Gastric Cancer Tissue Microarray Research Articles

Wnt/β-Catenin Signaling Axis Is Required for TFEB-Mediated Gastric Cancer Metastasis and Epithelial-Mesenchymal Transition.

Gastric cancer remains the third leading cause of cancer-related death, and tumor metastasis is the main risk factor for poor prognosis of patients with gastric cancer. Transcription factor EB (TFEB) is a MiT family member and has been found to drive tumorigenesis in a number of tissues, whereas few studies were focused on investigating its prometastasis role and mechanism in gastric cancer. Here, we found TFEB was upregulated in gastric cancer tissues compared with adjacent normal gastric epithelial tissues. IHC analysis from gastric cancer tissue microarray revealed that TFEB in gastric cancer was correlated with depth of tumor invasion, lymph node or distant metastasis, tumor tumor-node-metastasis stage, and overall survival. Gastric cancer cells with TFEB overexpression presented an increased cell migration or invasion, and epithelial-mesenchymal transition (EMT). Furthermore, gene correlation analysis and gene set enrichment analysis enriched Wnt/β-catenin signaling pathway members in TFEB high-expression group, and the TOP/FOPflash assay verified the effect of TFEB on β-catenin transcription activity. Besides, we found that TFEB could trigger the aggregation of β-catenin in nucleus and activate its transcription, as well as facilitate the expression of Wnt/β-catenin target genes and EMT-related markers, which could be reversed by the Wnt/β-catenin inhibitor XAV-939. Collectively, TFEB enhances gastric cancer metastatic potential by activating Wnt/β-catenin signaling pathway and may become a promising therapeutic target for gastric cancer metastasis. IMPLICATIONS: Overexpressed TFEB predicts a higher rate of metastasis and worse survival in patients with gastric cancer. Mechanistically, TFEB activates Wnt/β-catenin signaling to fuel migratory and invasive activities of gastric cancer cells, as well as EMT.

Bone Marrow-Derived Myofibroblasts Promote Gastric Cancer Metastasis by Activating TGF-β1 and IL-6/STAT3 Signalling Loop.

BackgroundMurine bone marrow-derived myofibroblasts (BMFs) have previously been shown to promote gastric cancer growth. However, whether BMFs promote gastric cancer cell metastasis remains largely unknown.MethodsWound healing assay, Transwell invasion and migration assay and 3D organotypic co-culture systems were conducted to study the effects of BMFs on invasion and migration of gastric cancer cells and the invasion and migration ability of gastric cancer stem cell-like cells (CSC-LCs) induced by BMFs. We employed two animal model to study the role of BMFs on the in vivo metastasis of gastric cancer cells and the metastatic ability of gastric BMF-induced CSC-LCs. A human gastric cancer tissue microarray and TCGA gastric cancer database were analysed to study the relationship between the expression of IL-6 and TGF-β1 and clinicopathological characteristics and survival in gastric cancer.ResultsWe found that BMFs promoted the in vitro migration and invasion of gastric cancer cells. BMFs promoted liver, lung, subcutaneous, and splenic metastases of MKN28 cells in the spleen injection liver metastasis model and co-injection of caudal vein (IOCV) mouse model. BMFs reprogrammed non-gastric cancer stem cell (CSC) to CSC-LCs and enhanced CSC-LC migration and metastasis. BMF-derived IL-6 and gastric cancer cell-secreted TGF-β1 mediated the interaction between BMFs and gastric cancer cells, promoting tumour metastasis. BMFs enhanced the expressions of STAT3 and p-STAT3 in co-cultured gastric cancer cells. A combination of Napabucasin and Galunisertib exhibited the strongest inhibition of cell migration compared to when administered alone. Gastric cancer tissue array and TCGA database indicated that the overexpression of IL-6 and TGF-β1 was associated with gastric cancer metastasis.ConclusionOur results demonstrated that BMFs promote gastric cancer metastasis through the activation of the TGF-β1 and IL-6/STAT3 signalling pathways. Targeting the inhibition of these interactions may be a potent therapeutic strategy for addressing gastric cancer metastasis.

