Tomohisa Okuno

CD9-positive exosomes from cancer-associated fibroblasts stimulate the migration ability of scirrhous-type gastric cancer cells

This corrects the article DOI: 10.1038/bjc.2017.85

Pyruvate kinase isozyme M2 and glutaminase might be promising molecular targets for the treatment of gastric cancer.

The aim of this study was to analyze the significance of glucose metabolism‐related enzymes in the proliferation of gastric cancer under hypoxia. Four hypoxia‐resistant gastric cancer cell lines and four parent cell lines were used. Reverse transcription–PCR was used to evaluate the mRNA expression levels of the following metabolism‐related enzymes: pyruvate kinase isozyme M2 (PKM2), glutaminase (GLS), enolase 1 (ENO1), glucose‐6‐phosphate dehydrogenase (G6PDH), and PKM1. The effects of these enzymes on the proliferation of gastric cancer cells were examined using siRNAs, shikonin as a PKM2 inhibitor, or BPTES as a GLS inhibitor, in vitro and in vivo. Levels of both PKM2 and GLS mRNA were significantly high in all hypoxia‐resistant cell lines, compared with those of their parent cells. Knockdown of PKM2 and GLS significantly decreased the proliferation of all hypoxia‐resistant cells. The combination of siPKM2 and siGLS significantly decreased proliferation compared with treatment by siPKM2 or siGLS alone. The knockdown of ENO1, G6PDH, or PKM1 did not decrease the proliferation of all hypoxia‐resistant cells. Combination treatment using shikonin and BPTES inhibited the proliferation of all hypoxia‐resistant cancer cells more than that by either agent alone. The in vivo study indicated that the tumor size treated by the combination of shikonin and BPTES was significantly smaller than that of vehicle‐treated group. These findings suggested that PKM2 and GLS might play important roles in the proliferation of hypoxic gastric cancer cells. A combination of PKM2 and GLS inhibitors could be therapeutically promising for the treatment of gastric cancer.

Clinico-pathological significance of exosome marker CD63 expression on cancer cells and stromal cells in gastric cancer

Background It has been reported that CD63, an exosome marker, is expressed in solid cancer tissues. However, its significance in patients with gastric cancer has not been clarified. Exosomes derived from cancer cells and stromal cells might play an important role in the intracellular communications involved in the development of carcinoma. This study aimed to clarify the relationship between CD63 expression in cancer cells and stromal cells and clinical-pathologic factors. Methods A total of 595 gastric cancer patients were enrolled in this study. CD63 expression in cancer cells and stromal cells was analyzed by immunohistochemistry. The correlations between CD63 expression and several clinicopathological factors were investigated. Results CD63 expression was mainly observed on the cell membranes of cancer cells, and in the cytoplasm of stromal cells. Of 595 patients, 247 cases had CD63-positive cancer cells, and 107 cases had CD63-positive stromal cells. Cases with CD63-positive cancer cells were significantly correlated with scirrhous-type gastric cancer, tumor depth, lymph node metastasis, lymphatic invasion, and tumor size. Cases with CD63-positive stromal cells were significantly correlated with age (≥65), tumor depth (T3-4), lymphatic invasion, and tumor size (≥ 5 cm). The 5-year survival rate was significantly lower (p<0.001) in patients with CD63-positive than CD63-negative tumors. Multivariate analysis showed that CD63 expression in cancer cells was a significant independent prognostic factor in patients with gastric cancer. Conclusion CD63 might be a prognostic marker for patients with gastric cancer. CD63-positive exosomes might be associated with the interaction between stromal cells and cancer cells.

Significance of the Lysyl Oxidase Members Lysyl Oxidase Like 1, 3, and 4 in Gastric Cancer

Background/Aims: Lysyl oxidase (LOX) family members play a key role in modifying the primary tumor microenvironment by crosslinking collagens and elastin in the extracellular matrix. The aim of this study was to analyze the LOX-like (LOXL)1, LOXL3, and LOXL4 expressions in gastric cancer tissue by immunohistochemical staining. Methods: The correlations between the clinicopathological features of 597 primary gastric carcinomas and LOX family members – LOXL1, LOXL3, and LOXL4 – were investigated by immunohistochemical studies. The effect of the transforming growth factor β1 (TGFβ1) on the expressions of LOXL1, LOXL3, and LOXL4 in gastric cancer was examined using diffuse-type gastric cancer cell lines in vitro. Results: The expressions of LOXL1, LOXL3, and LOXL4 were correlated with T invasion, lymph node metastasis, and lymphatic and venous invasion. LOXL1 expression was associated with histological intestinal-type and expanding growth patterns. The overall survival of patients with LOXL1-, LOXL3-, or LOXL4-positive cancer was poorer than those with negative cancer. LOXL3 and LOXL4 mRNA expressions were significantly high in diffuse-type gastric cancer cells with high invasion ability. TGFβ decreased the LOXL1 expression and increased LOXL3 and LOXL4 expression. Conclusion: LOXL1, LOXL3, and LOXL4 expressions are associated with distant metastasis of gastric cancer.

Abstract 3791: Clinical significance of EpCAM-negative and CEA-positive circulating tumor cells in gastric carcinoma

It has been reported that the examination of circulating tumor cells (CTCs) is beneficial for predicting tumor stage or treatment response. The epitherial cell adhesion molecule (EpCAM) and cytokeratin has been used as epithelial markers for the identification of CTCs. Since the epithelial-mesenchymal transition is one of malignant phenotype of cancer cells, it is necessary to investigate other markers which might detect mesenchymal CTCs. This study aims to clarify the clinical significance of EpCAM negative and CEA positive CTCs in patients with gastric cancer. Forty patients who underwent operation for gastric cancer were enrolled in this study. A total of 10ml peripheral blood samples were obtained preoperatively. Informed consent to participate in this study was obtained from all of the patients before their surgery. This study was approved by the Human Ethics Review Committee of our University. After collection of mononuclear cell fraction, these cells were stained by PAPI, anti-CD45, anti-EpCAM, anti-cytokeratin, and anti-CEA (CD66e) antibody. We enumerated the number of cells which showed EpCAM, cytokeratin, and CEA positive expression among CD45 negative cells. ROC analyses were performed for investigating predictive value of these cell counts for distant metastasis. We examined association between these cell counts and clinicopathological features. ROC analyses revealed that the AUC for predicting distant metastasis using EpCAM, Cytokeratin, and CEA positive cell counts were 0.583, 0.638, and 0.769, respectively. The median number of EpCAM negative and CEA positive cells was 231 (range: 0-61897). The cell counts of EpCAM negative and CEA positive cells were significantly associated with Tumor depth (p=0.0084), lymph node metastasis (p=0.0107), and tumor size (p=0.023). CEA might be useful marker for detecting high risk of recurrence in patients with gastric cancer. Citation Format: Yuichiro Miki, Masakazu Yashiro, Tomohisa Okuno, Kishu Kitayama, Tatsuro Tamura, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. Clinical significance of EpCAM-negative and CEA-positive circulating tumor cells in gastric carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3791. doi:10.1158/1538-7445.AM2017-3791