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Purpose: Cancer stem cells (CSCs) are responsible for cancer progression, metastasis, and recurrence. The identification of CSC markers may open a new therapeutic perspective on the basis of selectively targeting this small population of cancer cells, however, the specific markers of CSCs remain undiscovered. The aim of this study was to identify novel biomarkers of gastric CSCs for clinical diagnosis using proteomics technology. Materials and Methods: Since the side population (SP) cells of gastric cancer possess CSC-like properties, we used CSC-like SP cells, OCUM-12/SP cells and OCUM-2MD3/SP cells in this study. Their parent OCUM-12 cells and OCUM-2MD3 cells were also used. Protein lysates from each cell line were analyzed using QSTAR Elite Liquid Chromatography with Tandem Mass Spectrometry, coupled with isobaric tags for relative and absolute quantitation technology. Proteins overexpressed in SP cells were determined by comparing the proteomes among parent cells and CSC-like SP cells. Candidate proteins detected by proteomics technology were validated by RT-PCR, western blot, invasion assay, and immunohistochemical analysis of 300 gastric cancers. Results: Based on the results of LC-MS/MS, eight proteins, including RBBP6, GLG1, VPS13A, DCTPP1, HSPA9, HSPA4, ALDOA, and KRT18, were up-regulated in both OCUM-12/SP cells and OCUM-2MD3/SP cells when compared to their corresponding parent cells. RT-PCR analysis indicated that the expression level of RBBP6, HSPA4, DCTPP1, HSPA9, VPS13A, ALDOA, GLG1, and CK18 was high in OCUM-12/SP and OCUM-2MD3/SP, in compared with the control of parent OCUM-12 and OCUM-2MD3. These proteins were significantly associated with advanced invasion depth, lymph node metastasis, distant metastasis, or advanced clinical stage. RBBP6, DCTPP1, HSPA4, and ALDOA expression in particular were significantly associated with a poor prognosis in the 300 gastric cancer patients. RBBP6 was determined to be an independent prognostic factor. The motility-stimulating ability of OCUM-12/SP cells and OCUM-2MD3/SP cells was inhibited by RBBP6 siRNA. Conclusion: The eight proteins, RBBP6, GLG1, VPS13A, DCTPP1, HSPA9, HSPA4, ALDOA, and KRT18, utilizing comparative proteomics analysis, were perceived to be potential CSC markers of gastric cancer. Of the eight candidate proteins, RBBP6 was suggested to be a promising prognostic biomarker and a therapeutic target for gastric cancer. Citation Format: Masakazu Yashiro, Tamami Morisaki, Tsuyoshi Hasegawa, Anna Kakehashi, Haruhito Kinoshita, Tatsunari Fukuoka, Hiroaki Kasashima, Go Masuda, Katsunobu Sakurai, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Hideki Wanibuchi, Kosei Hirakawa. Novel biomarkers for gastric cancer stem cells utilizing comparative proteomics analysis. [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 1405. doi:10.1158/1538-7445.AM2015-1405

Purpose: Abnormalities in the subpopulations in white blood cells, such as neutrophilia and lymphopenia, have been reported in tumor-bearing patients, and the neutrophil/ lymphocyte ratio (NLR) has been found to associate with prognosis in patients following radical surgery. The NLR was reported to associate with prognosis in cancer patients by influencing both cancer progression and chemosensitivity. The correlation between NLR and the outcome of neoadjuvant chemotherapy (NAC) in breast cancer patients has not been elucidated yet. The aim of this study is to evaluate NLR as one of the possible markers to predict the outcome of NAC in patients with TNBC by a retrospective investigation of the consecutive patient series treated with a standardized single protocol in our institute. Experimental Design: NLR was evaluated in a total of 177 patients with early-stage breast cancer treated with NAC consisting FEC (5-fluorouracil, epirubicin, and cyclophosphamide) followed by weekly paclitaxel followed by curative surgery. The correlation between NLR and the prognosis including the efficacy of the NAC was evaluated retrospectively. NLR was calculated from the preoperative blood sample by dividing the absolute neutrophil count by the absolute lymphocyte count. On the basis of previous studies, an NLR value of 3.0 was used as the cut-off value to discriminate between high-NLR (3.0 and over) and low-NLR (less than 3.0) groups. Results: NLR distributed from 0.5 to 10.6 with a median of 2.0. Fifty-eight patients with low NLR (less than 3) had a higher pCR rate (p Conclusions: Low NLR in patients with TNBC could be expected to show high efficacy, and favorable outcome after NAC. Citation Format: Yuka Asano, Shinichiro Kashiwagi, Tamami Morisaki, Satoru Noda, Hidemi Kawajiri, Tsutomu Takashima, Naoyoshi Onoda, Kosei Hirakawa. Predictive value of neutrophil-lymphocyte ratio for the efficacy of preoperative chemotherapy in triple-negative breast 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 1824. doi:10.1158/1538-7445.AM2015-1824