Effects of miR-18a on proliferation and apoptosis of gastric cancer cells by regulating RUNX1.

The aim of this study was to explore the effects of micro ribonucleic acid (miR)-18a on the proliferation and apoptosis of gastric cancer (GC) cells, and to elucidate the possible underlying mechanism. In this study, the expression of miR-18a in GC tissues and para-cancer tissues was verified by in situ hybridization (ISH) of GC tissue microarray (TMA). Meanwhile, the effect of miR-18a expression on the prognosis of GC patients was evaluated. GC AGS cell line was selected and transfected with miR-18a mimic and mimic control (NC) to up-regulate miR-18a expression in vitro. Thereafter, changes in cell proliferation, apoptosis and migration after transfection were detected by biological functional assays. Luciferase reporter gene assay was carried out to verify the target gene Runt-related transcription factor 1 (RUNX1) modulated by miR-18a. Finally, the Spearman's grade correlation coefficient was calculated to explore the correlation between the expressions of miR-18a and RUNX1. ISH results of TMA showed that overexpression of miR-18a in GC tissues was significantly associated with low survival rate of patients (p<0.001). High expression of miR-18a remarkably enhanced the proliferation, migration and invasion of GC cells (p<0.05). Besides, it has been predicted in biology that RUNX1 is one of the target genes of miR-18a. Luciferase reporter gene assay showed that Luciferase activity in cells transfected with wild-type (WT) RUNX1 3' untranslated region (3'UTR) was significantly reduced (p<0.05). Moreover, the protein expression of RUNX1 decreased remarkably in GC cells with over-expression of miR-18a (p<0.05). All these findings indicated that the expression of miR-18a was negatively correlated with RUNX1 in GC cells (p<0.001, r=0.86). MiR-18a exerts a high predictive value for the prognosis of GC patients by directly targeting the transcription factor RUNX1. All our findings may provide therapeutic candidates for GC identification.

Lysine-222 succinylation reduces lysosomal degradation of lactate dehydrogenase a and is increased in gastric cancer

BackgroundLysine succinylation is an emerging posttranslational modification that has garnered increased attention recently, but its role in gastric cancer (GC) remains underexplored.MethodsProteomic quantification of lysine succinylation was performed in human GC tissues and adjacent normal tissues by mass spectrometry. The mRNA and protein levels of lactate dehydrogenase A (LDHA) in GC and adjacent normal tissues were analyzed by qRT-PCR and western blot, respectively. The expression of K222-succinylated LDHA was measured in GC tissue microarray by the K222 succinylation-specific antibody. The interaction between LDHA and sequestosome 1 (SQSTM1) was measured by co-immunoprecipitation (co-IP) and proximity ligation assay (PLA). The binding of carnitine palmitoyltransferase 1A (CPT1A) to LDHA was determined by co-IP. The effect of K222-succinylated LDHA on tumor growth and metastasis was evaluated by in vitro and in vivo experiments.ResultsAltogether, 503 lysine succinylation sites in 303 proteins were identified. Lactate dehydrogenase A (LDHA), the key enzyme in Warburg effect, was found highly succinylated at K222 in GC. Intriguingly, this modification did not affect LDHA ubiquitination, but reduced the binding of ubiquitinated LDHA to SQSTM1, thereby decreasing its lysosomal degradation. We demonstrated that CPT1A functions as a lysine succinyltransferase that interacts with and succinylates LDHA. Moreover, high K222-succinylation of LDHA was associated with poor prognosis in patients with GC. Finally, overexpression of a succinylation-mimic mutant of LDHA promoted cell proliferation, invasion, and migration.ConclusionsOur data revealed a novel lysosomal pathway of LDHA degradation, which is mediated by the binding of K63-ubiquitinated LDHA to SQSTM1. Strikingly, CPT1A succinylates LDHA on K222, which thereby reduces the binding and inhibits the degradation of LDHA, as well as promotes GC invasion and proliferation. This study thus uncovers a new role of lysine succinylation and the mechanism underlying LDHA upregulation in GC.

