Hiroaki Kasashima

Lysyl oxidase-like 2 (LOXL2) from stromal fibroblasts stimulates the progression of gastric cancer

The aim of this study was to clarify the role of fibroblast-derived Lysyl oxidase-like 2 (LOXL2) in the development of gastric cancer. The correlation between the clinicopathological features of 548 primary gastric carcinomas and LOXL2 expression in stromal cells was examined by immunohistochemistry. Two gastric cancer cell lines, OCUM-12 and NUGC-3, and cancer-associated fibroblasts (CAFs) were used in this in vitro study. The effect of fibroblast-derived LOXL2 on the motility of gastric cancer cells was analyzed by using a wound-healing assay, a double-chamber invasion assay, and western blot. LOXL2 expression in stromal cells was significantly associated with tumor invasion depth, lymph node metastasis, lymphatic invasion, venous invasion, and peritoneal dissemination. Multivariable logistic regression analysis revealed that LOXL2 expression in stromal cells could be an independent predictive parameter for the overall survival of patients. CAFs significantly stimulated the migration and invasion of OCUM-12 and NUGC-3 cells. This motility-stimulating ability of CAFs was inhibited by LOXL2 siRNA. Western blot analysis indicated that phosphorylation of focal adhesion kinase (FAK) in cancer cells was increased by the conditioned medium from CAFs, and was decreased by the conditioned medium from LOXL2 siRNA-treated CAFs. LOXL2 expression in stromal cells may be a useful prognostic factor for patients with gastric cancer. Fibroblast-derived LOXL2 may stimulate the motility of gastric cancer cells.

Lysyl oxidase is associated with the epithelial-mesenchymal transition of gastric cancer cells in hypoxia.

PurposeIt has been reported that lysyl oxidase (LOX) is a hypoxia-responsive factor and is associated with the malignant progression of carcinoma. The aim of this study was to clarify the relationship between the epithelial–mesenchymal transition (EMT) and LOX in gastric cancer cells under hypoxia.MethodsTwo gastric cancer cell lines, OCUM-2MD3 and OCUM-12, were used in an in vitro study. The effect of LOX small interfering RNA (siRNA) on the EMT and motility of gastric cancer cells under hypoxic condition was analyzed by reverse transcription PCR, Western blot, a wound-healing assay, and an invasion assay. Correlations between LOX expression and the clinicopathological features of 544 patients with gastric carcinoma were examined immunohistochemically.ResultsHypoxic conditions increased the number of polygonal or spindle-shaped cells resulting from EMT in gastric cancer cells. The EMT of cancer cells induced by hypoxia was inhibited by treatment with LOX siRNA. The number of migrating and invading gastric cancer cells in hypoxia was significantly decreased by LOX knockdown. LOX siRNA significantly increased the E-cadherin level and decreased the vimentin level of gastric cancer cells. LOX expression was significantly associated with invasion depth, tumor differentiation, lymph node metastasis, lymphatic invasion, venous invasion, and peritoneal metastasis. Multivariable analysis revealed that LOX was an independent parameter for overall survival.ConclusionLOX affects the EMT of gastric cancer cells in hypoxic conditions. LOX expression is a useful prognostic factor for patients with gastric cancer.

Comparative Proteomics Analysis of Gastric Cancer Stem Cells

Cancer stem cells (CSCs) are responsible for cancer progression, metastasis, and recurrence. To date, 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. CSC-like SP cells, OCUM-12/SP cells, OCUM-2MD3/SP cells, and their parent OCUM-12 cells and OCUM-2MD3 cells were used in this study. 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. Candidate proteins detected by proteomics technology were validated by immunohistochemical analysis of 300 gastric cancers. 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. These findings might suggest that 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.

Diffuse-type gastric cancer cells switch their driver pathways from FGFR2 signaling to SDF1/CXCR4 axis in hypoxic tumor microenvironments

Cancer-associated fibroblasts (CAFs) have been considered to play an important role for tumor progression of cancer. Solid tumors contain heterogeneous distribution of oxygen in their microenvironments. This study investigated the growth signaling of gastric cancer (GC) cells in focus on the interaction with CAFs and GC cells under normoxia and hypoxia. Four diffuse-type GC cell lines, two intestinal-type GC cell lines and three CAF cell lines were used. Cells were examined for expression of C-X-C chemokine receptor 4 (CXCR4), fibroblast growth factor receptor 2 (FGFR2) and stromal-derived factor 1 (SDF1) by RT-PCR, western blot, ELISA and immunohistochemical staining of xenografted tumors. GC cell proliferation was examined under hypoxia in the presence or absence of CAFs, a FGFR2 inhibitor, a CXCR4 inhibitor and HIF1α siRNA. Proliferation of diffuse-type GC cells, but not intestinal-type GC cells, was significantly increased by CAFs. CXCR4 expression by diffuse-type GC cells was significantly increased in hypoxia, while FGFR2 expression was decreased. CXCR4 expression was correlated with hypoxic microenvironment of xenografted tumor, but FGFR2 expression was not. FGFR2 inhibition significantly decreased the growth-stimulating activity of CAFs for diffuse-type GC cells in normoxia. In contrast, CXCR4 inhibition significantly decreased the growth-stimulating activity of CAFs in hypoxia. SDF1 production by CAFs was increased in hypoxia, while cancer cells did not produce SDF1. HIF1 siRNA significantly decreased both CXCR4 expression by diffuse-type GC cells and SDF1 production by CAFs. These findings suggest that diffuse-type GC cells might switch their driver pathways from FGFR2 signaling to SDF1/CXCR4 axis through HIF1 in hypoxic tumor microenvironments.

