extracellular matrix, Serine Proteinase Inhibitor, Clade E, Member 2 (SERPINE2) was found to be highly expressed in 58As9 compared with HSC58, and we focused on SERPINE2 as a candidate of potential regulators of peritoneal dissemination. In vitro experiments, the SERPINE2 knockdown was related with decrease in cell motility and invasion, but not with decrease in proliferation, spheroid formation. Immunohistochemistry analysis of the clinical samples revealed higher SERPINE2 expression was significantly related with invasion of serosa and lymphatic vessels, and lymph node matastasis, and peritoneal metastatic metastasis. Survival analysis also revealed SERPINE2 expression was related with poor peritoneal recurrence-free survival (p=0.038), and peritoneal metastasis-specific survival (p=0.036). Conclusion: We found SERPINE2 as a candidate of driving molecules of peritoneal metastasis partly because it promotes invasive potential. Further study including in vivo study is required to reveal relationship of SERPINE2 with the other molecules and pathways. Citation Format: Daisuke Kuroda, Junji Kurashige, Hiroshi Sawayama, Masaaki Iwatsuki, Tomoyuki Uchihara, Tasuku Toihata, Eri Oda, Taisuke Yagi, Tsugio Eto, Keisuke Miyake, Mayuko Ouchi, Kenichi Nakamura, Koichi Kinoshita, Kousuke Mima, Takatsugu Ishimoto, Yasuo Sakamoto, Yoshifumi Baba, Naoya Yoshida, Koshi Mimori, Hideo Baba. Significance of SERPINE2 expression in peritoneal metastasis in gastric carcinoma [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 1336. doi:10.1158/1538-7445.AM2017-1336

Background: Cancer cells consume large amount of energy and maintain high levels of anabolism to promote cell proliferation via metabolic reprogramming. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master transcription regulator of stress and promotes metabolic reprogramming to support cell proliferation in various type of cancers. In particular, esophageal cancer was regarded as one of the most aggressive tumors of gastrointestinal cancer. However, the effect of metabolic reprogramming in esophageal cancer tissues was not fully understood. In this study, we investigated the prognostic significance of Nrf2, impacts on metabolic reprogramming and reactive oxygen species (ROS) detoxification in esophageal cancer. Methods: We immunohistochemically evaluated the protein expression of Nrf2 in the surgically resected specimens in 201 esophageal cancer patients. By using esophageal cancer cell lines, we examined the change of phenotype and the regulation of ROS, compared control cells with Nrf2 depletion cells. Moreover, we measured the levels of metabolites in the esophageal cancer cell lines by using mass spectrometry and identified the metabolic pathway Nrf2 promoted. Results: We revealed that high expression of Nrf2 in esophageal cancer was associated with tumor recurrence (P=0.0004) and poor prognosis (P Conclusions: The expression of Nrf2 was up-regulated in some esophageal cancer cell lines and resected tumor specimen. And, high expression of Nrf2 in esophageal cancer was associated with poor prognosis. Our in vitro assay revealed that Nrf2 appeared to promote esophageal cancer cell proliferation via metabolic reprogramming and ROS detoxification. Therefore, strategies to pharmacologically manipulate the level and / or activity of Nrf2 may have potential to reduce tumor growth and improve the prognosis of esophageal cancer patients. Citation Format: Yuki Kitano, Yoshifumi Baba, Shigeki Nakagawa, Kosuke Mima, Hiroshi Sawayama, Masaaki Iwatsuki, Yasuo Sakamoto, Yo-ichi Yamashita, Naoya Yoshida, Hideo Baba. Nrf2 promotes esophageal cancer cell proliferation via metabolic reprogramming and ROS detoxification [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 3556. doi:10.1158/1538-7445.AM2017-3556

