CD90(+) Mesothelial-Like Cells in Peritoneal Fluid Promote Peritoneal Metastasis by Forming a Tumor Permissive Microenvironment

The peritoneal cavity is a common target of metastatic gastrointestinal and ovarian cancer cells, but the mechanisms leading to peritoneal metastasis have not been fully elucidated. In this study, we examined the roles of cells in peritoneal fluids on the development of peritoneal metastasis. We found that a minor subset of human intraperitoneal cells with CD90(+)/CD45(−) phenotype vigorously grew in culture with mesothelial-like appearance. The mesothelial-like cells (MLC) displayed the characteristics of mesenchymal stem cell, such as differentiating into adipocytes, osteocytes, and chondrocytes, and suppressing T cell proliferation. These cells highly expressed type I collagen, vimentin, α-smooth muscle actin and fibroblast activated protein-α by the stimulation with TGF-β, which is characteristic of activated myofibroblasts. Intraperitoneal co-injection of MLCs with the human gastric cancer cell line, MKN45, significantly enhanced the rate of metastatic formation in the peritoneum of nude mice. Histological examination revealed that many MLCs were engrafted in metastatic nodules and were mainly located at the fibrous area. Dasatinib, a potent tyrosine kinase inhibitor, strongly inhibited the proliferation of MLCs but not MKN45 in vitro. Nevertheless, oral administration of Dasatinib significantly inhibited the development of peritoneal metastasis of MKN45, and resulted in reduced fibrillar formation of metastatic nodules. These results suggest floating MLCs in the peritoneal fluids support the development of peritoneal metastasis possibly through the production of the permissive microenvironment, and thus the functional blockade of MLCs is a reasonable strategy to treat recurrent abdominal malignancies.     

Reg protein is overexpressed in gastric cancer cells,where it activates a signal transduction pathway that converges on ERK1/2 to stimulate growth

Reg is a growth factor with mitogenic effects on pancreatic β cells and gastric stem cells. To date, there has been no information available on Reg-mediated intracellular signal transduction pathways. The role of Reg in the gastric carcinogenesis is also unknown. In the current study, the Reg signaling pathway in gastric cancer cell was examined. Reg treatment of MKN45 gastric cancer cells resulted in tyrosyl-phoshorylation of several cellular proteins and subsequent activation of classical MAPK, ERK1/2. Reg also stimulated thymidine incorporation in MKN45 and AGS gastric cancer cells in a dose-dependent manner. Finally, Reg was shown to be highly expressed in a large number of gastric cancers in vivo. Taken together, these data suggest that gastric cancer cells have gained the ability to overexpress Reg protein, which confer upon themselves added proliferative capacities, resulting in a considerable growth advantage.     

Enhancement of Metastatic and Invasive Capacity of Gastric Cancer Cells by Transforming Growth Factor-β1

Transforming growth factor-β (TGF-β),a multifunctional cytokine,exerts contradictory rolesin different kinds of cells.A number of studies have revealed its involvement in the progression of many typesof tumors.To investigate the effect of TGF-β on gastric carcinoma,SGC7901,BGC823 and MKN28 (aTGF-β-resistant cell line) adenocarcinoma clones were used.After pretreatment in serum-free medium withor without 10 ng/ml TGF-β1,their experimental metastatic potential,chemotaxis,and invasive and adhesiveability were measured.Furthermore,zymography for gelatinase was processed.Liver colonies were alsomeasured 4 weeks after inoculation of SGC7901,BGC823 and MKN28 in Balb/c nude mice,and an increasein the number of surface liver metastases was seen in SGC7901 (from 11.0±3.0 to 53.3±3.3) and BGC823(from 9.3±2.5 to 60.0±2.8) groups,whereas there was no difference between MKN28 groups (from 35.2±3.8 to 38.5±2.7).In vitro experiments showed that TGF-β1 increased the adhesion capacity of SGC7901and BGC823 cells to immobilized reconstituted basement membrane/fibronectin matrices and promoted theirpenetration through reconstituted basement membrane barriers.Zymography demonstrated that enhancedinvasive potential was partly due to the increased type Ⅳ collagenolytic (gelatinolytic) activity,but there wasno difference in type Ⅳ collagenolytic activity and other biological behaviors between MKN28 groups.Theseresults suggested that TGF-β1 might modulate the metastatic potential of gastric cancer cells by promotingtheir ability to break down and penetrate basement membrane barriers and their adhesive and motile activities.We speculated that TGF-β1 might act as a progression-enhancing factor in gastric cancer.Therefore blockageof TGF-β or TGF-β signaling might prevent gastric cancer cells from invading and metastasizing.     