COX-2 promotes metastasis and predicts prognosis in gastric cancer via regulating mTOR.

Aim: Gastric cancer (GC) is one of the most common malignant tumors in the world. It is important to find accurate and reliable biomarkers in order to decrease whole morbidity and mortality. Results: We examined the expression of COX-2 and mTOR on GC tissue microarrays by immunohistochemistry. Multivariate COX regression analysis showed that the expression of COX-2 or mTOR was an independent factor in the prognosis of GC patients. In addition, COX-2 and mTOR have a potentially synergistic effect on predicting the prognosis of GC. Conclusion: The combined expression of COX-2 and mTOR could serve as efficient prognostic indicators and COX-2 could suppress GC metastasis via regulating mTOR.

The LINC00365/SCGB2A1 (Mammaglobin B) Axis Down-Regulates NF-κB Signaling and Is Associated with the Progression of Gastric Cancer.

PurposeA lack of early diagnostic biomarkers and therapeutic targets has led to poor prognosis for gastric cancer patients. However, the analysis of cancer-associated genomic data has been shown to be effective in identifying potential markers. Recently, the long non-coding RNA LINC00365 and SCGB2A1 gene (as known as mammaglobin B) were predicted to be co-expressed in gastric cancer based on the Gene Expression Omnibus database. However, their precise role in gastric cancer tumors is still not clear.MethodsThe expressions of LINC00365 and SCGB2A1 in gastric cancer tissues were investigated using qPCR and their expressions were detected in a gastric cancer tissue microarray by in situ hybridization and immunohistochemical staining. The functions of LINC00365 in BGC-823 and MGC-803 gastric cancer cells were tested using the MTT assay, flow cytometry, colony formation assay, EDU staining, immunofluorescence and luciferase assay.ResultsWe found that LINC00365 and SCGB2A1 mRNA were both expressed at low levels in 30 cases of gastric cancer. Gastric cancer tissue microarray analysis indicated that LINC00365 and SCGB2A1 were expressed at low levels in tumor tissue, and low expression of both factors correlated with shorter survival time. Functional studies showed that LINC00365 overexpression significantly inhibited gastric cancer cell viability through the impairment of proliferation rather than the promotion of apoptosis. Furthermore, overexpressed LINC00365 upregulated SCGB2A1 in gastric cancer cell lines. Immuno-fluorescence and luciferase assay analysis indicated that LINC00365 overexpression inhibited the NF-κB pro-survival signaling pathway. Consistent with the effects of LINC00365, SCGB2A1 upregulation also reduced cell survival and inactivated NF-κB.ConclusionCollectively, our findings revealed that SCGB2A1 may be the target coding protein regulated by LINC00365 in gastric cancer. LINC00365 and SCGB2A1 may function as tumor suppressors and may serve as potential prognostic and therapeutic markers in gastric cancer treatment.

Expression Status And Prognostic Value Of M6A-associated Genes in Gastric Cancer.

Purpose: Gastric cancer (GC) is a primary cause of cancer-associated mortality worldwide. N6-methyladenosine (m6A) is one of the most common RNA modifications that involves in the progression of numerous cancers. However, the expression status and function of m6A-related genes in gastric cancer is still not well understood. The current study is aimed to investigate the expression status and determinate prognostic value of m6A-related genes in gastric cancer.Methods: m6A-asssociated gene expression was evaluated via analyzing the expression data of GC patients from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database. The protein expression levels of m6A-associated molecules were further validated by immunohistochemical (IHC) staining data from GC tissue microarray (TMA) cohort and Human Protein Atlas (HPA) database. Kaplan-Meier analysis was performed to assess the prognostic value of m6A-associated genes in gastric cancer. Risk score model was established by lasso COX regression analysis and its prognostic predicted efficiency was assessed by the receiver-operator characteristic (ROC) curve. Cox regression analyses were used for exploring risk factors related to GC patient prognosis.Results: Most of m6A-related genes were upregulated at both mRNA and protein levels in gastric cancer tissues compared with that in normal gastric tissues. The expression levels of m6A-related genes were associated with clinicopathological features including race, age and TNM stage. High expression of WTAP and FTO predicted poor prognosis of GC patients. Survival analysis demonstrated that patients with high-risk scores had worse overall survival (OS) and ROC curves suggested the prediction performance for gastric patients. Moreover, Cox regression analyses indicated that m6A risk model score was a prognostic factor for OS and FTO upregulation might be a potential independent prognostic factor for recurrence-free survival (RFS) in gastric cancer patients.Conclusion: m6A-related genes were dysregulated in GC and were closely associated with prognosis of GC patients. FTO might serve as a novel prognostic biomarker for gastric cancer, while the m6A-related risk score might be informative for risk assessment and prognostic stratification.