Background: Colorectal cancer is one of the most general causes of cancer related death. Ring Finger Protein 43(RNF43) is an E3 ubiquitin ligase that suppresses the Wint signaling pathway and is known as a tumor suppressor gene. Previous studies reported RNF43 mutation leads to inactivation of RNF43 in pancreatic, ovarian, colorectal cancer and so on. Subsequently, Wnt signaling activation is associtated with tumor progression in these types of cancers. However, functional role of RNF43 for tumor formation is not clear in colorectal cancer. Aim: To investigate the functional role of RNF43 in colorectal cancer by using RNF43 knockout mice. Method: We prepared RNF43 knockout mice by using CRISPR/Cas9 system. To induce colorectal tumor in mice, we used Azoxymethane-Dextran sulfate sodium (AOM-DSS) model. First, we injected AOM 10mg/kg into the peritoneal cavity. After 7days from injection, drunk 2% DSS water to mice for 7days. We sacrificed the mice and evaluated the tumor incidence and tumor size after 8, 10, 12, and 16 weeks from injection. Next, we prepared the small intestine organoid from RNF43 wild and knockout mice. Then we compared the organoid size and formation rate. In vitro analysis, we prepared colon cancer cell lines (COLO205, SW620, HCT116) and evaluated the effect on cell proliferation in suppression of RNF43 and normal condition. Result: There was no significant difference in the tumor incidence and tumor size at 8, 10, and 12weeks after AOM injection. However, at 16 weeks, the tumor size of RNF43 knockout mice was significantly bigger than that of RNF43 wild type mice. (p Conclusion: Our results suggest that RNF43 expression suppresses epithelial cell growth in the intestine, and the dysregulation of Wint signaling via RNF43 disruption promote intestinal stem cell expansion and colorectal tumorigenesis. Citation Format: Tsugio Eto, Takatsugu Ishimoto, Eri Oda, Daisuke Kuroda, Kota Arima, Mayuko Ohuchi, Kenichi Nakamura, Hiroshi Sawayama, Koichi Kinoshita, Masaaki Iwatsuki, Yoshifumi Baba, Yasuo Sakamoto, Naoya Yoshida, Hideo Baba. Functional role of Ring Finger Protein 43 in intestinal stem cell during colorectal tumorigenesis [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 4825. doi:10.1158/1538-7445.AM2017-4825

CD44 is frequently overexpressed in a wide variety of epithelial malignancies including gastrointestinal cancer and causes resistance to currently available treatments. MicroRNAs (miRNAs) are non-coding RNAs that regulate molecular pathways in cancer by targeting various genes. The aim of this study was to investigate the regulation of CD44 expression by miRNAs and to develop new molecular targets in gastrointestinal cancer. We performed miRNA screening in six human gastrointestinal cancer cell lines and identified three candidate miRNAs that could regulate CD44 expression in gastrointestinal cancer. Among these, we focused on miR-328 and examined its functional relevance using growth assays and cytotoxicity assays. CD44 expression was reduced in gastrointestinal cancer cell lines forced to express miR-328, leading to inhibition of cancer cell growth in vitro and in vivo, and impaired resistance to chemotherapeutic drugs and reactive oxygen species (ROS). In contrast, induction of CD44 expression by miR-328 inhibitor led to promotion of cancer cell growth. Furthermore, we revealed that ROS produced by macrophages triggered CD44 expression through suppression of miR-328 in gastric cancer cells. Finally, tumor-infiltrating macrophages (CD68 and CD163) were closely related to both miR-328 downregulation and CD44 upregulation in 63 patients with surgically resected gastric cancer. These findings suggest that macrophages in the tumor microenvironment may cause increased CD44 expression through miR-328 suppression, resulting in tumor progression by enhancing ROS defense. miR-328-CD44 signaling mediated by macrophages may thus represent a potential target for the treatment of gastrointestinal cancer.

