Takafumi Nishii

Cancer stem cell-like SP cells have a high adhesion ability to the peritoneum in gastric carcinoma.

Cancer stem cells (CSCs) are considered to be responsible for cancer metastasis, but the evidence to conclusively prove this hypothesis remains uncertain. The side population (SP), as evaluated by a flow cytometric analysis using Hoechst 33342, has been known as CSC‐rich population. The aim of this study was to clarify the characterization of the SP cells in peritoneal metastasis of gastric carcinoma. Gastric cancer cell lines OCUM‐2M, OCUM‐2D, and OCUM‐2MD3 (a daughter cell line with high potential for peritoneal metastasis) were used. We isolated SP cells from OCUM‐2M and OCUM‐2D using flow cytometry. Serial sorting was performed three times to enrich SP cells, and they were designated as OCUM‐2M/SP and OCUM‐2D/SP cells. Flow cytometric analysis showed 0.46%, 0.29%, 5.24%, 6.49%, and 11.3% of the SP cells to be found in OCUM‐2M, OCUM‐2D, OCUM‐2MD3, OCUM‐2M/SP, and OCUM‐2D/SP cells, respectively. The intraperitoneal inoculation of SP cells and OCUM‐2MD3 cells produced peritoneal metastasis, but parent cells did not. The adhesion ability of SP and OCUM‐2MD3 cells was significantly high in comparison to that of parent cells. The expression level of adhesion molecules α2‐, α5‐, β3‐, and β5‐integrin, and CD44, was high in SP cells compared to parent cells. The expression of stemness markers, Oct3/4 and Sox2, increased in the SP‐cell‐injected tumors. These findings suggested that CSC‐like SP cells expressing α2‐, α5‐, β3‐, and β5‐integrin, and CD44, may play an important role for peritoneal metastasis in gastric carcinoma. Oct3/4 and Sox2 may be associated with CSC in gastric cancer. (Cancer Sci 2009)

Establishment and characterization of a new hypoxia-resistant cancer cell line, OCUM-12/Hypo, derived from a scirrhous gastric carcinoma

Background:Many kinds of solid tumour have heterogeneously a hypoxic environment. Tumour hypoxia reported to be associated with more aggressive tumour phenotypes such as high metastatic ability and resistance to various anti-cancer therapies which may lead to a poorer prognosis. However, the mechanisms by which hypoxia affects the aggressive phenotypes remain unclear.Methods:We established a scirrhous gastric carcinoma cell line (OCUM-12) from ascites associated with scirrhous gastric carcinoma, and a hypoxia-resistant cancer cell line (OCUM-12/Hypo) was cloned from OCUM-12 cells by continuous exposure to 1% oxygen.Results:Histologic findings from orthotopic tumours derived from parent OCUM-12 cells and daughter OCUM-12/Hypo cells revealed poorly differentiated adenocarcinoma with extensive fibrosis that resembled human scirrhous gastric cancer. Necrotic lesions were frequently detected in the OCUM-12 tumours but were rarely found in the OCUM-12/Hypo tumours, although both types had multiple hypoxic loci. Apoptosis rate of OCUM-12 cells was increased to 24.7% at 1% O2, whereas that of OCUM-12/Hypo was 5.6%. The OCUM-12/Hypo orthotopic models developed multiple metastases to the peritoneum and lymph nodes, but the OCUM-12 models did not. OCUM-12/Hypo cells showed epithelial-to-mesenchymal transition and high migratory and invasive activities in comparison with OCUM-12 cells. The mRNA expression levels of both E-cadherin and zonula occludens ZO-1 and ZO-2 decreased in OCUM-12/Hypo cells, and that of vimentin, Snail-1, Slug/Snail-2, Twist, ZEB-1, ZEB-2, matrix metalloproteinase-1 (MMP-1), and MMP-2 were increased in OCUM-12/Hypo cells.Conclusion:OCUM-12 and OCUM-12/Hypo may be useful for the elucidation of disease progression associated with scirrhous gastric cancer in the setting of chronic hypoxia.

