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Supplementary Figures S1-S5; Supplementary Table S1.

This figure shows p53 status in ESCC cells and the effect of p53 rerated signals after irradiation and SOCS1 over expression. ESCC cells in this study harbored p53 mutation and p53-related response by IR and/or SOCS1 over expression was not observed in our study.

This text is Figure Legends of Supplementary Figure S1 and S2.

STAT3 has been implicated recently in radioresistance in cancer. In this study, we investigated the association between STAT3 and radioresistance in esophageal squamous cell carcinoma (ESCC). Strong expression of activated phospho-STAT3 (p-STAT3) was observed in 16/22 ESCC patients with preoperative chemoradiotherapy (CRT), compared with 9 of 24 patients with surgery alone, where the prognosis of those with CRT was poor. Expression of p-STAT3 and the antiapoptotic proteins Mcl-1 and survivin was strongly induced in ESCC cells by irradiation. Ectopic STAT3 expression increased radioresistance, whereas expression of the STAT3 negative regulator SOCS1 via an adenoviral vector improved radioresponse. Inhibiting the STAT3–Mcl-1 axis by SOCS1 enhanced DNA damage after irradition and induced apoptosis. Combining SOCS1 with radiotherapy enhanced antitumor responses in a murine xenograft model compared with the individual therapies. Tumor repopulation occurred transiently after treatment by irradiation but not the combination SOCS1/radiotherapy. Tumors subjected to this combination expressed high levels of γH2AX and low levels of Ki-67, which was maintained after cessation of treatment. Overall, we demonstrated that inhibiting the STAT3–Mcl-1 signaling axis by ectopic SOCS1 improved radiosensitivity by inducing apoptosis and enhancing DNA damage after radiotherapy, offering a mechanistic rationale for a new ESCC treatment. Cancer Res; 77(24); 6975–86. ©2017 AACR.

Purpose: Cisplatin-based chemotherapy is widely used for esophageal cancer, sometimes in combination with surgery/radiotherapy, but poor response to chemotherapy is not uncommon. The aim of this study was to examine whether miRNA expression is useful to predict the response to chemotherapy in patients with esophageal cancer.Experimental Design: Using pretreatment biopsy samples from 98 patients with esophageal cancer who received preoperative chemotherapy, we measured the expression level of several miRNAs whose expression was altered in cisplatin-resistant esophageal cancer cell lines compared with those parent cell lines and examined the relationship between the miRNA expression and response to chemotherapy. In vitro assays were conducted to clarify the mechanism of miRNA-induced changes in chemosensitivity.Results: The expression levels of 15 miRNAs were altered in cisplatin-resistant cells. Of these, low expression of let-7b and let-7c in before-treatment biopsies from 74 patients of the training set correlated significantly with poor response to chemotherapy, both clinically and histopathologically. Low expression of let-7c also correlated with poor prognosis (P = 0.032). The relationship between let-7b and let-7c expression and response to chemotherapy was confirmed in the other 24 patients of the validation set. In in vitro assay, transfection of let-7c restored sensitivity to cisplatin and increased rate of apoptosis after exposure to cisplatin. Let-7c directly repressed cisplatin-activated interleukin (IL)-6/STAT3 prosurvival pathway.Conclusions: Let-7 expression in esophageal cancer can be potentially used to predict the response to cisplatin-based chemotherapy. Let-7 modulates the chemosensitivity to cisplatin through the regulation of IL-6/STAT3 pathway in esophageal cancer. Clin Cancer Res; 18(18); 5144–53. ©2012 AACR.

PDF file, 2332K, (A) Let-7c expression in different esophageal cell lines. (B) Stable expression of let-7c was confirmed until 72 hr after transfection of pre-let-7c. Data are mean�SD of three experiments.

This figure shows the association between STAT3 activation and chemotherapy. There were no differences of p-STAT3 and Mcl-1 expression after irradiation.

PDF file, 58K, Characteristics of participating patients (n=98).

Purpose: Cisplatin-based chemotherapy is widely used for esophageal cancer, sometimes in combination with surgery/radiotherapy, but poor response to chemotherapy is not uncommon. The aim of this study was to examine whether miRNA expression is useful to predict the response to chemotherapy in patients with esophageal cancer. Experimental Design: Using pretreatment biopsy samples from 98 patients with esophageal cancer who received preoperative chemotherapy, we measured the expression level of several miRNAs whose expression was altered in cisplatin-resistant esophageal cancer cell lines compared with those parent cell lines and examined the relationship between the miRNA expression and response to chemotherapy. In vitro assays were conducted to clarify the mechanism of miRNA-induced changes in chemosensitivity. Results: The expression levels of 15 miRNAs were altered in cisplatin-resistant cells. Of these, low expression of let-7b and let-7c in before-treatment biopsies from 74 patients of the training set correlated significantly with poor response to chemotherapy, both clinically and histopathologically. Low expression of let-7c also correlated with poor prognosis (P = 0.032). The relationship between let-7b and let-7c expression and response to chemotherapy was confirmed in the other 24 patients of the validation set. In in vitro assay, transfection of let-7c restored sensitivity to cisplatin and increased rate of apoptosis after exposure to cisplatin. Let-7c directly repressed cisplatin-activated interleukin (IL)-6/STAT3 prosurvival pathway. Conclusions: Let-7 expression in esophageal cancer can be potentially used to predict the response to cisplatin-based chemotherapy. Let-7 modulates the chemosensitivity to cisplatin through the regulation of IL-6/STAT3 pathway in esophageal cancer. Clin Cancer Res; 18(18); 5144–53. ©2012 AACR.