Background: Autophagy is an intracellular degradation system that is induced under starvation or hypoxia. Recently, autophagy has been reported to be associated with carcinogenesis and tumor progression. The significance of autophagy in gastric cancer progression remains controversial. Microtubule-associated protein light chain 3 (LC3) is a component of autophagosomes that stimulate autophagy. There are 3 types of LC3 gene family members, LC3A, LC3B and LC3C. LC3A and LC3B were recently reported to be associated with autophagy in LC3 members. The aim of this study was to evaluate the significance of an autophagy protein, LC3, in gastric cancer. Materials and Methods: A diffuse-type gastric cancer cell line, OCUM-2MD3, was used in this study. Effect of starvation on the expression level of LC3A and LC3B mRNA of OCUM-2MD3 cells was examined by RT-PCR under glucose free condition. A total of 528 patients who underwent resection of a primary gastric cancer were enrolled. The correlation between the clinicopathological features of 528 primary gastric carcinomas and LC3 expression was examined by immunohistochemistry. Results: Expression level of LC3A and LC3B mRNA significantly increased five and two times respectively in OCUM-2MD3 under glucose free conditions compared to that under high glucose conditions. Immunohistochemical study showed that LC3 expression was positive in 94 (17.8%) of 528 gastric cancers. There was a statistically significant correlation between LC3 positive-expression and histologic type (p = 0.002), lymphatic invasion (p Conclusion: LC3 might play an important role for autophagy of gastric cancer cells in starvation condition. LC3 is associated with progression of gastric cancer, and is a useful prognostic marker for patients with gastric cancer. Citation Format: Go Masuda, Masakazu Yashiro, Hiroaki Kasashima, Haruhito Kinoshita, Tatsunari Fukuoka, Tamami Morisaki, Tsuyoshi Hasegawa, Katsunobu Sakurai, Takahiro Toyokawa, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Kosei Chung. Significance of an autophagy protein, microtubule-associated protein light chain 3 (LC3), 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 992. doi:10.1158/1538-7445.AM2015-992

Background: Cancer-associated fibroblasts (CAF) have been suggested to be associated with the progression of gastric cancer cells. Hypoxic region was frequently found in cancer microenvironment of gastric cancer. The aim of our study was to clarify the interaction between CAF and gastric cancer cells under hypoxia. We found that hypoxia up-regulated the growth and interaction between the CAF and gastric cancer cells through SDF1α/CXCR4 dependent mechanism. Materials and Methods: Six gastric cancer cell lines and three fibroblasts cell lines were used. The expression level of CXCR4 under hypoxia was examined by RT-PCR, Western blot. SDF1 production from CAF under hypoxia wad examined by ELISA. The proliferation of diffuse type gastric cancer cells that were co-culture with CAF was examined under hypoxia, in comparison with that under normoxia. Proliferation activity of diffuse type gasitrc cancer cells was examined in the presence of CAF, CXCR4 inhibitor, FGFR2 inhibitor, HIF1α siRNA or CXCR4 siRNA under hypoxia, in comparison with that under normoxia. Results: The proliferation of diffuse type gastric cancer cells was significantly increased in the presence of CAF. The proliferation - stimulating effects by CAF were evident in hypoxia, in compared with normoxia. CXCR4 expression of diffuse type gastric cancer cells was significantly increased under hypoxia, but decreased FGFR2 expression under hypoxia. HIF1α siRNA significantly down-regulated CXCR4 expression level in hypoxia. SDF1 from CAF was increased under hypoxia, and was also decreased by HIF1α knockdown. FGFR2 inhibitor significantly decreased he proliferation-stimulating activity of CAF under normoxia, but not under hypoxia. Although, CXCR4 inhibitor significantly decreased the proliferation-stimulating activity of CAF under hypoxia, but not under normoxia. These findings suggested that the key signaling associated with the growth-interaction between CAF and gastric cancer cells in hypoxic condition might be different from those in normoxic condition. Conclusion: Hypoxic microenvironment switch the driver signaling from FGF7/FGFR2 to SDF-1/CXCR4 in diffuse type of gastric cancer. CXCR4/SDF1 axis might play an important role for the proliferation of gastric cancer in hypoxic tumor microenvironment. Citation Format: Haruhito Kinoshita, Masakazu Yashiro, Hiroaki Kasashima, Go Masuda, Tamami Morisaki, Tatsunari Fukuoka, Katsunobu Sakurai, Takahiro Toyokawa, Kenjiro Kimura, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Kosei Hirakawa. SDF1α / CXCR4 axis might be associated with growth-interaction between cancer-associated fibroblasts and gastric cancer cells in hypoxic tumor microenvironment. [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 436. doi:10.1158/1538-7445.AM2015-436