S100P is a molecular determinant of E-cadherin function in gastric cancer

BackgroundE-cadherin has been awarded a key role in the aetiology of both sporadic and hereditary forms of gastric cancer. In this study, we aimed to identify molecular interactors that influence the expression and function of E-cadherin associated to cancer.MethodsA data mining approach was used to predict stomach-specific candidate genes, uncovering S100P as a key candidate. The role of S100P was evaluated through in vitro functional assays and its expression was studied in a gastric cancer tissue microarray (TMA).ResultsS100P was found to contribute to a cancer pathway dependent on the context of E-cadherin function. In particular, we demonstrated that S100P acts as an E-cadherin positive regulator in a wild-type E-cadherin context, and its inhibition results in decreased E-cadherin expression and function. In contrast, S100P is likely to be a pro-survival factor in gastric cancer cells with loss of functional E-cadherin, contributing to an oncogenic molecular program. Moreover, expression analysis in a gastric cancer TMA revealed that S100P expression impacts negatively among patients bearing Ecad− tumours, despite not being significantly associated with overall survival on its own.ConclusionsWe propose that S100P has a dual role in gastric cancer, acting as an oncogenic factor in the context of E-cadherin loss and as a tumour suppressor in a functional E-cadherin setting. The discovery of antagonist effects of S100P in different E-cadherin contexts will aid in the stratification of gastric cancer patients who may benefit from S100P-targeted therapies.Graphical abstract

Malic enzyme 1 (ME1) is a potential oncogene in gastric cancer cells and is associated with poor survival of gastric cancer patients.

Background and objectiveGastric cancer is one of the most common cancers worldwide. However, the mechanisms associated with this disease are still not clear. Malic enzyme 1 (ME1) is a metabolic enzyme that is overexpressed in various cancers. Here, we examined whether it is involved in gastric cancer.MethodsME1 expression was knocked down in the gastric cancer cell line SGC7901. Cell growth and migration were measured using a real-time microelectronic cell sensor system. Cell invasion was measured using a Transwell assay. Cell cycle analysis was also performed to examine cell cycle arrest. A gastric cancer tissue microarray of gastric cancer was stained using immunohistochemistry. ME1 expression levels were also statistically analysed.ResultsME1 knockdown in gastric cancer SGC7901 cells significantly inhibited cell proliferation, migration, and invasion. Cell cycle arrest was induced in the G2 phase. Further, ME1 expression was significantly correlated with gastric cancer patient prognosis based on both univariable and multivariable survival analysis. No significant difference was found between ME1 expression in gastric cancer tissues and that in adjacent tissues.ConclusionOur results provide evidence that ME1 is a key factor for gastric cancer. ME1 might be pro-oncogenic during both the development and migration of gastric cancer; it also might be related to gastric cancer patient survival.

Activation of IGF1R by DARPP-32 promotes STAT3 signaling in gastric cancer cells.

Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32), is frequently overexpressed in earlt stages of gastric cancers. We utilized in vitro assays, 3D gastric gland organoid cultures, mouse models, and human tissue samples to investigate the biological and molecular impact of DARPP-32 on activation of IGF1R and STAT3 signaling and gastric tumorigenesis. DARPP-32 enhanced phosphorylation of IGF1R (Y1135), a step that was critical for STAT3 phosphorylation at Y705, nuclear localization, and transcription activation. By using proximity ligation and co-immunoprecipitation assays, we found that IGF1R and DARPP-32 co-existed in the same protein complex. Binding of DARPP-32 to IGF1R promoted IGF1R phosphorylation with subsequent activation of downstream SRC and STAT3. Analysis of gastric tissues from the TFF1 knockout (KO) mouse model of gastric neoplasia, demonstrated phosphorylation of STAT3 in the early stages of gastric tumorigenesis. By crossing the TFF1 KO mice with DARPP-32 (DP) knockout (KO) mice, that have normal stomach, we obtained double knockout (TFF1 KO/DP KO). The gastric mucosa from the double KO mice did not show phosphorylation of IGF1R or STAT3. In addition, the TFF1 KO/DP KO mice had significant delay in developing neoplastic gastric lesions. Analysis of human gastric cancer tissue microarrays, showed high levels of DARPP-32 and positive immunostaining for nuclear STAT3 in cancer tissues, as compared to non-cancer histologically normal tissues. In summary, the presence of a signaling axis mediated by DARPP-32–IGF1R is a critical step in gastric tumorigenesis, playing an important role in activation of STAT3.

Knockdown of STIL suppresses the progression of gastric cancer by down-regulating the IGF-1/PI3K/AKT pathway.

SCL/TAL1 interrupting locus (STIL) regulates the mitotic centrosome to promote the centriolar replication and cell cycling, and is associated with malignancies. However, the role and mechanism of STIL in gastric cancer (GC) remain elusive. STIL expression in GC tissue microarray was detected by immunohistochemistry (IHC). GC cells were transduced with control lentivirus or lentivirus for expression STIL‐specific shRNA and the effect of STIL silencing on the malignant behaviors of GC cells was measured in vitro and in vivo. The potential mechanisms underlying the action of STIL were analyzed by transcriptome microarray and bioinformatics. STIL expression was up‐regulated in GC tissues both in our cohort and the data from the cancer genome atlas, and positively associated with T stage and poor overall survival of GC patients. Knockdown of STIL significantly inhibited the proliferation and clonogenicity of human GC cells and attenuated the growth of implanted GC in vivo. Furthermore, STIL silencing induced cell cycle arrest in G2/M phase and apoptosis of GC cells. Transcriptome analysis indicated that STIL silencing modulated many gene expression, particularly for down‐regulating the IGF‐1/PI3K/AKT pathway. In addition, treatment with SC79, an AKT activator, significantly mitigated the effect of STIL‐silencing in GC cells. In conclusion, STIL promotes gastric carcinogenesis and progression by enhancing the IGF‐1/PI3K/AKT signaling, and STIL may be a novel target for intervention of GC.

The ubiquitin ligase CHIP modulates cellular behaviors of gastric cancer cells by regulating TRAF2