Introduction: Esophageal squamous cell carcinoma (ESCC) is the one of the deadliest gastrointestinal cancers. The important problem in treating ESCC is resistance to chemotherapy. A few previous reports showed that mitochondrial DNA (mtDNA) copy number was decreased in esophageal squamous cell carcinoma (ESCC). However, it is unclear whether alteration of mtDNA copy number affects the effect of chemotherapy for ESCC. In the present study, we investigated the relationship between mtDNA copy number and the effect of chemotherapy in ESCC. Methods: To analyze the mtDNA copy number of surgically resected primary tumors from ESCC patients, qPCR for Cytochrome Oxidase I was performed. Human ESCC cells (TE8 and TE11) with decreased mtDNA copy number were established by knockdown of mitochondrial transcription factor A (TFAM). Those cells were treated with 5FU, cisplatin and docetaxel. The response of chemotherapy was accessed by cell viability assay (WST assay) and apoptosis assay (flow cytometry). To investigate the mechanism of resistance of chemotherapy, expression of EMT-related genes such as CDH1, CDH2, vimentin and zeb1 was measured by qPCR. Wound healing assay and invasion assay were performed to access the cell migration and invasion. Results: Patients were categorized into higher and lower subgroups according to the median value of the mtDNA copy number. Lower mtDNA copy number group was significantly correlated with tumor depth and pathologic response of chemotherapy. ESCC patients with lower mtDNA copy number had significantly poorer 5-year recurrence-free and overall survival than higher group. By shRNA knockdown of TFAM gene expression, mtDNA copy number decreased to 40% in TE8 and 60% in TE11 compared with non-target control cells. Viability of mtDNA-depleted TE8 and TE11 treated with chemotherapy was higher than that of control cells. Apoptosis assay showed the percentages of apoptosis induced by chemotherapy in mtDNA-depleted TE8 and TE11 were lower than those of control cells. Invasion assay and wound healing migration assay showed that mtDNA-depleted TE8 and TE11 were significantly more invaded and migrated than the control-sh cells. Conclusions: These results suggest that low mitochondrial copy number induces resistance to chemotherapy in esophageal cancer. Citation Format: Moyuru Yamada, Koji Tanaka, Yasunori Masuike, Tomoki Makino, Tsuyoshi takahashi, Yukinori Kurokawa, Makoto Yamasaki, Kiyokazu Nakajima, Masaki Mori, Yuichiro Doki, Kabuki Odagiri. Low mitochondrial copy number induces resistance to chemotherapy in esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4344.

Alterations of mitochondrial DNA (mtDNA) copy numbers in various human cancers have been studied. However, those of esophageal squamous cell carcinoma (ESCC) is still unclear. In the present study, we investigated the correlation of mtDNA copy number with clinicopathologic features, prognosis and malignant potential of ESCC. MtDNA copy numbers of resected specimen from 80 patients treated with radical esophagectomy were measured by quantitative real-time PCR analyses. Human ESCC cells, TE8 and TE11, were cultured and depletion of mtDNA content was induced by knockdown of mitochondrial transcription factor A expression or treatment with ethidium bromide. The mRNA expression, proliferation, invasion and cell cycle were investigated. The results showed that mtDNA copy number of cancerous parts was 56.0 (37.4-234.5) % of non-cancerous parts and significantly lower (p Citation Format: Takeo Hara, Yasunori Masuike, Koji Tanaka, Tomoki Makino, Makoto Yamasaki, Yasuhiro Miyazaki, Tsuyoshi Takahashi, Yukinori Kurokawa, Hidetoshi Eguchi, Kiyokazu Nakajima, Masaki Mori, Yuichiro Doki. Low copy number of mtDNA is associated with cancer stemness in ESCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3073.