Enhanced Expression of Long Non-Coding RNA HOTAIR Is Associated with the Development of Gastric Cancer

The long non-coding RNA HOTAIR has been reported to be a poor prognostic biomarker in a variety of malignant tumors. However, little is known about the association of HOTAIR with gastric cancer. We examined the expression of HOTAIR in 68 gastric cancer samples using quantitative real-time RT-PCR and analyzed its correlation with the clinical parameters. The functional role of HOTAIR was examined by generating human gastric cancer cell lines with increased or suppressed HOTAIR expression. The anchorage -independent growth was assessed by soft agar assay. The increased or suppressed HOTAIR expressing gastric cancer cells were injected into the tail vein or peritoneal cavity of immunodeficient mice to examine the effect of this molecule on metastasis and peritoneal dissemination. The expression of HOTAIR was significantly higher in cancer lesions than in adjacent non-cancerous tissues in human gastric cancers. In the diffuse type of gastric cancer, the High-HOTAIR group (HOTAIR/GAPDH > 1) showed significantly more venous invasion, frequent lymph node metastases and a lower overall survival rate compared to the Low-HOTAIR group (HOTAIR/GAPDH < 1). Colony formation on the soft agar was enhanced in a HOTAIR-dependent manner. HOTAIR-expressing MKN74 formed more liver metastasis compared to control when they were injected into the tail vein of mice. In addition, reduced expression of HOTAIR in KATO III suppressed peritoneal dissemination. These results suggest that HOTAIR plays a pivotal role in the development of gastric cancer.     

Silencing of Discoidin Domain Receptor-1 (DDR1) Concurrently Inhibits Multiple Steps of Metastasis Cascade in Gastric Cancer

Accumulating evidence suggests that a unique set of receptor tyrosine kinases, known as discoidin domain receptors (DDRs), plays a role in cancer progression by interacting with the surrounding collagen matrix. In this study, we investigated the expression and role of DDR1 in human gastric cancer metastasis. Proliferation, migration, invasion, and tube formation assays were conducted in DDR1-expressing MKN74 gastric cancer cells and corresponding DDR1-silenced cells. The effects of DDR1 on tumor growth and metastasis were examined in orthotopically implanted and experimental liver metastasis models in nude mice. The expression of DDR1 in surgical specimens was analyzed by immunohistochemistry. DDR1 was expressed in human gastric cancer cell lines, and its expression in human gastric tumors was associated with poor prognosis. Among seven gastric cancer cell lines, MKN74 expressed the highest levels of DDR1. DDR1-silenced MKN74 cells showed unaltered proliferation activity. In contrast, migration, invasion, and tube formation were significantly reduced. When examined in an orthotopic nude mouse model, DDR1-silenced implanted tumors significantly reduced angiogenesis and lymphangiogenesis, thereby leading to reductions in lymph node metastasis and liver metastasis. In a model of experimental liver metastasis, DDR1-silenced cells almost completely inhibited liver colonization and metastasis. DDR1 deficiency led to reduced expression of the genes encoding vascular endothelial growth factor (VEGF)-A, VEGF-C, and platelet-derived growth factor-B. These results suggest that DDR1 is involved in gastric cancer tumor progression and that silencing of DDR1 inhibits multiple steps of the gastric cancer metastasis process.     

Inhibition of PRL-3 gene expression in gastric cancer cell line SGC7901 via microRNA suppressed reduces peritoneal metastasis

High expression of PRL-3, a protein tyrosine phosphatase, is proved to be associated with lymph node metastasis in gastric carcinoma from previous studies. In this paper, we examined the relationship between PRL-3 expression and peritoneal metastasis in gastric carcinoma. We applied the artificial miRNA (pCMV-PRL3miRNA), which is based on the murine miR-155 sequence, to efficiently silence the target gene expression of PRL-3 in SGC7901 gastric cancer cells at both mRNA and protein levels. Then we observed that, in vitro, pCMV-PRL3miRNA significantly depressed the SGC7901 cell invasion and migration independent of cellular proliferation. In vivo, PRL-3 knockdown effectively suppressed the growth of peritoneal metastases and improved the prognosis in nude mice. Therefore, we concluded that artificial miRNA can depress the expression of PRL-3, and that PRL-3 might be a potential therapeutic target for gastric cancer peritoneal metastasis.     