Bmi1 is overexpressed in a variety of human cancers including gastrointestinal cancer. The high expression level of Bmi1 protein is associated with poor prognosis of gastrointestinal cancer patients. On the other hand, tumor-associated macrophages (TAMs) contribute to tumor growth, invasion, and metastasis by producing various mediators in the tumor microenvironment. The aim of this study was to investigate TAM-mediated regulation of Bmi1 expression in gastrointestinal cancer. The relationship between TAMs and Bmi1 expression was analyzed by immunohistochemistry and quantitative real-time PCR (qRT-PCR), and results showed a positive correlation with tumor-infiltrating macrophages (CD68 and CD163) and Bmi1 expression in cancer cells. Co-culture with TAMs triggered Bmi1 expression in cancer cell lines and enhanced sphere formation ability. miRNA microarray analysis of a gastric cancer cell line co-cultured with macrophages was conducted, and using in silico methods to analyze the results, we identified miR-30e* as a potential regulator of Bmi1 expression. Luciferase assays using miR-30e* mimic revealed that Bmi1 was a direct target for miR-30e* by interactions with the putative miR-30e* binding sites in the Bmi1 3′ untranslated region. qRT-PCR analysis of resected cancer specimens showed that miR-30e* expression was downregulated in tumor regions compared with non-tumor regions, and Bmi1 expression was inversely correlated with miR-30e* expression in gastric cancer tissues, but not in colon cancer tissues. Our findings suggest that TAMs may cause increased Bmi1 expression through miR-30e* suppression, leading to tumor progression. The suppression of Bmi1 expression mediated by TAMs may thus represent a possible strategy as the treatment of gastrointestinal cancer.

741 Background: The number of patients with colorectal cancer has been increasing in all over the world. Approximately 10 percent of CRC is complicated by obstructive symptoms at the time of their diagnosis. Obstructive colorectal cancer (OCRC) could be a fatal because of perforation peritonitis or sepsis, and it is necessary to immediate treatment. The aim of this study was to explore the treatment strategies for patients with OCRC. Methods: Between April 2008 and December 2014, six hundred seventy two patients underwent surgery in out institute. The numbers of OCRC were 82 cases (12.2%). We evaluated the feasibility and effectiveness of our treatments for OCRC. Results: Treatment strategies were adopted according to the location of CRC. For patients with right-sided OCRC, we performed one-stage surgery. They underwent primary tumor resection and anastomosis at the same time. For left-sided OCRC, patients were treated by two-stage surgery. At first, they were managed by decompression of their colon with colostomy or transanal ileus tube, and then, underwent colectomy. The cases of right-side OCRC were 23 cases, and all of them underwent one-stage surgery. We could find no cases of anastomotic leakage in these cases. The cases of left-side OCRC were 59 cases, and 50 patients (86%) of them were treated two-stage surgery. Stage IV OCRC patients tended to undergo colostomy only. Conclusions: One-stage surgery was feasible for patients with right-sided OCRC. For left-sided OCRC cases, two-stage surgery was effective to prevent perioperative complications.

The purpose of this study was to evaluate the mesh repair for an incarcerated groin hernia. A total of 110 patients who underwent emergency surgery for incarcerated hernias were retrospectively analyzed using a multivariate analysis. The postoperative complications were associated with bowel resection, odds ratio (OR) 2.984, and 95 % confidence interval (CI) 1.273 to 6.994. The risk factors for bowel resection were femoral hernia, (OR 5.621, 95 % CI 2.243 to 14.082), and late hospitalization (24 h

The purpose of this study was to predict the postoperative organ derangement before surgery and to select the optimal surgical methods for a perforation due to colorectal cancer. The Mannheim Peritonitis Index (MPI) was used to determine the preoperative status and Sequential Organ Failure Assessment (SOFA) score for postoperative status, retrospectively. There were 25 cases of colorectal cancer-related colon perforation. These patients were classified as having a preoperative MPI ≤ 29 or ≥ 30 and the mean postoperative SOFA scores were 2.30 ± 2.45 and 7.93 ± 3.45 (p = 0.0002), respectively. Seven of 20 patients who underwent tumor resection received a bowel anastomosis without a stoma. The MPI of these seven patients was low (23.1 ± 7.47) and there were no severe postoperative organ disorders (SOFA score 1.86 ± 2.26). Eighteen of the 25 patients had no distant metastasis. Eight of these 18 patients underwent lymph node (LN) dissection, including intermediate LNs, while the other 10 cases only had the pericolic/perirectal LNs dissected. The average preoperative MPI was 23.9 ± 7.68 and 32.8 ± 5.77, and the postoperative SOFA scores were 3.75 ± 3.01 and 7.10 ± 5.34, respectively. The MPI could be used to predict postoperative organ disorders and thus represents a useful index that can be used to determine the optimal surgical methods.