Background: Various monoclonal antibodies targeting tumor antigens have recently been reported. Antitumor monoclonal antibodies greatly depend on antibody-dependent cellular cytotoxicity (ADCC) via tumor-infiltrating natural killer cells and macrophages. We have reported significant antitumor activity of anti Glypican1 (GPC1) monoclonal antibody. The toll-like receptor 9 (TLR9) agonist CpG oligodeoxynucleotides (ODN) has been widely used as a cancer vaccine adjuvant. Because CpG ODN induce intratumor infiltration of natural killer cells and macrophages, combination therapy with CpG ODN with antitumor monoclonal antibody depending on ADCC may show synergistic antitumor activity. The aim of this study is to evaluate the synergistic antitumor activity of combination therapy with anti GPC1 antibody and CpG ODN. Materials and Method: We examined K3-SPG, which is composed of Schizophyllan (SPG) and K3-CpG ODN. The established human esophageal cancer cell line TE14 was subcutaneously transfected into SCID mice. Xenograft mice were treated by monotherapy with anti GPC1 antibody by intraperitoneal (i.p.) injection and K3-SPG by intratumoral (i.t.) injection twice a week for 4 weeks. Xenograft mice were also treated with combination therapy as follows: (i.p./ i.t.) injection of (A) PBS/ PBS, (B)PBS/ K3-SPG, (C) anti GPC1 antibody/ PBS, (D) anti GPC1 antibody/ K3-SPG, respectively, twice a week for 4 weeks. Antitumor activity was evaluated by measuring the tumor volume twice a week. Tumors were resected 2 weeks after the final treatment and tumor weight was measured. For FACS analysis, agents were administrated twice a week for 2 weeks, and tumors were resected 1 day after the final treatment. Result: Anti GPC1 monoclonal antibody and K3-SPG monotherapy had a significant dose-dependent antitumor activity, respectively. Combination therapy had significant antitumor activity in terms of tumor volumes (A/B/C/D: 605/370/485/247) (mm3) and tumor weight (A/B/C/D: 416/191/317/137) (mg), respectively. FACS analysis revealed that they induced a significant increase in number of macrophage (p=0.02) and high ratio of activated NK cells (p=0.08). Conclusions: Combination therapy with K3-SPG and anti GPC1 monoclonal antibody depending on ADCC may represent a new treatment option for cancer. Citation Format: Yurina Saito, Tsuyoshi Takahashi, Kosuke Hiramatsu, Satoshi Serada, Minoru Fujimoto, Koji Tanaka, Yasuhiro Miyazaki, Tomoki Makino, Yukinori Kurokawa, Kiyokazu Nakajima, Makoto Yamasaki, Ken J. Ishii, Masaki Mori, Yuichiro Doki, Tetsuji Naka. CpG oligodeoxynucleotides potentiate the antitumor activity of anti-GPC1 antibody [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B034.

Purpose: The prognosis of hepatocellular carcinoma (HCC) is very poor, particularly in patients with tumors that have invaded the major branches of the portal vein. Combination chemotherapy with intra-arterial 5-fluorouracil and subcutaneous interferon-α has shown promising results for such advanced HCC, but it is important to develop the ability to accurately predict chemotherapeutic responses. Experimental Design: We analyzed the expression of 3,080 genes using a polymerase chain reaction-based array in 20 HCC patients who were treated with combination chemotherapy after reduction surgery. After unsupervised analyses, a supervised classification method for predicting chemotherapeutic responses was constructed. To minimize the number of predictive genes, we used a random permutation test to select only significant (P < 0.01) genes. A leave-one-out cross-validation confirmed the gene selection. We also prepared an additional 11 cases for validation of predictive performance. Results: Hierarchical clustering analysis and principal component analysis with all 3,080 genes revealed distinct gene expression patterns in responders (those with complete response or partial response) and nonresponders (those with stable disease or progressive disease) to the combination chemotherapy. Using a weighted-voting classification method with either all genes or only significant genes as assessed by permutation testing, the objective responses to treatment were correctly predicted in 17 of 20 cases (accuracy, 85%; positive predictive value, 100%; negative predictive value, 80%). Moreover, patients in the validation dataset could be classified into two distinct prognostic groups using 63 predictive genes. Conclusions: Molecular analysis of 63 genes can predict the response of patients with advanced HCC and major portal vein tumor thrombi to combination chemotherapy with 5-fluorouracil and interferon-α.