Bone marrow-derived stromal cells are associated with gastric cancer progression

Background:The aim of this study was to clarify the role of bone marrow-derived stromal cells (BM-SCs) expressing CD271 in the development of gastric cancer.Methods:The effect of human BM-SCs on the proliferation and motility of six gastric cancer cell lines, OCUM-2M, OCUM-2MD3, OCUM-12, KATO-III, NUGC-3, and MKN-74, was examined. CD271 expression levels in BM-SCs were analysed by flow cytometry. We also generated a gastric tumour model by orthotopic inoculation of OCUM-2MLN cells in mice that had received transplantation of bone marrow from the CAG-EGFP mice. The correlation between the clinicopathological features of 279 primary gastric carcinomas and CD271 expression in tumour stroma was examined by immunohistochemistry.Results:Numerous BM-SCs infiltrated the gastric tumour microenvironment; CD271 expression was found in ∼25% of BM-SCs. Conditioned medium from BM-SCs significantly increased the proliferation of gastric cancer cell lines. Furthermore, conditioned medium from gastric cancer cells significantly increased the number of BM-SCs, whereas migration of OCUM-12 and NUGC-3 cells was significantly increased by conditioned medium from BM-SCs. CD271 expression in stromal cells was significantly associated with macroscopic type-4 cancers, diffuse-type tumours, and tumour invasion depth. The overall survival of patients (n=279) with CD271-positive stromal cells was significantly worse compared with that of patients with CD271-negative stromal cells. This is the first report of the significance of BM-SCs in gastric cancer progression.Conclusions:Bone marrow-derived stromal cells might have an important role in gastric cancer progression, and CD271-positive BM-SCs might be a useful prognostic factor for gastric cancer patients.

Clinicopathologic significance of the CXCL1-CXCR2 axis in the tumor microenvironment of gastric carcinoma

Purpose It was reported that the chemokine (C-X-C motif) ligand 1 (CXCL1) from cancer cells stimulated the recruitment of bone marrow-derived mesenchymal cells (BM-MCs) into tumor stroma via chemokine (C-X-C motif) receptor 2 (CXCR2) signaling. We conducted this retrospective study to determine the clinicopathologic significance of the CXCL1-CXCR2 axis in human gastric cancer. Methods The correlations between the clinicopathological features of 270 primary gastric carcinomas and CXCL1 in cancer cells and CXCR2 in stromal cells were analyzed in immunohistochemical studies. The effect of gastric cancer cells on the expression of CXCR2 in BM-MCs was examined using diffuse-type gastric cancer cell lines in vitro. Results The expression of CXCL1 in cancer cells was correlated with T invasion (T2–T4), lymph node metastasis, lymphatic invasion, venous invasion, peritoneal cytology, peritoneal metastasis and CXCR2 expression in stromal cells. The expression of CXCR2 in stromal cells was correlated with macroscopic type-4 cancers, histological type, T invasion (T2–T4), lymph node metastasis, lymphatic invasion, infiltration, peritoneal cytology, peritoneal metastasis and CD271 expression in stromal cells. The overall survival of patients with CXCL1 and CXCR2-positive cancer was poorer than that of the patients with negative cancer. Both CXCL1 expression in cancer cells and CXCR2 expression in stromal cells were independent prognostic factors for gastric cancer patients. Conclusion The expressions of CXCL1 in cancer cells and CXCR2 in stromal cells are useful prognostic factors for gastric cancer patients.

A case of retroperitoneal liposarcoma after delivery with expression of estrogen receptor: Report of a case.

Highlights • Retroperitoneal liposarcoma related pregnancy is rare.• Liposarcoma is sometimes positive for hormone receptor.• Additional therapy for liposarcoma is important to improve prognosis.

Identification of tumour-reactive lymphatic endothelial cells capable of inducing progression of gastric cancer

Background:Tumour cells and stromal cells interact in the tumour microenvironment; moreover, stromal cells can acquire abnormalities that contribute to tumour progression. However, interactions between lymphatic endothelial cells (LECs) and tumour cells are largely unexamined. In this study, we aimed to determine whether tumour-specific LECs inhabit the tumour microenvironment and examine their influence on this microenvironment.Methods:We isolated normal LECs (NLECs) from a non-metastatic lymph node and tumour-associated LECs (TLECs) from cancerous lymph nodes. We examined proliferative and migratory potency, growth factor production, and gene expression of each type of LEC. Moreover, we developed a co-culture system to investigate the interactions between gastric cancer cells and LECs.Results:When compared with NLEC, TLECs had an abnormal shape, high proliferative and migratory abilities, and elevated expression of genes associated with inflammation, cell growth, and cell migration. NLECs co-cultured with gastric cancer cells from the OCUM12 cell line acquired TLEC-like phenotypes. Also, OCUM12 cells co-cultured with TLECs expressed high levels of genes responsible for metastasis.Conclusions:Our results demonstrated that LECs interacted with tumour cells and obtained abnormal phenotypes that could have important roles in tumour progression.

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.

Abstract 3863: The stemness of gastric cancer stem cells is sustained by TGFβ produced from cancer-associated fibroblasts

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