The central venous access port (CV-port) system was examined in a series of colorectal cancer (CRC) patients.One hundred and one CRC patients underwent chemotherapy with the 5-fluorouracil + oxaliplatin (FOLFOX) or 5-fluorouracil + irinotecan regimen. The complications of the CV-port system were retrospectively assessed.The CV-port system was placed in a total of 101 patients. The patients received a total of 1020 [corrected] courses of these regimens. Eight complications occurred in the 101 patients (7.9%). The complications included three instances of catheter rupture, two thrombotic events around the catheter, and three infections at the site of the port or catheter. The complications were identified after a median of nine courses (range 6-16) and 135 days after the placement of the CV-port system. Sixty-six of the 101 patients switched their regimen from FOLFOX to another regimen, and 4 of these 66 patients (6.1%) experienced complications associated with the CV-port system. There were 25 subjects who were admitted to the hospital emergency wing during the chemotherapeutic regimens, and 4 of these patients (16%) had complications associated with the CV-port system.The complications of the CV-port system occurred at a defined rate, therefore the early diagnosis and the appropriate treatment to address these complications is crucial.

The quantitative expansion and the qualitative changes of the short-term financial market in Japan since the Japan-U.S. Dollar-Yen Committee Report, published in May 1984, has indeed been remarkable.

Purpose: Gastric cancer (GC) is the fourth most commonly diagnosed cancer and the third leading cause of cancer mortality worldwide. The range of therapeutic strategies available for the treatment of GC has improved in recent decades. However, the prognosis of patients with advanced GC remains poor even after curative resection, mainly because of recurrence, such as peritoneal dissemination and liver metastases. Previous studies have shown that the interaction between cancer cells and their surrounding stroma plays an important role in tumor development. Cancer-associated fibroblasts (CAFs) have been reported to be involved in invasion and metastasis in cancers, including gastric cancer (GC), via their stimulation of CXCL12/CXCR4 signaling. However, the mechanisms underlying the tumor-promoting effects and the potential of developing therapeutic targets using CXCL12/CXCR4 signaling activated by CAFs are not fully understood. Experimental Design: We first analyzed CXCL12 expression in resected GC tissues of 110 patients by immunohistochemistry (IHC). We next established primary normal fibroblasts (NFs) and CAFs from GC tissues and examined the functional differences between these primary fibroblasts using co-culture assays with GC cell lines. Furthermore, we evaluated the efficacy of a CXCR4 antagonist (AMD3100) and a FAK inhibitor (PF-573,228) on the invasive ability of GC cells. Results: We determined that high CXCL12 expression was significantly associated with larger tumor size, deeper tumor depth, lymphatic invasion and poor prognosis in GC. We established new primary CAFs and their adjacent NFs from resected stomach tissue in GC patients. We evaluated the change in mobility during the direct co-culture of GC cells and fibroblasts using real-time imaging. The motility of GC cells significantly increased upon direct co-culture with CAFs more than it did in cells with NFs compared with control RPMI during observation for 24 hours. The motility of GC cells with CAFs was effectively suppressed by AMD 3100 compared to GC cells with NFs. CXCL12/CXCR4 activation by CAFs mediated the clustering of integrin β1 on the cell surface and promoted the invasive ability of GC cells. Notably, AMD3100 was more efficient at inhibiting GC cell invasion through the suppression of integrin β1/FAK signaling compared with PF-573,228. Conclusions: These results suggest that CXCL12 derived from CAFs promotes invasive ability by enhancing the clustering of integrin β1 in GC cells and results in GC progression. The current data provide evidence that CXCL12/CXCR4 inhibition by AMD3100 is more efficient in suppressing GC cell invasion by not only inhibiting intracellular signaling but also by attenuating the interaction between cancer cells and the extracellular matrix compared with PF-573,228. Our present study represents the rationale for establishing a therapeutic strategy targeting CXCL12/CXCR4 signaling. Citation Format: Daisuke Izumi, Takatsugu Ishimoto, Hidetaka Sugihara, Eto Kojiro, Hiroshi Sawayama, Keisuke Miyake, Yuki Kiyozumi, Keisuke Kosumi, Ryuma Tokunaga, Kazuto Harada, Junji Kurashige, Masaaki Iwatsuki, Shiro Iwagami, Yoshifumi Baba, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Masayuki Watanabe, Hideo Baba. CXCL12/CXCR4 activation by cancer-associated fibroblasts promotes integrin β1 clustering and invasive ability in gastric 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 423. doi:10.1158/1538-7445.AM2015-423