[Background] Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the digestive tract and are reported to harbor gain-of-function mutations in the KIT gene, which contribute to the development of sporadic GISTs. This knowledge has facilitated the development of targeted therapies with tyrosine kinase inhibitors and the revolutionary chemotherapeutic drug imatinib mesylate (IM). In clinical trials, the disease control rate was nearly 85%, and corresponding 2-year overall survival rates ranged from 70-80%, indicating markedly improved patient outcomes compared with anecdotal data in the pre-IM era. Despite its effectiveness, half of GISTs treated with IM develop resistance within 2 years, largely due to the accumulation of additional kinase domain mutations accompanied by concomitant re-activation of the KIT tyrosine kinase, even in the presence of IM. Heat shock protein 90 (HSP90) is one of chaperon molecules required for the proper folding, function, and stability of various client proteins such as KIT. The aim of this study is to clarify the efficacy of HSP90 inhibitor against IM resistant GIST. [Material and Method] We used the established human GIST cell line GIST-T1, and two IM-resistant cell lines (GIST-T1R8, GIST-T1R9), which had additional kinase domain mutations accompanied by concomitant re-activation of the KIT tyrosine kinase same as clinical samples, by exposure to IM. These resistant cell lines exhibited imatinib IC50 values (>10 μM) that were >1000-fold higher than the parental cell line. We investigated the cytotoxicity and signaling inhibition by HSP90 inhibitor using by the WST-8 assay, caspase3/7 apoptosis assay and western blotting. Immunobolts for KIT and for KIT-depending signaling pathways were observed at 12 hours of treatment. Caspase 3/7 apoptosis assay was measured after 24h. [Result] HSP90 inhibitor showed growth inhibition not only for GIST T1 but also GIST-T1R8 and GIST-T1R9. Apoptosis induction of these cell lines were also confirmed by the exposure to HSP90 inhibitor in a dose-dependent manner. To find the mechanism of apoptosis induced by HSP90 inhibitor, we investigated the signaling pathway depending on KIT. HSP90 inhibitor showed inhibition of phosphorylated-KIT, a client for HSP90, in both IM sensitive and resistant GIST cell lines. And, it also inhibited downstream of KIT signaling, e.g. p-ERK and p-AKT. [Conclusions]HSP90 inhibitor showed the anti-tumor efficacy for imatinib resistant GIST in vitro. It might have a possibility to apply for clinical use. Citation Format: Yurina Saito, Tsuyoshi Takahashi, Satoshi Serada, Minoru Fujimoto, Koji Tanaka, Yasuhiro Miyazaki, Tomoki Makino, Yukinori Kurokawa, Makoto Yamasaki, Kiyokazu Nakajima, Shuji Takiguchi, Masaki Mori, Yuichiro Doki, Tetsuji Naka. HSP90 inhibitor has a possibility to overcome imatinib resistance in gastrointestinal stromal tumors [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 1330. doi:10.1158/1538-7445.AM2017-1330

INTRODUCTION Radiation therapy (RT) is one of the main strategies for esophageal squamous cell carcinoma (ESCC) besides surgery and chemotherapy. Although RT has shown remarkable clinical benefits for ESCC patients, there remains the resistance for RT. Recently, some reports show that STAT3 activation is induced by irradiation and can result in treatment-resistance for radiotherapy in various cancer. Suppressor of cytokine signaling-1 (SOCS-1) has been cloned as a negative regulator of various cytokine signaling. We previously reported that overexpression of SOCS-1 showed a potent anti-tumor effect for ESCC through targeting of JAK/STAT and FAK/ERK signaling pathway. Since these results, SOCS-1 might have a possibility to overcome the resistance of RT. The aim of this study is to evaluate the antitumor effect of SOCS-1 gene therapy using adenoviral vector (AdSOCS-1) combined with RT. METHODS At first, we evaluated RT resistance by the STAT3 in ESCC cell lines by using pEB-Multi-constitutive-STAT3(c-STAT3) vector. We examined the colony forming assay between parent (TE8, TE9, TE11, TE14) and stably expression of c-STAT3 cells. Next, we evaluated cell growth inhibition effect of AdSOCS-1 gene therapy combined with RT in vitro and in vivo. As in vivo model, we examined the combined effect of RT (2Gy) and AdSOCS-1 gene therapy by using TE14 xenograft mice (ICR nu/nu mice). RESULTS At first, ESCC cell with stably expression of c-STAT3 showed a significant increase in colony forming ability after RT as compared with parent and mock cell. It suggested that activation of STAT3 might be related to RT resistance in ESCC cell. Furthermore, ESCC cells with RT were induced the secretion of IL-6, and the activation level of STAT3 was elevated by multiple RT. In addition, they increased the expression of anti-apoptosis protein (mcl-1, survivin), and might have a RT resistance. Next, we confirmed that combination therapy with RT and AdSOCS-1 had better proliferation inhibitory effect and decreasing of colony forming ability. In addition, we showed that AdSOCS-1 gene therapy inhibited the induction of the expression of anti-apoptosis protein (mcl-1, survivin) by RT and induced apoptosis synergistically. As in vivo model, the combination therapy group showed a significant anti-tumor effect compared to the RT alone group and AdSOCS-1 gene therapy alone group. CONCLUSIONS The activation of STAT3 is involved in resistance to radiation therapy in esophageal squamous cell carcinoma, and SOCS-1 gene therapy combined with radiation therapy may have a potent anti-tumor effect. Note: This abstract was not presented at the meeting. Citation Format: Takahito Sugase, Tsuyoshi Takahashi, Satoshi Serada, Minoru Fujimoto, Koji Tanaka, Yashuhiro Miyazaki, Tomoki Makino, Yukinori Kurokawa, Makoto Yamasaki, Kiyokazu Nakajima, Shuji Takiguchi, Masaki Mori, Yuichiro Doki, Tetsuji Naka. SOCS-1 gene therapy combined with radiation therapy has a potent antitumor effect for 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 5085. doi:10.1158/1538-7445.AM2017-5085