Background: Cancer-associated fibroblasts (CAF) have been suggested to be associated with the scirrhous gastric cancer (SGC) progression. SGC have a heterogeneously hypoxic environment which has been currently considered to be associated with aggressive tumor phenotypes cancer. Stromal cell-derived factor (SDF)-1 and C-X-C chemokine receptor type 4 (CXCR4) form an important chemokine/receptor pair, and are supposed to be up-regulated by hypoxia inducible factor-1 (HIF-1). The aim of our study was to clarify the effect of hypoxic environment on the CAF and CXCR4/SDF-1 axis in SGC. Experimental Design: Proliferation of SGC cells grown in monoculture and co-culture with CAF under hypoxia. SGC and CAF expression level of CXCR4 and SDF-1 under hypoxia were examined by RT-PCR and ELISA. Proliferation and migration of SGC cells with SDF-1 or hepatocyte growth factor (HGF) or conditioned medium from CAF under hypoxia, furthermore, added c-Met inhibitor or CXCR4 inhibitor. Results: The proliferation that co-culture of SGC cells with CAF was significantly increased under hypoxia. The expression level of CXCR4 mRNA was significantly increased under hypoxia in all of SGC cells. SDF-1 concentration level was significantly increased under hypoxia. The proliferation of SGC cells with SDF-1 and CAF was significantly increased. The proliferation of SGC cells with CAF was inhibited by CXCR4 inhibitor under hypoxia. The migration of SGC cells with HGF and CAF was significantly increased. The migration of SGC cells with CAF was inhibited by c-Met inhibitor. Conclusion: CAF might up-regulate the proliferation and migration of SGC cells under hypoxia, in comparison with that under normoxia. CXCR4/SDF-1 axis might play an important role for the proliferation of SGC cells under hypoxia. Citation Format: Haruhito Kinoshita, Masakazu Yashiro, Go Masuda, Hiroaki Kasashima, Tamami Morisaki, Tatsunari Fukuoka, Masatune Shibutani, Sadaaki Yamazoe, Katsunobu Sakurai, Hisashi Nagahara, Kenjiro Kimura, Takahiro Toyokawa, Ryosuke Amano, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Hiroshi Otani, Kiyoshi Maeda, Masaichi Ohira, Kosei Hirakawa. Effect of cancer associated fibroblast on gastric cancer cells under hypoxia. [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 3612. doi:10.1158/1538-7445.AM2014-3612

Object: Lysyl oxidase (LOX) family is the extracellular matrix-modifying enzymes comprising of LOX and LOX-like (LOXL) 1-4. These enzymes have intracellular functions involved in the regulation of motility, migration, differentiation, and transcription in various types of cells. LOX is associated with mesenchymal reconstruction that is essential for cancer invasion and metastasis. LOX overexpression is crucial to promoting tumor growth and metastasis in several cancers such as breast cancer and colorectal cancer, however, role of LOX in gastric cancer remain unknown. Here, we investigated the clinicopathological significance of LOX and LOXL2 in gastric cancer. Materials and Methods: A total of 549 patients who had undergone surgery for gastric cancer at Osaka-City University were enrolled. LOX and LOXL2 expressions were evaluated by intensity of staining and percentage of stained tumor cells at the invading tumor front. This study was approved by the Osaka City University ethics committee. Informed consent was obtained from all patients. Results: LOX and LOXL2 expression were positive in 265 (48.3%) and 342 (58.5%) of the 549 gastric tumors. Both LOX and LOXL2 expression in cancer cells were significantly associated with macroscopic type (p Conclusions: LOX and LOXL2 expression might be associated with metastatic activity of gastric cancer cells. LOX and LOXL2 might be useful predictive prognostic factors for patients with gastric cancer. Citation Format: Hiroaki Kasashima, Masakazu Yashiro, Yukie Go, Go Masuda, Haruhito Kinoshita, Mao Tokumoto, Tamami Morisaki, Tatsunari Fukuoka, Takahiro Toyokawa, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Kosei Hirakawa. Role of lysyl oxidase and lysyl oxidase-like 2 in gastric carcinoma. [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 1980. doi:10.1158/1538-7445.AM2014-1980

Background: Eribulin mesylate (eribulin) is a microtubule dynamics inhibitor having an action mechanism that differs from taxane and vinca alkaloid. Eribulin does not have cross resistance due to an action mechanism that differs from other taxane drugs. Moreover, because it was effective against TNBC upon sub-analysis at phase III clinical trial 301, it is expected to become the key drug for chemotherapy against TNBC in the future. In this study, we investigated predictive factors regarding the therapeutic effect in eribulin chemotherapy using factors such as TLE3, beta-tubulin class III, GSTP1, etc. previously reported as being predictive factors of the therapeutic effect of taxane drugs. Methods: The subjects were 52 inoperable or metastasis / recurrent breast cancer cases that underwent chemotherapy using eribulin. The clinical benefit rate (CBR) was calculated regarding the efficacy thereof. Morphologies such as tissue-type, nucleus grade, etc. were compared by hematoxylin and eosin staining and the expression of ER, PR, HER2, Ki67, beta-tubulin class III, GSTP1, and TLE3 was compared by immunostaining regarding a needle biopsy specimen infiltrate prior to treatment. Results: The clinical effects were CBR 46.2 per cent. According to the intrinsic subtype classification: Luminal A: 16 cases: Luminal B: 12 cases: Luminal HER2: 2 cases: HER2 enriched: 3 cases: and TNBC: 19 cases. When the clinicopathological background and expression of each factor were investigated, TNBC was observed with significantly higher expression of TLE3 compared to non-TNBC; however, no significant difference was observed regarding other factors. Further, breast cancer patients with TLE3 expression had significantly higher CBR. Meanwhile, no significant difference was observed in beta-tubulin class III and GSTP1 expression. Conclusions: Our findings therefore suggest that TLE3 may be useful as a marker for predicting the therapeutic effect in eribulin chemotherapy for TNBC.