Cancer cells exhibit altered glucose metabolism, mitochondrial dysfunction, anaerobic glycolysis and upregulation of the pentose phosphate pathway (PPP). Recent genetic and metabolic analyses have provided insights into the molecular mechanisms of genes that are involved in the alteration of cancer metabolism and tumorigenesis. Hypoxic induced factor 1 regulates the reciprocal relationship between glycolysis and oxidative phosphorylation, and p53 also modulates the balance between the glycolytic pathway and oxidative phosphorylation. Mitochondria function in cancer differs from that in normal cells owing to mutations of mitochondrial DNA and alterations of metabolism. Overexpression of transcription factors, metabolite transporters and glycolytic enzymes is observed and associated with poor prognosis, and it may be associated with chemoradiotherapy resistance in multiple cancer cell types. The PPP plays a critical role in regulating cancer cell growth by supplying cells with ribose-5-phosphate and nicotinamide adenine dinucleotide phosphate for detoxification of intra-cellular reactive oxygen species (ROS), reductive biosynthesis and ribose biogenesis. ROS levels increase during carcinogenesis owing to metabolic aberrations. This review discusses alterations of mitochondrial metabolism, anaerobic glycolysis, the PPP and control of ROS levels by the endogenous anti-oxidant system in cancer, as well as the novel small molecules targeting these enzymes or transporters that exert anti-proliferative effects.

Objectives: Gastric cancer is one of the most threatening malignancies with a high incidence and metastasis rate. Many patients are diagnosed in an advanced stage sometimes with multi-organ metastasis, which leads a poor prognosis of gastric tumors. Chemokine and their receptors have been originally demonstrated as essential mediators of leukocyte directional migration, particularly during infection and inflammation, and have further emerged as crucial players in all stages of tumor development. It has been reported that an important CXCL12-CXCR4 signaling is involved in tumor development and metastasis in several types of cancer including gastric cancer. Cancer associated fibroblasts (CAF) are reported to communicate microenvironment-derived signals through chemokine/chemokine receptor interaction such as CXCL12-CXCR4 signaling. The aim of our study was to evaluate the role of CXCL12-CXCR4 signaling in crosstalk between gastric cancer and CAF. Methods and Results: We evaluated CXCL12 and CXCR4 expression status by immunohistochemistry using a nonbiased database of 89 curatively resected gastric cancers. As a result, CXCL12/ CXCR4 expression are significant correlation with metastasis. Furthermore, we performed Western blotting analysis for CXCL12 and CXCR4 in human gastric cancer cell lines. CXCR4 expression was detectable in all gastric cancer cell lines. We isolated CAF from human gastric cancer tissue resected in our institute. We performed Western blotting analysis for alpha-SMA and CXCL12 in CAF, and confirmed higher expression in CAF than normal fibroblast (NF) isolated from normal gastric mucosa of same patients. Then we established stable AGS gastric cancer cell line expressing GFP. Invasion assay revealed that AGS (gastric cancer cell line) cells with CAF showed more invasive phenotype than that without CAF. We also found the motility of GFP-expressing AGS was elevated during direct co-culture of GFP-expressing AGS cells and CAF compared with NF. Conclusions: These findings revealed that cancer associated fibroblasts were implicated in cancer cell invasion and metastasis through CXCL12-CXCR4 signaling in gastric cancer. The therapeutic blockade of this pathway with CXCR4 antagonist might abrogate gastric cancer cell invasion and metastasis. Citation Format: Daisuke Izumi, Takatsugu Ishimoto, Hietaka Sugihara, Hiroshi Sawayama, Ryuichi Karashima, Satoshi Ida, Yu Imamura, Shiro Iwagami, Yoshifumi Baba, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. Cancer associated fibroblasts stimulates cancer cell invasion through CXCL12-CXCR4 signaling 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 185. doi:10.1158/1538-7445.AM2014-185