Background/Purpose: For esophageal cancer patients, combination chemotherapy with docetaxel, cisplatin, and 5FU (DCF therapy) is adopted for neoadjuvant chemotherapy (NAC). Although the response rate is high (72.5%), further investigation of indicators to predict its therapeutic effects is needed because of its side effects, and few have been reported. In this study, we obtained peripheral blood before and after the DCF therapy, analyzed various immune related cells by flow cytometry and tried to identify indicators to predict its therapeutic effects. Subjects/Methods: Peripheral blood were obtained from 20 esophageal cancer patients before and after preoperative DCF therapy. During two cycles of one-week-on and three-week-off administration, peripheral blood was collected before and after each “on” and before and after the surgical treatment followed by. By flow cytometry, CD15+CD14− granulocytes and CD15−CD14+ monocytes were observed. The effectiveness of NAC was determined hisitopathologically in accordance with the grading system of the Japanese Classification of Esophageal Carcinoma. Grading was as follows: Grade 0, no part of the tumor was affected; Grade1, less than two-thirds of the tumor was affected; Grade2, between two-thirds and the entire tumor was affected; and Grade 3, no residual tumor was present (Pathologically complete response) and clinically RECIST. Then their association were analyzed. Results/Discussion: Of 20 patients with esophageal cancer, 13 patients were responders and 7 patients were non-responders to the DCF therapy. Just after the first “on” course, the ratio of monocytes to myelocytes was observed in 25.4 ± 32.0% in the non-responders and 7.8 ± 10.3% in the responders (p = 0.073). The numbers of monocytes calculated were 229.4 ± 212.2 /μL in the non-responders and 51.1 ± 71.1 /μL in the responders (p = 0.01). Patients with better pathological or clinical responses showed lower ratio of monocytes to myelocytes and fewer number of monocytes in peripheral blood. No relationship was observed between granulocytes and the therapeutic response. Conclusion: These results suggest that analyzing immune related cells in peripheral blood might be useful for identification of predictive indicators for the response of preoperative DCF therapy. Citation Format: Tomohira Takeoka, Kumiko Goto, Mitsunobu Matsumoto, Yasuhiro Miyazaki, Tomoki Makino, Tsuyoshi Takahashi, Yukinori Kurokawa, Makoto Yamasaki, Shuji Takiguchi, Masaki Mori, Yuichiro Doki, Hisashi Wada. Chemotherapy for esophageal cancer: Off-target effect and biomarker [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B135.

[Introduction]Despite recent improvements in multi-treatment approaches, including surgery, radiotherapy and chemotherapy, the prognosis for patients with Esophageal squamous cell cancer (ESCC) remains unsatisfactory. In addition, predicting a prognosis and treatment response for ESCC had been constantly being required but remains difficult, even when using the TNM system. We identified Glypican-1(GPC1) as a novel disease specific antigen of ESCC by quantitative proteomic analysis focused on cell surface membrane proteins. GPC1 is one of the cell-surface heparan sulfate proteoglycans which has been reported as a prognostic factor in pancreatic cancer. [Objectives]This study aimed to clarify correlations between GPC1 expression and the prognosis in patient with ESCC and also reveal the association of GPC1 with the drug resistance to chemotherapeutic agents in squamous cancer cell lines and esophageal cancer patients treated with chemotherapy. [Methods]ESCC tissues were obtained from 175 patients who underwent esophagectomy at Osaka University Hospital(Osaka, Japan), from 2001 to 2013 and were subjected to immunohistochemistry. We performed immunohistochemical (IHC) assessment using anti-GPC1 antibody and investigated the association between GPC1 expression and the clinicopathological parameters. We divided the patients into two groups such as high expression group (HG) and low expression group (LG). Furthermore, we investigated associations between GPC1 expression and clinical response of neo-adjuvant chemotherapy for patients with ESCC. We established GPC1 knocked out cells of TE-14 using Crisper-Cas9 system and stable GPC1-transfected cells of LK-2(Lung squamous cell carcinoma cell line) using pcDNA3.1-GPC1 plasmid transfection. To investigate the relationship between GPC1 expression and sensitivity to cisplatin (CDDP), we examined sensitivity to CDDP by the WST-8 assay. [Results]The patients characteristics as follows, age (median) = 64yrs(39-80), gender (Male: Female) = 155:20. By the IHC analysis, 99 patients were classified into HG and 76 were LG. HG patients exhibited a poorer prognosis compared with the LG patients. (p We clarified there was a significant negative correlation between histological responses and GPC1 expression in patient with neoadjuvant chemo therapy. Furthermore, in vitro experiments showed that the IC50 values for CDDP of GPC1 expressing cells (TE-14 and GPC1 transfected LK-2) were significantly higher than that of GPC1 no-expressing cells (GPC1 knocked out TE-14 and LK-2). [Conclusion]Our study showed that GPC1 was an independent prognostic factor in esophageal cancer. Furthermore, we showed that GPC1 was a critical molecule for chemoresistance to cisplatin. Citation Format: Takahiko Nishigaki, Tsuyoshi Takahashi, Satishi Serada, Minoru Fujimoto, Hisashi Hara, Yasuhiro Miyazaki, Tomoki Makino, Yukinori Kurokawa, Makoto Yamasaki, Kiyokazu Nakajima, Shuji Takiguchi, Masaki Mori, Yuichiro Doki, Tetsuji Naka. Glypican-1 is a potential marker of prognosis and involved in chemoresistance of cisplatin in esophageal squamous cell 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 3133.