BackgroundCHIP is an E3 ubiquitin ligase that plays contrast roles in diverse human malignancies, depending on its targets. To date, the mechanisms underlying the function of CHIP in gastric cancer remains unclear. Here, we aim to further clarify the effects of CHIP on the development and progression of gastric cancer and explore its potential target.MethodsStably transfected CHIP-shRNA and TRAF2-shRNA AGS gastric cancer cell lines were established using Lipofectamine 2000. Cell growth was measured by an xCelligence real-time monitoring system and colony formation assay. Cell proliferation was detected using CCK-8, Ki-67, or CFSE assays. Apoptosis was detected by TUNEL assay or Annexin V/PI-staining followed by flow cytometric analysis. Cell cycle distribution was detected by PI-staining followed by flow cytometric analysis. Cell migration and invasion abilities were measured by a real-time xCelligence system, Transwell insert, and scratch assays. The expression of cell cycle-related proteins, apoptosis-related proteins, AKT, ERK, NF-κB signaling subunits, MMP2, MMP9, and Integrin β-1 were detected by Western blotting analysis. NF-κB DNA-binding capability was quantified using an ELISA-based NF-κB activity assay. Gastric cancer tissue microarray was analyzed to investigate the expression of both CHIP and TRAF2, and their clinical significance.ResultsThe CHIP-silencing in the AGS cells was oncogenic evidenced by the appearance of capable of anchorage-independent growth. The CHIP-silencing significantly enhanced the AGS cell proliferation capability likely due to the induced phosphorylation of ERK. The CHIP-silencing significantly inhibited apoptosis due to increased expression of Bcl-2. The CHIP-silencing promoted the AGS cell migration and invasion abilities, likely by regulating the expression of Integrin β-1. TRAF2 expression was markedly decreased in the CHIP-overexpressing cells at protein level, but not at mRNA level. The TRAF2-silencing markedly inhibited the proliferation ability of the AGS cells, the defected cell proliferation and enhanced apoptosis were involved in. The TRAF2-silencing also attenuated the cell migration and invasion capacities of the AGS cells. Furthermore, the expression of CHIP was downregulated while the expression of TRAF2 was upregulated in gastric cancer tissues. TRAF2 expression is independent prognostic factors of gastric cancer. The expression of CHIP and TRAF2 was negatively correlated in the gastric cancer tissue. Lower CHIP or higher TRAF2 was significantly linked to shorter overall survival in gastric cancer patients.ConclusionsTRAF2 influenced diverse aspects of cellular behavior of gastric cancer cells, including cell growth, migration, and invasion, which was in contrast to the functions of CHIP. TRAF2 could be considered as an independent prognostic factor in gastric cancer patients. It is possible that TRAF2 was a substrate of CHIP and CHIP regulated the TRAF2/NF-κB axis, which modulated diverse cellular behaviors in the AGS gastric cancer cells.

Expression of DNA Damage Response Markers in Early-Onset or Familial Gastric Cancers

Background:Early-onset or familial gastric cancer (GC) is known to have clinicopathologic profiles different from those of sporadic GC. We aimed to compare DNA damage response marker expression between early-onset or familial GC and sporadic GC.Methods:GC samples were obtained from patients who underwent gastrectomy for GC at Seoul National University Hospital. Immunohistochemical analyses of various DNA damage response markers, including BRCA1, BRCA2, MRE11, RAD51C, and γH2AX, were performed using 54 early-onset GC, 59 familial GC, and 337 sporadic GC tissue microarray samples. Correlations between marker expression and clinicopathologic features were evaluated by univariate and multivariate analyses, and overall survival was analyzed.Results:The rate of γH2AX positivity was significantly higher (p < 0.001) in early-onset or familial GC than in sporadic GC. In contrast, the rates of MRE11 negativity and RAD51C negativity were significantly higher in sporadic GC than in early-onset or familial GC. BRCA1 negativity was associated with decreased overall survival in sporadic GC (p = 0.002), and MRE11 negativity was associated with decreased overall survival in sporadic GC (p = 0.012).Conclusion:Our results show significant differences in DNA damage response marker expression between early-onset or familial GC and sporadic GC.