Background: CD44 has recently been identified as one of the cell surface markers associated with cancer stem cells (CSCs) in several types of tumor. We have reported the functional role of CD44 variant isoform in the maintenance of low reactive oxygen species (ROS) levels in gastrointestinal cancer cells. While, microRNA (miRNA) is involved in the pathogenesis of many cancers and the networks of miRNA regulate CSCs properties. However, the mechanism underlying the miRNA in the regulation of CSC marker is mostly unrevealed. Objectives: The aim of this study was to investigate the regulation of CD44 expression by miRNAs and to develop newmolecular targets in gastrointestinal cancer. Methods and Results: To investigate the mechanism in the regulation of CD44, We performed miRNA screening in six human gastrointestinal cancer cell lines and identified three candidate miRNAs that could regulate CD44 expression in gastrointestinal cancer. Among these, we focused on miR-328 and examined its functional relevance using growth assays and cytotoxicity assays. CD44 expression was reduced in gastrointestinal cancer cell lines forced to express miR-328, leading to inhibition of cancer cell growth in vitro and in vivo, and impaired resistance to chemotherapeutic drugs and reactive oxygen species (ROS). In contrast, induction of CD44 expression by miR-328 inhibitor led to promotion of cancer cell growth. Furthermore, we revealed that ROS produced by macrophages triggered CD44 expression through suppression of miR-328 in gastric cancer cells. Finally, tumor-infiltrating macrophages (CD68 and CD163) were closely related to both miR-328 down-regulation and CD44 up-regulation in 63 patients with surgically-resected gastric cancer. Conclusions: Macrophages in the tumor microenvironment may cause increased CD44 expression through miR-328 suppression, resulting in tumor progression by enhancing ROS defense. miR-328-CD44 signaling mediated by macrophages may thus represent a potential target for the treatment of gastrointestinal cancer. Citation Format: Takatsugu Ishimoto, Naoya Yoshida, Hidetaka Sugihara, Daisuke Izumi, Keisuke Miyake, Hiroshi Sawayama, Yu Imamura, Shiro Iwagami, Yoshifumi Baba, Hideo Baba. Macrophage-derived reactive oxygen species suppress miR-328 targeting CD44 in gastrointestinal cancer cells and promote redox adaptation. [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 3598. doi:10.1158/1538-7445.AM2014-3598