[INTRODUCTION] Esophageal squamous cell carcinoma (ESCC) is the major histological form of esophageal cancer in East Asian countries. Despite improvements of multimodality treatment, including surgery, radiotherapy and chemotherapy, the prognosis for patients with ESCC remains unsatisfactory compared to other cancer. Therefore, development of novel therapy is urgently required. Constitutive activation of JAK/STAT pathway has been reported in various tumor including ESCC, and associated with progression of tumor. Suppressor of cytokine signaling-1 (SOCS-1) has been cloned as a negative regulator of various cytokine signaling including JAK/STAT pathway. Recently, lines of evidences suggest that overexpression of SOCS-1 has been a promising therapeutic approach for various cancer. This study was aimed to evaluate the therapeutic effect of ESCC using SOCS-1 overexpressed by adenoviral vector (AdSOCS-1). [METHODS] For ten ESCC cell line, we evaluated cell growth inhibition via adenotransfection with various dose of AdSOCS-1 by WST-8 assay and Western blotting. Induction of apoptosis was also evaluated. In addition, it was also compared to AdSOCS-1 and JAK inhibitor for antiprolificative effect. To evaluate the therapeutic efficacy of AdSOCS-1, ESCC cell line (TE-14) was subcutaneously implanted into the ICR nu/nu mice. In addition, patient-derived tumor xenografts (PDX) using human ESCC subcutaneously transplanted in NOD-SCID mice were also assessed. Mice were intra-tumorally injection with AdSOCS-1 or control adenovirus vector (AdLacZ) twice a week for 4weeks. [RESULTS] AdSOCS-1 markedly suppressed proliferation of all ESCC cell lines in vitro. AdSOCS-1 strongly induced apoptosis via inhibiting not only JAK/STAT pathway but also MAPK and FAK signaling, especially in TE14 which was not significantly suppressed in JAK inhibitor. In TE14 xenograft model, the volume of tumors with AdSOCS-1 therapy was significantly lower than that with AdLacZ. Also, in PDX mice, injection with AdSOCS-1 significantly inhibited the tumor growth compared to AdLacZ. [CONCLUSIONS] Our results indicated that overexpression of SOCS-1 inhibited the progression of ESCC both in cell lines and xenograft model mice. Gene therapy by SOCS-1 can be a promising novel approach for intra-lesional therapy in ESCC. Citation Format: Takahito Sugase, Tsuyoshi Takahashi, Serada Satoshi, Eri Nakatsuka, Minoru Fujimoto, Yasuhiro Miyazaki, Tomoki Makino, Yukinori Kurokawa, Makoto Yamasaki, Kiyokazu Nakajima, Shuji Takiguchi, Masaki Mori, Yuichiro Doki, Tetsuji Naka. Antitumor effect of gene therapy with SOCS-1 in esophageal squamous cell carcinoma. [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 3740.