Overexpression of Lin28 Decreases the Chemosensitivity of Gastric Cancer Cells to Oxaliplatin,Paclitaxel, Doxorubicin,and Fluorouracil in Part via microRNA-107

Higher Lin28 expression is associated with worse pathologic tumor responses in locally advanced gastric cancer patients undergoing neoadjuvant chemotherapy. However, the characteristics of Lin28 and its mechanism of action in chemotherapy resistance is still unclear. In this study, we found that transfection of Lin28 into gastric cancer cells (MKN45 and MKN28) increased their resistance to the chemo-drugs oxaliplatin (OXA), paclitaxel (PTX), doxorubicin (ADM), and fluorouracil (5-Fu) compared with gastric cancer cells transfected with a control vector. When knockdown Lin28 expression by Lin28 small interfering RNA (siRNA) was evaluated in vitro, we found that the resistance to chemo-drugs was reduced. Furthermore, we found that Lin28 up-regulates C-myc and P-gp and down-regulates Cylin D1. Finally, we found that miR-107 is a target microRNA of Lin28 and that it participates in the mechanism of chemotherapy resistance. Our results suggest that the Lin28/miR-107 pathway could be one of many signaling pathways regulated by Lin28 and associated with gastric cancer chemo-resistance.     

AEG‐1 induces gastric cancer metastasis by upregulation of eIF4E expression

Gastric cancer is the third leading cause of cancer‐related deaths worldwide, and patients with lymph node, peritoneal and distant metastasis have a poor prognosis. Overexpression of Astrocyte‐elevated gene‐1 (AEG‐1) has been reported to be correlated with the progression and metastasis of gastric cancer. However, its mechanisms are quite unclear. In this study, we found that elevated expression of AEG‐1 was correlated with metastasis in human gastric cancer tissues. Moreover, gain‐ or loss‐of‐function of AEG‐1, respectively, promoted or suppressed epithelial–mesenchymal transition (EMT), migration and invasion of gastric cancer cells. AEG‐1 positively regulated eIF4E, MMP‐9 and Twist expression. Manipulating eIF4E expression by transfection of overexpression constructs or siRNAs partially eliminated AEG‐1‐regulated EMT, cell migration and invasion. In addition, overexpression or knockdown of eIF4E promoted or suppressed EMT, cell migration and invasion in parallel with upregulation of MMP‐9 and Twist expression, while manipulating eIF4E expression partially abrogated AEG‐1‐induced MMP‐9 and Twist. Finally, silencing of AEG‐1 expression not only inhibited tumour growth in parallel with downregulation of eIF4E, MMP‐9 and Twist expression in a xenograft nude mouse model, but also suppressed lymph node and peritoneal metastasis of gastric cancer in an orthotopic nude mouse model. These findings suggest that AEG‐1 promotes gastric cancer metastasis through upregulation of eIF4E‐mediated MMP‐9 and Twist, which provides new diagnostic markers and therapeutic targets for cancer metastasis.     

The calcium pump PMCA4 prevents epithelial-mesenchymal transition by inhibiting NFATc1-ZEB1 pathway in gastric cancer

Epithelial-mesenchymal transition (EMT) is considered as the key mechanism involved in cancer metastasis. Several studies showed that various cell membrane calcium channels play different roles in cancer metastasis. In the present study, the potential role of ATPase plasma membrane Ca²⁺ transporting 4 (PMCA4) in regulating EMT in gastric cancer (GC) was investigated. GC patients who underwent radical surgery were enrolled in this study. In vitro human GC cell lines MKN45 and NCI-N87 were used, and MKN45 cells were injected in nude mice to evaluate tumor development. Our results showed that low PMCA4 expression was associated with advanced TNM stage and poor prognosis in GC patients. Knockdown of PMCA4 suppressed E-cadherin, grainyhead like 2 (GRHL2) and ovo-like 1 (OVOL1) expression, up-regulated vimentin expression, increased migration and invasion ability, and promoted the resistance to cytotoxic drug. Furthermore, GC cells displayed an elongated fibroblastoid morphology when PMCA4 was knockdown. PMCA4 overexpression resulted in an up-regulated E-cadherin expression and decreased migration and invasion ability. In vivo metastasis assay showed that PMCA4 overexpression resulted in a decreased incidence of lung metastasis. PMCA4 inhibition increased ZEB1 expression and nuclear accumulation of nuclear factor of activated T-cell isoform c1 (NFATc1). EMT induced by PMCA4 inhibition could be prevented by the knockdown of NFATc1 or ZEB1. In addition, cyclosporine A prevented EMT induced by PMCA4 inhibition by suppressing the NFATc1-ZEB1 pathway. Our data identified a novel mechanism in the regulation of EMT in GC, and provided a novel target in the treatment of EMT subtype in GC.     