Introduction: Prostaglandins and cyclooxygenase2 have been suggested to play an important role for the proliferation and migration of cancer cells. But Prostaglandins (PG) have been suggested to affect cancer cells in various directions. For example, PGF2α and PGD2 has been demonstrated to have anti-tumor effects against some types of cancer cells. But PGE2 has been demonstrated to promote the proliferation and migration of cancer. Therefore, the investigation of the involvement of PG signals for cancer in lower level of arachidonic acid cascade is very important. The aim of this study is to investigate the effects of PGE2 and PGE2 receptor inhibitor (EP2 inhibitor) on the growth-interaction between cancer cells and fibroblasts. Methods: Three human gastric cancer cell lines (OCUM-2M, OCUM-2MD3, OCUM-12) and a human gastric fibroblast (CAF45) cell line were used in this study. Serum-free conditioned medium (SF-CM) from CAF-45 stimulated with PGE2 or EP2 inhibitor was prepared and used in study. Cell proliferation was studied by MTT assay and cell count in the presence or absence of CAF45. Cell migration was studied by wound healing assay in the presense or absence of SF-CM. In vivo study, We examined the effect of EP2 inhibitor on subcutaneous and orthotopically transplanted tumors. Results: In proliferation, PGE2 significantly stimulated the proliferation and migration of cancer cells. PGE2 significantly stimulated the proliferation and migration of cancer cells co-cultured with CAF45 or SF-CM more in comparison with single culture. EP2 inhibitor significantly decreased the proliferation and migration of cancer cells, EP2 inhibitor significantly decreased the proliferation migration of cancer cells co-cultured with CAF-45 or SF-CM more in comparison with single culture. In vivo study, EP2 inhibitor significantly suppressed the subcutaneous tumor in nude mice and suppressed orthotopic tumor growth and lymph node metastasis. Conclusion: PGE2 is associated with progression of gastric cancer. PGE2 receptor inhibitors might be promising anti-cancer drug for gastric carcinoma. Citation Format: Tatsunari Fukuoka, Masakazu Yashiro, Hiroshi Takeda, Takayuki Maruyama, Hiroaki Kasashima, Go Masuda, Haruhito Kinoshita, Tamami Morisaki, Katsunobu Sakurai, Masatsune Shibutani, Sadaaki Yamazoe, Kenjiro Kimura, Hisashi Nagahara, Takahiro Toyokawa, Ryosuke Amano, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Hiroshi Otani, Kiyoshi Maeda, Masaichi Ohira, Kosei Hirakawa. The effect of PGE2 receptor inhibitor on the microenviroment of scirrhous 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 3606. doi:10.1158/1538-7445.AM2014-3606

Purpose: Cancer-associated fibroblasts (CAFs) have recently been implicated in tumor growth and metastasis in gastric cancer. Cancer stem cells. (CSCs) have been proposed to have an important role in cancer progression. CSCs are known to display stem cell properties, “stemness”, the ability to sustain self-renewal. Because CAFs are abundant in the stroma of gastric cancer, CAFs are supposed to affect CSC characteristics of gastric cancer. However, the effects of CAFs on the characteristics of gastric cancer CSCs have not been clarified. The aim of this study was to clarify the effect of CAFs on CSCs characteristics in gastric tumor microenvironment. Experimental Design: Four gastric cancer cell lines, OCUM-12, OCUM-2MD3, MKN-45, and MKN-74, were used. Side population (SP) cells were sorted by flow cytometry as CSC-rich cells from each parent cell line. CaF-37 was established from the tumoral gastric specimens as CAFs. Flow cytometric analysis of SP fraction, spheroid colony assay, and RT-PCR analysis of CSC markers, ABCG2, ALDH1, CD44, CD133, NANOG, and OCT3/4, were performed to identify CSCs properties. Effect of CAFs on the tumorigenicity by OCUM-12/SP cells was examined using nude mice. Results: CaF-37 significantly increased the percentages of the SP fraction of scirrhous-type of gastric cancer cells, OCUM-12/SP and OCUM-2MD3/SP cells, but not that of non-scirrhous-type of gastric cancer cells, MKN-45/SP and MKN-74/SP cells. Taken together, CaF-37 significantly increased the number of spheroid colonies and the expression level of CSC markers of scirrhous-type of gastric cancer cells. These stimulating-activities by CaF-37 were significantly decreased by TGFβ inhibitors, but not FGFR and cMet inhibitor. Also, SP fractions of scirrhous-type of gastric cancer cells were significantly increased by TGFβ, but not by FGF2 and HGF. Tumorigenicity by subcutaneous co-inoculation of OCUM-12/SP cells with CaF-37 was significantly high in comparison with that by OCUM-12/SP cells alone. Phospho-Smad2 expression level was significantly increased by co-inoculation with CaF-37. Conclusions: TGFβ from CAFs in the tumor microenvironment might sustain the stemness of CSCs of gastric cancer cells, especially in scirrhous-type of gastric cancer. Citation Format: Masakazu Yashiro, Tsuyoshi Hasegawa, Tatsunari Fukuoka, Haruhito Kinoshita, Tamami Morisaki, Hiroaki Kasashima, Go Masuda, Naoshi Kubo, Kosei Hirakawa. The stemness of gastric cancer stem cells is sustained by TGFβ produced from cancer-associated fibroblasts. [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 3863. doi:10.1158/1538-7445.AM2014-3863