NY-ESO-1 is expressed in wide range of human malignancies expect testis in normal tissues and induces spontaneous antibody in patients with cancers expressing NY-ESO-1. Because of its unique expression and high immunogenicity, NY-ESO-1 is promising as a target of cancer vaccine. We have conducted serial clinical trials with the NY-ESO-1 protein since 2004 and antigen-specific immune responses were observed in most of patients immunized. Some patients showed feasible clinical responses however they were limited and novel immune modulators which could make NY-ESO-1 protein vaccination effective are needed. Recently, stimulation through the Toll-like receptors (TLRs) enhance the induction of antigen-specific immune responses upon cancer vaccine has become apparent. In this study, we conducted a phase I cancer vaccine clinical trial using NY-ESO-1 protein combined with Poly ICLC and Picibanil OK-432 as TLR 3 and 4 agonist, respectively. This trial was designed to evaluate the safety, induction of immune response and clinical response. Patients with advanced cancers expressing NY-ESO-1 were eligible. 200μg NY-ESO-1 protein mixed with Montanide in combination of Poly ICLC and/or Picibanil OK-432 was administered subcutaneously once every 2 weeks in six times. Seven patients with esophageal cancer, three with gastric cancer, two with lung cancer, two with ovarian cancer and one with malignant melanoma were enrolled in four groups, i) NY-ESO-1 protein alone (n=3), ii) NY-ESO-1 protein with OK-432 (n=3), iii) NY-ESO-1 protein with Poly ICLC (n=3), iv) NY-ESO-1 protein with OK-432 and Poly ICLC (n=6). All patients developed an injection-site reaction and fever (grade1 or 2) but no severe adverse events related to the drug were observed. An increase in the NY-ESO-1 antibody response was observed in all patients after vaccination. Earlier induction and higher frequency of NY-ESO-1 responsible T cells were observed in patients immunized with Poly ICLC than those without Poly ICLC. NY-ESO-1 protein vaccine with Poly ICLC and OK432 was safe and antigen-specific immune responses were induced in all patients. TLR agonists would be useful and effective immune modulators for cancer vaccines. Citation Format: Tomohira Takeoka, Hirotsugu Nagase, Yasuhiro Miyazaki, Tsuyoshi Takahashi, Yukinori Kurokawa, Tomoki Makino, Makoto Yamasaki, Shuji Takiguchi, Masaki Mori, Yuichiro Doki, Hisashi Wada. NY-ESO-1 protein cancer vaccine with TLR 3 and 4 agonists. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A169.

Objective Esophageal squamous cell carcinoma (ESCC) is the major histological form of oesophageal cancer in East Asian countries. Despite recent improvements in multimodality treatment, including surgery, radiotherapy and chemotherapy, the prognosis for patients with ESCC remains unsatisfactory. Therefore, development of novel therapy is urgently required. Suppressor of cytokine signaling-1 (SOCS-1) has been cloned as a negative regulator of various cytokine signaling. Recently, lines of evidences suggest that overexpression of SOCS-1 has been considered as a promising therapeutic approach for treatment of cancer. This study was aimed to evaluate the therapeutic effect of the overexpression of SOCS-1 by adenoviral vector (AdSOCS-1) against ESCC. METHODS By WST-8 assay, cell growth inhibition via infection with various dose of AdSOCS-1 was evaluated in 11 ESCC cell lines in vitro. We also evaluated the induction of apoptosis by overexpression of SOCS-1 in vitro. To evaluate the therapeutic efficacy of AdSOCS-1, ESCC cell line (TE-8) was subcutaneously implanted into the ICR nu/nu mice. In addition, therapeutic efficacy was also assessed using human xenografts subcutaneously transplanted in imuune-deficient mice. Mice were intra-tumorally injection with AdSOCS-1 or control adenovirus vector (AdLacZ) twice a week for 4weeks. RESULTS Among 11 ESCC cell lines, AdSOCS-1, markedly suppressed proliferation of 8 ESCC cell lines in vitro. AdSOCS-1 strongly induced apoptosis in vitro, via inhibiting JAK/STAT3 pathway. In the TE8 xenograft model, the volume of tumors after therapy in AdSOCS-1 group (1,580 ± 1,005mm3) were lower than that in control group (2,690 ± 1,309mm3). Furthermore, compared with AdLacZ, injection with AdSOCS-1 also inhibited the tumor growth of patient derived ESCC xenografts. CONCLUSIONS Our results indicated that overexpression of SOCS-1 inhibited the progression of ESCC both in ESCC cell lines and ESCC xenograft model derived from patient. SOCS-1 can be a promising novel therapeutic approach for intra-lesional therapy in ESCC. Citation Format: Hisashi Hara, Rie Nakatsuka, Tsuyoshi Takahashi, Satoshi Serada, Minoru Fujimoto, Yasuhiro Miyazaki, Tomoki Makino, Yukinori Kurokawa, Makoto Yamasaki, Hiroshi Miyata, Kiyokazu Nakajima, Shuji Takiguchi, Masaki Mori, Yuichiro Doki, Tetsuji Naka. Gene therapy for esophageal squamous cell carcinoma using SOCS-1 expressing adenoviral vector. [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 4213. doi:10.1158/1538-7445.AM2015-4213