Background: Bmi1 is a member of the polycomb-repressive complex 1 with an essential role in maintaining chromatin silencing. Bmi1 plays a function in the self-renewal of neuronal and hematopoietic stem cells through repression of the INK4A/ARF locus. Furthermore, Bmi1 is overexpressed in a variety of human cancers. The expression of Bmi1 is associated with poor prognosis of gastrointestinal cancer patients. On the other hand, solid tumors consist of cancer cells and various types of stromal cells, fibroblasts, endothelial cells and hematopoietic cells, mainly macrophages and lymphocytes. Tumor-associated macrophages (TAMs) contribute to tumor progression by producing various mediators. This study was performed to identify the tumor-associated macrophages (TAMs)-mediated regulation of Bmi1 expression in gastrointestinal cancers. Method: The relationship between the expression of Bmi1 and TAMs was assessed by immunohistochemistry and quantitative real-time PCR (qRT-PCR), and 3D sphere culture. Next, miRNAs microarray in gastric cancer cell co-cultured with macrophages was conducted. To examine the functional relevance of miR-30e* expression, we analyzed the relationship between miR-30e* and Bmi1 expression in high Bmi1 expressing cancer cell lines transfected with miR-30e* mimics, and low Bmi1 expressing cancer cell lines transfected with miR-30e* inhibitors. And we investigated if miR-30e* directly targets the 3′ UTR of Bmi1 using constructs containing the putative miR-30e* target site or a mutated sequence of the 3′ UTR of Bmi1 cloned immediately downstream of a luciferase gene. Furthermore Bmi1 and miR-30e* expression were assessed in cancer tissues. Results: We revealed the positive relationship between with tumor-infiltrating macrophages and Bmi1 expression in cancer cells. We showed co-culture with TAMs triggered Bmi1 expression in cancer cell lines and enhanced sphere formation ability. Based on microRNA screening analysis and in silico microRNA search, we focused on miR-30e* that could regulate Bmi1 expression. Western blot analysis revealed significantly reduced Bmi1 protein levels in high Bmi1 expressing cancer cell lines transfected with miR-30e* mimics compared with controls, and increased levels in low Bmi1 expressing cancer cell lines cells transfected with miR-30e* inhibitors compared with controls. And Bmi1 was a direct target for miR-30e* by interactions with the putative miR-30e* binding sites. MiR-30e* expression was down-regulated in tumor region compared with in non-tumor one. Furthermore Bmi1 expression was inverse correlation with miR-30e* expression. Conclusions: TAMs may cause increased Bmi1 expression through miR-30e* suppression, leading to tumor progression. Citation Format: Hidetaka Sugihara, Takatsugu Ishimoto, Daiauke Izumi, Hiroshi Sawayama, Yu Imamura, Satoshi Ida, Shiro Iwagami, Yoshifumi Baba, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. Novel discovery of miR-30e* regulating Bmi1 expression induced by tumor-associated macrophages in gastrointestinal 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 4346. doi:10.1158/1538-7445.AM2014-4346

Purpose: Glucose transporter type 1 (Glut1) plays a crucial role in cancer specific metabolism to adapt to the rapid growth and tumor microenvironment in diverse malignant tumors. We recently reported the relationship between Glut1 status and 18F-FDG uptake. PET-positive lesions had higher Glut1 expression than PET-negative lesions in both primary tumors and metastatic lymph nodes. In this study, it was showed that the clinical, pathological and prognostic features of Glut1 expression on primary lesions of esophageal squamous cell carcinoma. Methods: Immunohistochemical staining of Glut1 and CD34 was performed using paraffin sections of tissues obtained from 145 resectable esophageal squamous cell carcinoma patients without preoperative treatment. Microvessel density was calculated from CD34 staining. Results: Glut1 positivity was observed in 41 patients (28.2%) and associated with depth of invasion (odds ratio (OR)=2.984; 95% confidence interval (CI): 1.208-7.371; P=0.018) and vascular invasion (OR=2.771; 95% CI: 1.118-6.871; P=0.028) in multivariate analysis. Glut1 positivity was a significant disadvantage to both relapse-free survival (hazard ratio (HR)=2.021; 95% CI: 1.100-3.712; P=0.023) and esophageal cancer-specific survival (HR=2.223; 95% CI: 1.121-4.411; P=0.022) in univariate Cox hazard analysis, but was not independently associated with relapse-free survival or cancer-specific survival in multivariate analysis. The relationship between Glut1 expression and first relapse site was investigated. Glut1 positivity was not associated with lymph node recurrence (HR 1.009; 95% CI: 0.402-2.530; P=0.985) but was significantly associated with hematogenous recurrence (HR=3.701; 95% CI: 1.655-8.273; P=0.001) in univariate Cox hazard analysis. Microvessel density was calculated to evaluate angiogenesis and high microvessel density was associated with invasion depth (P=0.044) and vascular invasion (P=0.016). The median microvessel densities for Glut1-negative tumors and Glut1-positive tumors were 38.0 and 54.0, respectively. The microvessel density for Glut1-positive tumors was significantly higher than that of Glut1-negative tumors (Mann-Whitney U-test: P Conclusions: Glut1 expression was associated with hematogenous recurrence. The relationship between Glut1 expression and hematogenous recurrence may stem from an acquired survival ability resulting from upregulated Glut1 and angiogenesis to adapt to the tumor microenvironment. The findings provide evidence of the significance of Glut1 expression as a biomarker in esophageal squamous cell carcinoma. Citation Format: Hiroshi Sawayama, Takatsugu Ishimoto, Masayuki Watanabe, Naoya Yoshida, Yoshifumi Baba, Hidetaka Sugihara, Daisuke Izumi, Hideo Baba. Glucose transporter 1 positivity on primary lesions of esophageal squamous cell carcinoma is associated with hematogenous recurrence. [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 3357. doi:10.1158/1538-7445.AM2014-3357