Cancer‐associated fibroblasts might sustain the stemness of scirrhous gastric cancer cells via transforming growth factor‐β signaling

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. The aim of this study was to clarify the effect of CAFs on CSCs characteristics in gastric carcinoma. Scirrhous gastric cancer cell lines, OCUM‐12 and OCUM‐2MD3, and non‐scirrhous gastric cancer cell lines, MKN‐45 and MKN‐74, were used. OCUM‐12/side population (SP) cells and OCUM‐2MD3/SP cells were sorted by flow cytometry as CSC‐rich cells from the parent cells. 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 were performed to identify CSCs properties. Effect of CAFs on the tumorigenicity by OCUM‐12/SP cells was examined using nude mice. CAF CM significantly increased the percentages of the SP fraction of OCUM‐12/SP and OCUM‐2MD3/SP cells, but not that of MKN‐45/SP and MKN‐74/SP cells. Taken together, CM from CaF‐37 significantly increased the number of spheroid colonies and the expression level of CSC markers of OCUM‐12/SP and OCUM‐2MD3/SP cells. These stimulating‐activities by CM were significantly decreased by TGFβ inhibitors, but not FGFR and cMet inhibitor. Tumorigenicity by subcutaneous coinoculation of OCUM‐12/SP cells with CAFs was significantly high in comparison with that by OCUM‐12/SP cells alone. Phospho‐Smad2 expression level was significantly increased by co‐inoculation with CAFs. These findings suggested that CAFs might regulate the stemness of CSCs in scirrhous gastric cancer by TGFβ signaling.

A c-Met inhibitor increases the chemosensitivity of cancer stem cells to the irinotecan in gastric carcinoma

Background:Cancer stem cells (CSCs) may be postulated mediators of the chemoresistance. This study aimed to determine an effective signal inhibitor with effects on the proliferation of CSCs in combination with anticancer drugs.Methods:We used three gastric cancer cell lines and three side population (SP)-enriched CSC cell lines. We examined the combined effects of inhibitors against stemness signals, including c-Met inhibitor SU11274, and five anticancer drugs on the CSC proliferation and mRNA expression of chemoresistance-associated genes.Results:The IC50 of irinotecan in SP-enriched CSC was 10.5 times higher than parent OCUM-2M cells, whereas that of oxaliplatin, taxol, gemcitabine, and 5-fluorouracil was 2.0, 2.8, 2.0, and 1.2, respectively. The SP cell lines had higher expression levels of UGT1A1, ABCG2, and ABCB1 than their parent cell lines. There was a synergistic antiproliferative effect with a combination of SU11274 and SN38 in SP cells, but not other inhibitors. The SU11274 significantly decreased the expression of UGT1A1, but not ABCG2 and ABCB1. The SN38 plus SU11274 group more effectively suppressed in vivo tumour growth by OCUM-2M/SP cells than either group alone.Conclusion:Cancer stem cells have chemoresistance to irinotecan. The c-Met inhibitor may be a promising target molecule for irinotecan-based chemotherapy of gastric cancer.

Phosphorylated Smad2 in Advanced Stage Gastric Carcinoma

BackgroundTransforming growth factor β (TGFβ) receptor signaling is closely associated with the invasion ability of gastric cancer cells. Although Smad signal is a critical integrator of TGFβ receptor signaling transduction systems, not much is known about the role of Smad2 expression in gastric carcinoma. The aim of the current study is to clarify the role of phosphorylated Smad2 (p-Smad2) in gastric adenocarcinomas at advanced stages.MethodsImmunohistochemical staining with anti-p-Smad2 was performed on paraffin-embedded specimens from 135 patients with advanced gastric adenocarcinomas. We also evaluated the relationship between the expression levels of p-Smad2 and clinicopathologic characteristics of patients with gastric adenocarcinomas.ResultsThe p-Smad2 expression level was high in 63 (47%) of 135 gastric carcinomas. The p-Smad2 expression level was significantly higher in diffuse type carcinoma (p = 0.007), tumours with peritoneal metastasis (p = 0.017), and tumours with lymph node metastasis (p = 0.047). The prognosis for p-Smad2-high patients was significantly (p = 0.035, log-rank) poorer than that of p-Smad2-low patients, while a multivariate analysis revealed that p-Smad2 expression was not an independence prognostic factor.ConclusionThe expression of p-Smad2 is associated with malignant phenotype and poor prognosis in patients with advanced gastric carcinoma.