Background: Triple-negative breast cancer (TNBC), a subtype of breast tumor with ER negative, PR negative, and HER2 negative, shows a poor prognosis because of a frequent recurrence. Androgen receptor (AR) is involved in the pathogenesis of breast cancer, but its role is not clearly defined. This study was to explore the expression of AR and its relationship with clinicopathologic parameters in TNBC. Methods: This study investigated a consecutive series of 1036 cases of sporadic invasive breast carcinoma. Immunohistological assays were performed to determine the expression of AR in 190 of TNBC. The relationships between AR expression and clinicopathologic data and prognosis were analyzed. Results: Of informative 1036 breast cancer cases, triple-negative phenotype was 190 (18.3%). TNBC patients experienced a poorer prognosis in comparison to non-TNBC patients (p Conclusions: AR might be a prognostic marker and possible endocrine target for therapy in patients with TNBC.

Aim: The recently developed reagent, eribulin mesylate (eribulin), is a microtubule dynamics inhibitor with a mechanism of action that differs from that of taxanes and vinca alkaloids. This drug is considered to be a promising chemotherapeutic agent for the treatment of locally advanced or metastatic breast cancer (MBC). In this study, we investigated predictive factors with respect to the therapeutic effect of eribulin chemotherapy among variables such as b-tubulin class III, GSTP1 and TLE3, which have previously been reported to be predictive factors of the therapeutic effect of taxanes, in an aim to identify possible biomarkers for predicting the efficacy of eribulin. Methods: The subjects included 52 patients with MBC who underwent chemotherapy using eribulin. The median follow-up time was 431 days (range, 50-650 days). The overall response rate (ORR), clinical benefit rate (CBR), disease control rate (DCR), overall survival (OS), time to treatment failure (TTF) and progression-free survival (PFS) were calculated regarding the efficacy of this regimen. Moreover, breast cancer was classified into intrinsic subtypes according to the status of the ER, PR, HER2 and Ki67 expression. Results: The clinical effects were as follows: overall ORR= 34.6%, CBR = 44.2%, DCR= 51.9%; median OS = 334 days; median TTF = 81 days; and median PFS = 275 days. TLE3, b-tubulin class III and GSTP1 were expressed in 24 cases, 21 cases and 24 cases, respectively, among the 52 patients investigated. The expression of TLE3 was found significantly more frequently in the TNBC lesions than in the non-TNBC lesions (p = 0.030). The patients with TLE3-negative tumors experienced significantly poorer outcomes in terms of progression-free survival when comparing the prognosis within the group of patients with triple-negative breast cancer (TNBC) lesions (p = 0.011). Based on a multivariate logistic regression analysis including 22 patients with TNBC also showed that a positive TLE3 expression significantly correlated with a better progression-free survival (p = 0.037). Conclusions: Our findings suggest that TLE3 is a useful marker for predicting the therapeutic effect of eribulin chemotherapy for TNBC. Disclosure: All authors have declared no conflicts of interest.

Aim: Fulvestrant (FUL) has been used to treat postmenopausal women with hormone receptor-positive metastatic or recurrent breast cancer after the failure of treatment attempts with tamoxifen (TAM) or aromatase inhibitor (AI). The factors predicting the outcome of treatment by FUL have not yet been identified. We retrospectively investigated our experience in an attempt to determine possible predictive factors of FUL treatment. Methods: 42 patients were treated by FUL at a dose of 500 mg/ month from November 2011 to April 2014 in our institute. All cases were postmenopausal women at average age of 67.2 years, showed resistance to previous treatments with TAM and/ or AI. Estrogen receptor expression was positive in 41 cases and was unknown in one case. Six cases had unresectable locally advanced disease, 6 cases had stage IV disease, and 30 cases had postoperative recurrence. Results: Responses were determined as PR 10%; SD 33%; PD 57%, with a clinical benefit rate of 29%. The clinical benefit of FUL was significantly more commonly observed in patients with higher age (67 years or more), greater number of prior endocrine therapy (3 or more), and having demonstrated clinical benefit by prior AI treatment. In addition to these three factors, PFS was significantly prolonged in the group of patients who had shown clinical benefit of prior TAM treatment compared with those without. Patients with high BMI (>25) had a tendency to extend PFS (p = 0.05). However, no predictive factor of better PFS could be identified by multivariate analysis. Conclusions: In responders to prior endocrine therapy, (especially AI treatment,) the effect of FUL might be expected even when conducted in a late line. Although higher BMI had been reported to have a negative impact on the outcome in postmenopausal women with breast cancer, treatment with FUL might be efficient in these patients. We believe this pilot study demonstrated the need for further validation. Disclosure: All authors have declared no conflicts of interest.