Objective: The peritoneal metastasis is one of the most common recurrence style of gastric cancer (GC) and an effective treatment for their patients has not been established. Suppressor of cytokine signaling-1 (SOCS-1) is a negative regulator of various cytokine signaling and has been recently focused on as a therapeutic target in treatment for cancer. The aim of this study was to evaluate the effect with the enforced SOCS-1 expression by adenoviral vector (AdSOCS-1). METHODS: We evaluated the therapeutic effect of SOCS-1 in 6 GC cell lines with proliferation assay. Moreover, we examined the mechanism of antitumor effect of SOCS-1 focusing on the cell signalling and cell cycle in vitro. In vivo study, we evaluated the therapeutic effect in mice model. MKN45-Luc cell line was intraperitoneally injected into ICR nu/nu mice. And, we evaluated the peritoneal dissemination by the in vivo imaging system using Xenogen IVIS ® Imaging System. We treated these mice using the intraperitoneal injection of AdSOCS-1, twice a week for 4weeks. We evaluated the peritoneum dissemination by the photon intensity with time course repeatedly. Finally, we examined these tumor specimen pathologically and the overall survival (N=22). RESULTS: In 3 GC cell lines among 6 GC cell lines, AdSOCS-1 suppressed the proliferation. SOCS-1 showed an anti-proliferative potential in the GC cell proliferation via the inhibition of JAK/STAT and p38 MAPK signaling pathways and also induced apoptosis in vitro. Additionally, SOCS-1 is proven to influence the cell cycle in G2/M phase activating the Chk2 in a JAK/STAT independent manner. In Xenograft peritoneal dissemination model, the total count of photon after therapy in AdSOCS-1 group was significantly lower than that in control group. By TUNEL immunohistochemical analysis, tumor specimen in AdSOCS-1 group included more apoptosis area comparing with that in the control group. To assess whether overexpression of SOCS-1 also reduced proliferation of MKN-45 cells in vivo, Ki-67 expression was determined by immunohistochemistry. AdSOCS-1 group showed decreased Ki-67 positive cells compared to control group. We compared the overall survival of the peritoneal dissemination mice in two groups. AdSOCS-1 group showed better outcome than control group significantly. CONCLUSIONS: Our results indicated that SOCS-1 inhibited the progression of gastric cancer in Xenograft peritoneal dissemination model. SOCS-1 can be a novel therapeutic application for peritoneal metastasis in gastric cancer. Citation Format: Rie Nakatsuka, Tsuyoshi Takahashi, Satoshi Serada, Minoru Fujimoto, Yoshihito Souma, Yasuhiro Miyazaki, Yukinori Kurokawa, Makoto Yamasaki, Hiroshi Miyata, Kiyokazu Nakajima, Shuji Takiguchi, Masaki Mori, Yuichiro Doki, Yuichiro Doki, Tetsuji Naka. Gene therapy for peritoneal dissemination model of gastric cancer using SOCS-1 by Adenoviral Vector. [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 722. doi:10.1158/1538-7445.AM2014-722

Despite the initial effectiveness of imatinib for gastrointestinal stromal tumors (GISTs), patients must continue lifelong imatinib therapy, and may eventually relapse owing to acquired drug resistance. Pathology reveals the persistence of viable cells during imatinib therapy, even in the disappearance region upon radiological examination. These cells are thought to cause acquired drug resistance and relapse. To uncover the mechanisms underlying these clinical issues, we used GIST-T1 cells to investigate the phosphoproteomic alterations induced by imatinib treatment. Because some tyrosine kinases might be activated after imatinib exposure, it is important to reveal the increased phosphorylation of proteins on a proteomic scale, and elucidate drug-resistant proteins. Proteins extracted from imatinib-treated or untreated GIST-T1 cells were digested with trypsin. The resulting tyrosine-phosphorylated peptides were purified using an immunoaffinity-enriched and iTRAQ-labeled reagent, and analyzed using mass spectroscopy. Using this phosphoproteomic approach, we identified 171 tyrosine phosphorylation sites spanning 134 proteins. We also detected increased tyrosine phosphorylation of Fyn (Y420) and focal adhesion kinase (FAK) (Y576). Increased phosphorylation was also confirmed by Western blot analysis, and knockdown of Fyn or FAK increased cells’ sensitivity to imatinib. Furthermore, the anti-proliferative effect of imatinib significantly enhanced imatinib-resistant GIST-T1 cells, which exhibited constitutive phosphorylation of FAK when treated with a FAK-selective kinase inhibitor. Moreover, FYN was proved to be a signal of FAK of upper reaches. These results indicate that an iTRAQ-based quantitative phospho-tyrosine-focused phosphoproteomic approach is a powerful method for screening phosphoproteins associated with drug resistance. Fyn or FAK might be potential therapeutic targets to overcome acquired resistance to imatinib in GISTs. Citation Format: Maiko Ako, Tsuyoshi Takahashi, Satoshi Serada, Minoru Fujimoto, Yasuaki Miyazaki, Kazuki Shimada, Takayuki Ikezoe, Takahiro Taguchi, Yukinori Kurokawa, Makoto Yamasaki, Hiroshi Miyata, Kiyokazu Nakajima, Shuji Takiguchi, Masaki Mori, Yuichirou Doki, Tetsuji Naka, Toshirou Nishida. New findings of kinase switch in gastrointestinal stromal tumor under imatinib using phosphoproteomic analysis. [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 3392. doi:10.1158/1538-7445.AM2013-3392