Targeting AURKA in treatment of peritoneal tumor dissemination in gastrointestinal cancer

Intraperitoneal (i.p.) tumor dissemination and the consequent malignant ascites remain unpredictable and incurable in patients with gastrointestinal (GI) cancer, and practical advances in diagnosis and treatment are urgently needed in the clinical settings. Here, we explored tumor biological and immunological mechanisms underlying the i.p. tumor progression for establishing more effective treatments.We established mouse tumor ascites models that murine and human colorectal cancer cells were both i.p. and subcutaneously (s.c.) implanted in mice, and analyzed peritoneal exudate cells (PECs) obtained from the mice. We then evaluated anti-tumor efficacy of agents targeting the identified molecular mechanisms using the ascites models. Furthermore, we validated the clinical relevancy of the findings using peritoneal lavage fluids obtained from gastric cancer patients.I.p. tumor cells were giant with large nuclei, and highly express AURKA, but less phosphorylated TP53, as compared to s.c. tumor cells, suggesting polyploidy-like cells. The i.p. tumors impaired phagocytic activity and the consequent T-cell stimulatory activity of CD11b⁺Gr1⁺PD1⁺ myeloid cells by GDF15 that is regulated by AURKA, leading to treatment resistance. Blocking AURKA with MLN8237 or siRNAs, however, abrogated the adverse events, and induced potent anti-tumor immunity in the ascites models. This treatment synergized with anti-PD1 therapy. The CD11b⁺PD1⁺ TAMs are also markedly expanded in the PECs of gastric cancer patients.These suggest AURKA is a determinant of treatment resistance of the i.p. tumors. Targeting the AURKA-GDF15 axis could be a promising strategy for improving clinical outcome in the treatment of GI cancer.     

Dual mode regulation of migration by lysophosphatidic acid in human gastric cancer cells

Lysophosphatidic acid (LPA), which interacts with at least three G protein-coupled receptors (GPCRs), LPA1/Edg-2, LPA2/Edg-4, and LPA3/Edg-7, is a lipid mediator with diverse effects on various cells. Here, we investigated the expression profiles of LPA receptors and patterns of LPA-induced migration in gastric cancer cells. Northern blot analysis revealed that various gastric cancer cells expressed variable levels of LPA1, LPA2, and LPA3 without a consistent pattern. Using a Boyden chamber assay, LPA markedly increased cell migration of LPA1-expressing cells, the effects of which were almost totally abrogated by Ki16425, an LPA antagonist against LPA1 and LPA3. In contrast, LPA by itself did not significantly induce migration in MKN28 and MKN74 cells, which exclusively expressed LPA2. However, when hepatocyte growth factor (HGF) was placed with LPA in the lower chamber, LPA induced migration of these cells in a dose-dependent manner. Immunoprecipitation analysis revealed that LPA induced transient tyrosine phosphorylation of c-Met in LPA2-expressing cells, which suggests that the transactivation of c-Met by LPA causes a cooperative migratory response with HGF to these cells. Our results indicate that LPA regulates the migration of gastric cancer cells in a receptor-specific manner and suggest that the expression pattern of LPA receptors may affect the metastatic behavior of gastric cancer.     

二氢杨梅素对人胃癌MKN45细胞迁移和侵袭的影响及其机制*

目的:探讨二氢杨梅素(DHM )对人胃癌MKN45细胞迁移和侵袭的作用及其分子机制。方法:培养人低分化胃癌MKN45细胞,用不同浓度的DHM(0,10,20,30,40,50 μmol/L)分别处理细胞24及48 h,每组实验重复3次,采用CCK8实验检测癌细胞增殖活力;划痕实验检测细胞迁移能力;Transwell小室检测细胞侵袭能力;免疫印迹分析细胞迁移和侵袭相关蛋白表达情况。结果:不同浓度DHM干预可降低MKN45细胞活力。20,30及40 μmol/L的DHM处理48 h可明显抑制细胞的迁移能力(P＜0.01)和侵袭能力(P＜0.05及0.01)。20及30 μmol/L的DHM处理48 h可增加E-cadherin蛋白表达(P＜0.01)、降低Vimentin表达(P＜0.01),从而逆转EMT过程;10,20及30 μmol/L的DHM处理48 h可明显降低pJNK的活性表达水平(P＜0.05及0.01),及MMP-2蛋白表达(P＜ 0.01);JNK通路特异性抑制剂SP600125预处理可明显促进DHM对癌细胞侵袭能力的抑制作用(P＜0.01)及降低MMP-2表达(P＜0.01)。结论:DHM具有抑制人胃癌MKN45细胞的迁移及侵袭的作用,其机制可能与通过JNK通路下调MMP-2蛋白表达水平、逆转上皮间质转化有关。     