Background: Various types of solid tumors, including gastric cancer, have a heterogeneously hypoxic environment which has been currently considered to be associated with aggressive tumor phenotypes cancer. Stromal cell-derived factor (SDF)-1 and C-X-C chemokine receptor type 4 (CXCR-4) form an important chemokine/receptor pair, and are supposed to be up-regulated by hypoxia inducible factor-1 (HIF-1). Although the chemokine signaling is associated with malignant potential of various carcinomas, few studies have addressed the expression and function of CXCR4 and SDF-1 under hypoxia in gastric cancer. The aim of our study was to clarify the significance of CXCR4 and SDF-1 under hypoxia in gastric carcinoma by evaluating the expression of a hypoxic marker, a carbonic anhydrase 9 (CA-9). Experimental Design: A total of 297 patients who had undergone a resection of the primary tumor and were confirmed histologically to have sporadic gastric cancer were enrolled in this study. Expressions of CXCR4, SDF-1, and CA-9, were assessed by immunohistochemistry. We examined the association between the expression levels of CXCR4, SDF-1, and CA-9 and clinicopathologic variables of patients with gastric cancers. Furthermore, using three diffuse-type gastric cancer cell lines, OCUM-2M, OCUM-2MD3, and OCUM-12, expression level of CXCR4 and SDF-1 in hypoxia (1% O2) were examined by RT-PCR. Results: CA-9 expression was found in 48% (143/297) of gastric cancers, CXCR4 was 47% (141/297), and SDF-1 was 41% (122/297). The CA9 expression was significantly correlated with CXCR4 expression and SDF-1. The CA9 expression level was significantly high in cases diffuse-type carcinoma (p=0.028). The prognosis for CA-9-positive patients was significantly poorer than that of CA-9-negative patients (p Conclusion: CXCR4/SDF-1 expression of gastric cancer cells might be regulated with by hypoxic condition. CXCR4/SDF-1 axis might play an important role for the progression of diffuse-type gastric carcinoma with hypoxic environment. Citation Format: Haruhito Kinoshita, Masakazu Yashiro, Tamami Morisaki, Tatsunari Fukuoka, Tsuyoshi Hasegawa, Toshiki Hirakawa, Naoki Aomatsu, Katsunobu Sakurai, Kenjiro Kimura, Hisashi Nagahara, Takahiro Toyokawa, Ryosuke Amano, Eiji Noda, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Hiroshi Otani, Kiyoshi Maeda, Masaichi Ohira, Kosei Hirakawa. Significance of CXCR4/SDF-1 axis expression in gastric cancer under hypoxic enviroment. [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 4054. doi:10.1158/1538-7445.AM2013-4054

Purpose: Gastric cancer remains a major global health threat and most patients with advanced stage disease require chemotherapy. The development of drug resistance is a major obstacle in the treatment of gastric cancer and only few effective therapies for combating chemoresistance are currently available. It has been demonstrated that a small subset of cancer cells with stem cell properties, referred to as “cancer stem cells” (CSCs), survive intensive anticancer therapies better than proliferating progenitor cells or differentiated tumor cells. CSCs have been reported to be postulated mediators of chemoresistance, so it might be important to comprehend the drug resistance mechanisms of CSCs to develop a promising therapy to combat chemoresistance. The stemness signal might be associated with the chemosensitivity of CSCs. In the present study, we analyzed the effect of c-Met inhibitors on the chemosensitivity of stem-like cancer cells in gastric cancer. We demonstrated that a c-Met inhibitor synergistically increased the antitumor activity of SN38 in CSCs. To determine mechanisms underlying this observed synergism, we observed that a c-Met inhibitor combined with SN38 also led to a significant increase in UGT1A1 and its subsequent interaction with apoptosis-related genes, i.e., bcl-2 and caspase-6. Experimental Design: We used three gastric cancer cell lines and three side population (SP)-enriched cell lines. We used three signal inhibitors, c-Met inhibitor SU11274, GSK3β inhibitor AR-A014418, and mTOR inhibitor rapamycin, and five anticancer drugs. We examined the combined effects of signal inhibitors and anticancer drugs on proliferation, mRNA expression, and cell cycle. Results: The IC50 of irinotecan, oxaliplatin, taxol, and gemcitabine in SP cells were 10.5, 2.0, 2.8, and 2.0 times higher than their parent cells, respectively. In contrast, the IC50 of 5-fluorouracil did not differ between the two cell lines. There was a synergistic anti-proliferative effect with a combination of c-Met inhibitor and SN38 in SP cells. In contrast, the GSK3β inhibitor and mTOR inhibitor had no synergistic effects in combination with any anticancer drugs. The SP cell lines had higher expression levels of UGT1A1, ABCG2, and ABCB1 than their parent cell lines, while the c-Met inhibitor significantly decreased the expression of UGT1A1, but not ABCG2 and ABCB1. G0-phase of SP cells was higher than their parent cells. c-Met inhibition induced S-phase arrest in SP cells. Conclusions: CSCs are associated with multiresistance during chemotherapy. c-Met inhibitors could be a novel strategy to overcome chemoresistance. c-Met inhibitor may be a promising target molecule for irinotecan-based chemotherapy of gastric cancer. A UGT1A1 alteration might be involved in the irinotecan refractory process in CSCs. Citation Format: Masakazu Yashiro, Tatsunari Fukuoka, Haruhito Kinoshita, Takafumi Nishii, Tsuyoshi Hasegawa, Taro Matsuzaki, Tamami Morisaki, Kosei Hirakawa. A c-Met inhibitor increases the chemosensitivity of cancer stem cells to the irinotecan active metabolite SN38 in gastric carcinoma. [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 225. doi:10.1158/1538-7445.AM2013-225