e15020 Background: Bmi1 is a member of the polycomb-repressive complex 1 with an essential role in maintaining chromatin silencing. The expression of Bmi1 is associated with poor prognosis of gastr...

Background: Peroxisome proliferator-activated receptor gamma (PPARγ) inhibits the proliferation of several cancers. Efatutazone, a novel third-generation thiazolidinedione PPARγ agonist, demonstrated acceptable tolerability with evidence of disease control in patients with advanced alignancies in the phase 1 study. However, the status and function of PPARγ in esophageal squamous cell carcinoma (ESCC) remains unclear and the effects of the PPARγ agonist are unknown. Methods: Immunohistochemical staining was performed using paraffin sections of specimens obtained from 145 ESCC patients who underwent curative resection. The anti-tumor effects of Efatutazone were investigated in ESCC cell lines both in vitro and in vivo. Results: The expression of PPARγ was detected in the nucleus of the normal esophageal squamous epithelium in all patients, while positive staining in ESCC was observed in 24.8% of the patients. The expression of PPARγ decreases according to the depth of invasion and exhibits an inverse relationship with the expression of ki-67. Efatutazone inhibited the proliferation of the ESCC cell lines in a time- and dose-dependent manner in vitro. The anti-proliferation effect of Efatutazone was more evident in cell lines with high PPARγ expression than in those with low PPARγ expression. Efatutazone inhibited the sphere formation of TE-4, which is one of the high PPARγ expression cell lines. Efatutazone upregulated the cell cycle kinase inhibitor p21 WAF1 / CIP1 and induced G1 arrest. We confirmed that the antitumor effect of this drug occurred in a PPARγ dependent manner using si-RNA for PPARγ. In a mouse xenograft model established with TE-4, we compared three arms: the control group (n=8), the Troglitazone group (10 mg/kg, n=8) and the Efatutazone group (10 mg/kg, n=8). Although tumor volume did not differ between the control group and the Troglitazone group, 50.4±13.6% (average ± SD) reduction in tumor volume was observed in the Efatutazone group compared with the control group. The mRNA expression of PLIN-2, a transcription product of PPARγ, was two-fold higher in the Efatutazone group than in the control group. Immunohistochemical staining demonstrated enhanced p21 WAF1 / CIP1 expression and decreased the ki-67 expression in the Efatutazone group in comparison to either the control or Troglitazone group. Conclusions: The expression of PPARγ decreased in ESCC compared with normal esophageal epithelium. The anti-proliferation effect of the third-generation PPARγ agonist, Efatutazone, for ESCC was observed both in vitro and in vivo. Efatutazone is a candidate for novel anti-cancer agent against ESCC. Citation Format: Hiroshi Sawayama, Takatsugu Ishimoto, Masayuki Watanabe, Naoya Yoshida, Yoshifumi Baba, Masaaki Iwatsuki, Hidetaka Sugihara, Hiroshi Onuma, Yoshinobu Shiose, Hideo Baba. The proliferation-inhibiting effects of Efatutazone, a novel third-generation peroxisome proliferator-activated receptor gamma agonist, on esophageal squamous cell carcinoma in vitro and in vivo. [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 5511. doi:10.1158/1538-7445.AM2013-5511