Low expression of PDHA1 predicts poor prognosis in gastric cancer

PDH E1 component subunit alpha (PDHA1) has been reported to be biologically significant in several human tumors. The aim of this study was to investigate the expression of PDHA1 in gastric cancer (GC) and its relationship with clinicopathological characteristics and prognosis. Oncomine analysis of neoplastic vs. normal tissue showed that the mRNA levels of PDHA1 were significantly underexpressed in different types of GC across three analyses. Underexpression of PDHA1 was found in intestinal-type GC (P = 0.009), diffuse-type GC (P = 0.036), and mixed-type GC (P = 0.025). Immunohistochemical staining of the 174 GC tissue microarray showed that PDHA1 staining is much stronger in normal mucosa than in GC samples (P = 0.040). Furthermore, PDHA1 expression levels were found to be significantly lower in 69.05% (87/126) of poorly differentiated GCs as compared to the well or moderately differentiated ones (P = 0.037). Intriguingly, PDHA1 expression was significantly correlated with depth of invasion (P < 0.001), lymph node metastasis (P < 0.001), TNM stage (P < 0.001), and nerve invasion (P = 0.006). However, it was not correlated with gender, age, Lauren classification, and lymphovascular invasion (P > 0.05 for all). Kaplan-Meier analysis revealed that low tumor expression of PDHA1 was significantly correlated with a poorer overall survival in patients with GC (5-year overall survival rates for patients with low vs high PDHA1 expression = 49.8% vs 72.7%, hazard ratio of death from GC = 2.594, 95% CI = 1.527 to 4.408, P < 0.001). Multivariate analysis showed that PDHA1 (P = 0.025) was an independent predictor of overall survival. These findings are of potential clinical utility and merit further validation.

Overexpression of Ubiquitous Mitochondrial Creatine Kinase Denotes a Worse Prognosis for Gastric Cancer Patients

Background: Ubiquitous mitochondrial creatine kinase (uMtCK) transfers high-energy phosphates from mitochondrially generated ATP to creatine to generate phosphocreatine. uMtCK overexpression has been reported in several malignant tumours, however, the clinical significance of uMtCK in gastric cancer (GC) has not been comprehensively studied. Methods: We first examined uMtCK expression in GC by qPCR and western blot. Then the clinicopathological significance of aberrant uMtCK expression was determined by immunohistochemical staining in a GC tissue microarray. Kaplan-Meier analysis was used for survival analysis. By virus transfection, we analyzed the biological function of uMtCK in GC cell in vivo and in vitro. Findings: uMtCK expression was substantially elevated in GC, significantly associated with a poor prognosis in GC patients. In univariate and multivariate analyses, uMtCK emerged as an independent prognostic factor for both disease-free survival and overall survival. In vitro, uMtCK overexpression increased GC cell proliferation, induced cell cycle transition, promoted cell migration and invasion. In vivo, uMtCK overexpression stimulated the formation of subcutaneous xenografts and tumour metastasis in nude mice. Moreover, we also revealed that high uMtCK expression promoted tumour chemoresistance to 5-fluorouracil chemotherapy. In the end, we verified that uMtCK activated the transforming growth factor-β signalling pathway, which was followed by increased levels of pSmad2 and pSmad3. Interpretation: uMtCK enhanced GC cell malignance via activating the transforming growth factor-β pathway, providing a novel independent prognostic biomarker for GC. Funding Statement: National Natural Science Foundation of China (81802420,81803073), Young Scientists of The Second Hospital of Shandong University (2018YT30). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: This research was approved by the Ethics Committee of Shanghai General Hospital. Written informed consent was obtained from all subjects, and they were subjected to close clinical follow-up observation.

MiR-17-92 functions as an oncogene and modulates NF-κB signaling by targeting TRAF3 in MGC-803 human gastric cancer cells.

The miR-17-92 cluster plays either an oncogenic or anti-oncogenic role in cancer progression in diverse human cancers. However, the underlying mechanisms of the miR-17-92 cluster in gastric cancer have not yet been fully elucidated. In this study, the function of the miR-17-92 cluster in diverse aspects of MGC-803 gastric cancer cells was systematically elucidated. The enforced introduction of the miR-17-92 cluster into the MGC-803 cells significantly promoted cell growth due to the increased cellular proliferation and decreased cellular apoptosis, which were detected by CCK-8, cell viability and TUNEL assays. Moreover, the results of western blot analyses revealed that the activated protein kinaseB(AKT), extracellular-signal-regulated kinase(ERK) and nuclear factor(NF-κB) signaling pathways were activated in these processes. Moreover, the overexpression of the miR-17-92 cluster markedly enhanced the migratory and invasive abilities of the MGC-803 cells, which was associated with the occurrence of epithelial-mesenchymal transition(EMT). Tumor necrosis factor receptor associated factor3(TRAF3), which negatively regulates the NF-κB signaling pathway, was identified as a direct target of miR-17-92. Furthermore, TRAF3 silencing enhanced the oncogenic functions of the miR-17-92 cluster in the MGC-803 cells, including the increased cellular proliferation, migration and invasion. Moreover, immunohistochemical staining and survival analyses of a gastric cancer tissue microarray revealed that TRAF3 functioned as a tumor suppressor in gastric cancer. Taken together, the findings of this study provide new insight into the specific biological functions of the miR-17-92 cluster in gastric cancer progression by directly targeting TRAF3.