Introduction: Prostaglandins (PGs) have been suggested to play an important role for the proliferation and migration of cancer. Cyclooxygenase2(COX-2) stimulates PG synthesis. The selective COX-2 inhibitors have reported to show antitumor effects on various types of cancers, including colorectal cancer and gastric cancer. Gastric cancer remains to be poor prognosis because few anti-cancer drugs are available for advanced gastric cancer. PGE2 binds to PGE2 receptor, EP1, EP2, EP3 and EP4 and activates MAPKinase. The aim of this study is to investigate the effects of the EP2 inhibitors on the proliferation and motility of gastric cancer cells. Methods: Human gastric cancer cell lines (OCUM-2M and OCUM-12) were used in this study. Cell proliferation was studied by 3-(4,5-Dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Cell migration was studied by a wound healing assay. Results: PGE2 significantly stimulated proliferation and migration of gastric cancer cells, but not PGE1. EP2 and EP4 inhibitors significantly decreased the proliferation and migration ability of gastric cancer cells in a dose dependent manner, but not EP1, EP3. Conclusion: PGE2 is associated with progression of gastric cancer cells.PGE2 receptor inhibitors might be promising anti-cancer drug for gastric carcinoma. 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 1211. doi:1538-7445.AM2012-1211

Background:Current studies have demonstrated that neoadjuvant chemotherapy (NAC) improves survival outcomes and increases the chance for breast conservation when applied preoperatively. However, no reliable maker to predict chemosensitivity of NAC is not available. CD133 has been considered as a cancer stem cells (CSCs) marker in many kinds of tumors such as breast cancer, colorectal cancer and brain cancer. The aim of this study was to evaluate the value of CD133 as a surrogate maker to predict chemosensitivity of NAC for breast cancer. Experimental Design: A total of 102 patients with breast cancer of stage II and III, as evaluated by core needle biopsy and ultrasonography, was treated with NAC. Expressions of CD133, estrogen receptor, progesterone receptor, and HER2 were assessed by immunohistochemistry. Results: The clinical complete response (CR) rate of all patients was 18% (18/102), partial response (PR) was 61% (62/102), no change (NC) was 20% (20/102), and progressive disease (PD) was 2% (2 /102). The response rate (RR) was 78% (80/102). The pathological complete response rate (pCR) was 29%. The pCR rate and pathological response of CD133-positive cases was significantly lower than that of CD133-negative cases. The recurrence rate of CD133-positive cases was significantly higher than that of CD133-negative cases. CD133 expression showed a poor disease free survival and overall survival time. A multivariate logistic regression analysis showed that CD133 expression was an independent prognostic factor in breast cancers treated with NAC. Conclusion: CD133 is a useful surrogate maker to predict chemosensitivity of NAC chemotherapy for breast cancer. 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 707. doi:1538-7445.AM2012-707

Background: Acquired drug resistance to irinotecan is one of the significant obstacles in the treatment of advanced gastric cancer. This study was performed to clarify the effect of epidermal growth factor receptor (EGFR) inhibitors in combination with SN38, an active metabolite of irinotecan, on the proliferation of irinotecan-refractory gastric cancer. Methods: Two irinotecan-resistant gastric cancer cell lines, OCUM-2M/SN38 and OCUM-8/SN38, were respectively established by stepwise exposure to SN38 from the parent gastric cancer cell lines OCUM-2M and OCUM-8. The combination effects of two EGFR inhibitors, gefitinib and lapatinib, with SN38 on proliferation, apoptosis, and cell-cycle on gastric cancer cells were examined. Results: Gefitinib or lapatinib showed synergistic anti-tumor effects against OCUM-2M/SN38 and OCUM-8/SN38 cells when used in combination with SN38, but not against OCUM-2M or OCUM-8 cells. SN38 increased the expression of EGFR and HER2 in OCUM-2M/SN38 and OCUM-8/SN38 cells. The combination of an EGFR inhibitor and SN38 significantly increased the levels of apoptosis-related molecules, caspase-6, p53, and DAPK-2, and resulted in the induction of apoptosis of irinotecan-resistant cells. The EGFR inhibitors increased the S-phase and decreased the UGT1A1 and ABCG expression in irinotecan-resistant cells. The SN38 plus Lapatinib group more effectively suppressed in vivo tumor growth by OCUM-2M/SN38 cells than either alone group.Conclusion: The combination treatment with an EGFR inhibitor and irinotecan might produce synergistic anti-tumor effects for irinotecan-refractory gastric cancer cells. The regulation of SN38 metabolism-related genes and cell cycle by EGFR inhibitors might be responsible for the synergism. 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 780. doi:1538-7445.AM2012-780