Deficiency of the complex I of the mitochondrial respiratory chain but improved adenylate control over succinate-dependent respiration are human gastric cancer-specific phenomena

The purpose of study was to comparatively characterize the oxidative phosphorylation (OXPHOS) and function of respiratory chain in mitochondria in human gastric corpus mucosa undergoing transition from normal to cancer states and in human gastric cancer cell lines, MKN28 and MKN45. The tissue samples taken by endobiopsy and the cells were permeabilized by saponin treatment to assess mitochondrial function in situ by high-resolution oxygraphy. Compared to the control group of endobiopsy samples, the maximal capacity of OXPHOS in the cancer group was almost twice lower. The respiratory chain complex I-dependent respiration, normalized to complex II-dependent respiration, was reduced that suggests deficiency of complex I, but the respiratory control by ADP in the presence of succinate was increased. Similar changes were observed also in mucosa adjacent to cancer tissue. The respiratory capacity of MKN45 cells was higher than that of MKN28 cells, but both types of cells exhibited a deficiency of complex I of the respiratory chain which appears to be an intrinsic property of the cancer cells. In conclusion, human gastric cancer is associated with decreased respiratory capacity, deficiency of the respiratory complex I of mitochondria, and improved coupling of succinate oxidation to phosphorylation in tumor tissue and adjacent atrophic mucosa. Detection of these changes in endobiopsy samples may be of diagnostic value.     

Inhibition of NF‐κB is required for oleanolic acid to downregulate PD‐L1 by promoting DNA demethylation in gastric cancer cells

Gastric cancer is one of the most common causes of cancer‐related death worldwide. Immunotherapy via programmed cell death protein 1 (PD‐1)/programmed cell death‐ligand 1 (PD‐L1) blockade has shown benefits for gastric cancer. Epigenetic DNA methylation critically regulates cancer immune checkpoints. We investigated how the natural compound oleanolic acid (OA) affected PD‐L1 expression in gastric cancer cells. Interleukin‐1β (IL‐1β) at 20 ng/mL was used to stimulate human gastric cancer MKN‐45 cells. IL‐1β significantly increased PD‐L1 expression, which was abolished by OA. Next, OA‐treated MKN‐45 cells were co‐cultured with activated and PD‐1‐overexpressing Jurkat T cells. OA restored IL‐2 levels in the co‐culture system and increased T cell killing toward MKN‐45 cells. Overexpression of PD‐L1 eliminated OA‐enhanced T cell killing capacity; however, PD‐1 blocking antibody abrogated the cytotoxicity of T cells. Moreover, OA abolished IL‐1β‐increased DNA demethylase activity in MKN‐45 cells. DNA methyltransferase inhibitor 5‐azacytidine rescued OA‐reduced PD‐L1 expression; whereas DNA demethylation inhibitor gemcitabine inhibited PD‐L1 expression, and, in combination with OA, provided more potent inhibitory effects. Furthermore, OA selectively reduced the expression of DNA demethylase TET3 in IL‐1β‐treated MKN‐45 cells, and overexpression of TET3 restored OA‐reduced PD‐L1 expression. Finally, OA disrupted nuclear factor κB (NF‐κB) signaling IL‐1β‐treated MKN‐45 cells, and overexpression of NF‐κB restored OA downregulation of TET3 and PD‐L1. The cytotoxicity of T cells toward MKN‐45 cells was also weakened by NF‐κB overexpression. Altogether, OA blocked the IL‐1β/NF‐κB/TET3 axis in gastric cancer cells, leading to DNA hypomethylation and downregulation of PD‐L1. Our discoveries suggested OA as an epigenetic modulator for immunotherapy or an adjuvant therapy against gastric cancer.     

Beclin 1 augmented cis-diamminedichloroplatinum induced apoptosis via enhancing caspase-9 activity

Beclin 1, identified as a Bcl-2-interacting protein, is known to enhance autophagy. However, the effect of Beclin 1 on apoptotic signaling has remained unclear. Here, we show that overexpression of Beclin 1 in MKN28 human gastric cancer cells augmented cis-diamminedichloroplatinum (CDDP)-induced apoptosis. Conversely, "knockdown" of Beclin 1 by a small inhibitory RNA in MKN 1 cells attenuated this cytotoxicity. Furthermore, not only caspase-3/7 activities, but also caspase-9 activity was increased in Beclin 1 gene transfectants treated with CDDP, and caspase-9 inhibitor completely abolished augmentation of CDDP-induced apoptosis by Beclin 1 as did a caspase-3 inhibitor. Thus, Beclin 1 augments CDDP-induced apoptosis through enhancing caspase-9 activity and functions as a pro-apoptotic molecule.