In vitro and in vivo evidence that a combination of lapatinib plus S-1 is a promising treatment for pancreatic cancer

Lapatinib is a small molecule inhibitor of both HER2 and the epidermal growth factor receptor (EGFR). We investigated the effect of treatment with lapatinib alone or in combination with a fluoropyrimidine derivative S‐1 against pancreatic cancer. The HER2/EGFR expression in each of the four pancreatic cancer cell lines MiaPaca‐2, PANC‐1, Capan‐1 and Capan‐2 was measured by flow cytometry. The anti‐tumor effects of lapatinib (30 mg/kg) and/or S‐1 (10 mg/kg) were evaluated using female BALB/c nude mice xenografts generated using these four cell lines. Synergy between lapatinib and S‐1 was examined by median effect analysis in vitro. Resected pancreatic cancer tissues from 137 patients were immunohistochemically stained with anti‐human HER2 and EGFR antibodies. The administration of lapatinib as a single agent substantially suppressed tumor growth in vivo of all pancreatic cancer cell lines examined. A strong correlation was observed between HER2 expression and the anti‐tumor effect of lapatinib in vivo. Lapatinib synergized with S‐1 to inhibit the tumor growth of MiaPaca‐2 and PANC‐1 xenografts. When used as a single agent in vitro, lapatinib barely inhibit the cell growth of any cell line. However, lapatinib synergized with the anti‐tumor activity of the S‐1 components 5‐fluorouracil and 5‐chloro‐2,4‐dihydrogenase against all cell lines. Immunohistochemical staining demonstrated that 70% of the pancreatic cancers overexpressed HER2 and/or EGFR. Both lapatinib monotherapy and combined treatment with S‐1 may be promising treatments for patients with pancreatic cancers; the majority these cancers express lapatinib target molecules. (Cancer Sci 2009; 00: 000–000)

The Preoperative Geriatric Nutritional Risk Index Predicts Postoperative Complications in Elderly Patients with Gastric Cancer Undergoing Gastrectomy

Background/Aim: The relationship between the preoperative Geriatric Nutritional Risk Index (GNRI) and morbidity of patients with gastric cancer (GC) undergoing gastrectomy has not yet been reported. Our study aimed to investigate whether preoperative GNRI is associated with short-term outcomes in elderly patients with GC. Patients and Methods: This study enrolled 348 elderly patients with GC who were more than 75 years old and underwent curative gastrectomy for GC at our Institution between January 2006 and December 2015. GNRI was invoked to stratify patients as high (GNRI≥92; n=190) or low (GNRI<92; n=158) GNRI nutritional status. The clinicopathologic features and short-term outcomes were compared. Results: In multivariate analysis, low GNRI emerged as an independent predictor of postoperative complications (Clavien Dindo classification grade II≤). Low GNRI demonstrated significantly more frequent extra-surgical complications than high GNRI. Significantly more patients with low GNRI suffered from postoperative pneumoniae than patients with high GNRI (p=0.013). On the other hand, the incidence of surgical field complications such as leakage, pancreatic fistula and intraabdominal abscess did not differ significantly between the groups. Conclusion: GNRI is useful in predicting postoperative complications of elderly patients with GC undergoing gastrectomy. Preoperative GNRI has merit as a gauge of postoperative complications in the extra-surgical field, especially pneumonia. There was no relationship between preoperative GNRI and surgical field complications in this setting.

Neoadjuvant Radiotherapy with Capecitabine Plus Bevacizumab for Locally Advanced Lower Rectal Cancer: Results of a Single-institute Phase II Study.

Background/Aim: A single-arm phase II clinical trial was conducted to evaluate the safety and efficacy of adding bevacizumab to standard capecitabine-based neoadjuvant chemoradiotherapy (CRT) for the treatment of locally advanced rectal cancer (LARC). Patients and Methods: Twenty-five patients were enrolled. Patients received capecitabine-based CRT for 5 weeks and 3 days. Bevacizumab was administered every 2 weeks during CRT. Within 6-10 weeks after completion of CRT, surgery was performed. Results: With regard to CRT-related acute toxicities, most of the adverse events were limited to grade 1. A pathological complete response was obtained in four (16%) patients. In total, six patients (24%) developed postoperative complications. Six out of five (83%) patients healed without the need for surgical intervention. Conclusion: Although acute toxicity during CRT with bevacizumab was minimal and postoperative complications do not seem to increase, the addition of bevacizumab apparently offers no clinically-significant benefit for patients with LARC.

Abstract 225: A c-Met inhibitor increases the chemosensitivity of cancer stem cells to the irinotecan active metabolite SN38 in gastric carcinoma.

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