Introduction: Cancer Stem Cells (CSCs) are thought to possess tumor initiation and self-renewal and are associated with tumor progression. The niche cells of microenvironment, such as fibroblast might play an important role for the progression of cancer cells. The aim of this study is to examine the effect of cancer-associated fibroblasts on the stemness of CSCs. Material and Methods: A gastric cancer cell line, OCUM-12, and cancer-associated gastric fibroblast, CaF-37, were used. SP cells, known as CSCs rich population, were isolated from OCUM-12 cells by flowcytometry using Hoechest33342, and were named as OCUM-12/SP. The percentage of SP cells was evaluated by flowcytometry, after it was cultured with condition medium from CaF-37 for three days. SP fraction was evaluated by flowcytometry, in the presence or absence of TGF-β receptor inhibitor, FGF receptor inhibitor, and c-Met Receptor inhibitor. In vivo, tumorigenicity of OCUM-12/SP cells were evaluated by subcutaneous inoculation in the presence or abcence of CaF-37. Results: The inoculation of 1.5x105, 5x104, and 1.5x104 OCUM-12/SP cells with 5x105 CaF-37 fibroblasts resulted in tumor formation in all of mice, respectively. However, the inoculation of 1.5x105, 5x104, and 1.5x104 OCUM-12/SP cells alone results in poor tumor formation at 3(60%) of 5 mice, 0(0%) of 5 mice and 0(0%) of 5 mice, respectively. The percentage of SP fraction of OCUM-12/SP was significantly increased in the presence of condition medium from CaF-37. The increase of SP fraction of OCUM-12/SP cells by condition medium from CaF-37 was inhibited by TGF-β receptor inhibitor, but not FGF receptor inhibitor and c-Met inhibitor. Conclusion: TGF-β from the niche, fibroblasts, might sustain the stemness of CSCs and might be associated with the progression of Cancer Cells. 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 3337. doi:1538-7445.AM2012-3337

Introduction: Epithelial mesenchymal transition (EMT) is a cellular process during which epithelial cells acquire mesenchymal properties while losing cell-cell interactions and apicobasal polarity. EMT is considered to be correlated with malignancy of cancer cells and responsible for cancer invasion and metastasis. Patients with gastric cancer show a poor prognosis because of frequent metastasis. We previously reported that distant metastasis was associated with hypoxia in gastric cancer. Therefore, we investigated the effect of hypoxic condition on EMT of gastric cancer cells. Materials and Methods:Seven gastric cancer cell lines derived from gastric cancer were used. Cells were cultured in normoxia (21% O2) or hypoxia (1% O2) for 24h. EMT rate was evaluated as the percentage of spindle-shaped cells of total cells. The expression level of TGFβ and TGFβ receptor was evaluated by real time RT-PCR. Effect of several inhibitors on the EMT was evaluated by cell morphology. The TGFβ1 level of the cell culture supernate was measured by a quantitative sandwich enzyme immunoassay technique. Results: Hypoxia stimulated EMT of OCUM-2MD3 and OCUM-12 cells, but not that of other cell lines. The level of TGFβ expression was increased significantly (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 2411. doi:1538-7445.AM2012-2411

Intro Cancer stem cells (CSCs) are considered to be responsible for cancer metastasis, and hypoxia is supposed to be an important regulator of CSCs differentiation. Furthermore, tumor hypoxia was reported to be associated with more aggressive tumor phenotypes such as high metastatic ability and resistance to various anti-cancer therapies which may lead to a poorer prognosis. This raises the question of whether there might be proteins representing similar alterations which are responsible for the correlation between hypoxic and CSCs phenotypes. We have established a diffuse-type of gastric carcinoma cell line (OCUM-12), and a hypoxia-resistant cancer cell line (OCUM-12/Hypo) cloned from parent OCUM-12 cells by continuous exposure to 1% oxygen. The side population (SP), as evaluated by a flow cytometric analysis using Hoechst 33342, has been known as CSC-rich population. To investigate and compare the proteomes of the hypoxic (OCUM-12/Hypo), stem cell (OCUM-12SP) and parent OCUM-12 diffuse-type gastric carcinoma cell lines, protein lysates from those cell lines were analyzed using QSTAR Elite LC MS/MS. Method Triplicate pooled samples (10 ug protein each) from stomach cancer cell lines were prepared and labeled with iTRAQ reagents. MS/MS data were searched against the Swiss Protein database (HUMAN) using ProteinPilot™ 2.0 software (AB Sciex). The Ingenuity Pathway analysis program was utilized, to assign biological significance, and to identify networks of interacting differentially expressed proteins, functional groups and pathways. Result In both OCUM-12SP and OCUM-12/Hypo cells, significant overexpression of Wnt signaling pathway was obvious. Furthermore, elevation of MTHFD1, a protein controlling histidine and purine nucleotide metabolism, cytokeratins 18 (CK18), and 19 (CK19) regulating cell cycle, apoptosis and angiogenesis, transcriptional regulator ANP32A, and cellular chaperones heat shock proteins A9 and STIP was observed as compared to the parent OCUM-12 cell line. Furthermore, anterior gradient homolog 2 (AGR2)was down-regulated in both cell lines. Conclusion Wnt signal, MTHFD1, CK18, CK19, ANP32A, HSPA9 and STIP, AGR2 might be responsible for the stem-like phenotype, hypoxia resistance and higher invasiveness of gastric cancer. 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 3380. doi:1538-7445.AM2012-3380