PTEN lipid phosphatase inactivation links the hippo and PI3K/Akt pathways to induce gastric tumorigenesis

BackgroundPhosphatase and tensin homolog (PTEN) is an important tumor suppressor gene, and its encoded protein has activities of both a protein phosphatase and a lipid phosphatase. However, the substitution effect of protein phosphatase activity remains unclear. PI3K/Akt is the most common pathway negatively regulated by PTEN. The Hippo and PI3K/Akt pathways have a joint effect in regulating cell proliferation and apoptosis. Therefore, how PTEN lipid phosphatase inactivation contributes to the occurrence and development of gastric cancer and the potential role of the Hippo and PI3K/Akt pathways in PTEN lipid phosphatase inactivation mediated gastric tumorigenesis remain to be explored.MethodsImmunohistochemical staining was performed to detect the expression of p-PTEN and YAP in a gastric cancer tissue microarray. Stable cell lines expressing a wild-type or dominant-negative mutant PTEN were established. The proliferation and migration of stable cells were detected by MTT, BrdU, and colony-formation, transwell assay and high content analysis in vitro, and tumor growth differences were observed in xenograft nude mice. Changes in the expression of key molecules in the Hippo and Akt signaling pathways were detected by western blot. Nuclear-cytoplasm separation, immunofluorescence and coimmunoprecipitation analyses were conducted to explore the dysregulation of Hippo in the stable cell lines.ResultsPTEN lipid phosphatase inactivation strongly promoted the proliferation and migration of gastric cancer cells in vitro and tumor growth in vivo. A immunohistochemical analysis of gastric cancer tissues revealed a significant correlation between phosphorylated PTEN and nuclear YAP expression, and both were determined to be independent prognostic factors for gastric cancer. Mechanistically, PTEN lipid phosphatase inactivation abolished the MOB1-LATS1/2 interaction, decreased YAP phosphorylation and finally promoted YAP nuclear translocation, which enhanced the synergistic effect of YAP-TEAD, thus inducing cell proliferation and migration. Moreover, PTEN lipid phosphatase inactivation promoted the PI3K/Akt pathway, and disruption of YAP-TEAD-driven transcription decreased the activation of Akt in a dose-dependent manner.ConclusionsTaken together, our findings indicate that PTEN lipid phosphatase inactivation links the Hippo and PI3K/Akt pathways to promote gastric tumorigenesis and cancer development.

SNRPA enhances tumour cell growth in gastric cancer through modulating NGF expression.

SNRPA is a protein component of U1 small nuclear ribonucleoprotein (U1 snRNP) complex, which takes part in the splicing of pre-mRNAs. Its expression and function in tumour remain unknown. Herein, we elucidated the functional contribution of SNRPA to the progression of gastric cancer (GC). SNRPA expression was investigated in a GC tissue microarray by immunohistochemical staining. Cell proliferation was evaluated by CCK-8, colony formation and EdU incorporation assays. A mouse xenograft model was used to detect the tumourigenicity. Gene expression profiling was performed and then the potential target genes were verified by quantitative real-time PCR and western blot analyses. The functional relevance between SNRPA and its target gene was examined by cell growth assays. SNRPA expression was higher in tumour tissues than in matched normal gastric mucosa tissues, and it was positively correlated with the tumour size and progression. High SNRPA expression indicated poor prognosis of GC patients. Silencing SNRPA in GC cells markedly inhibited cell proliferation in vitro and tumour growth in a xenograft model, while overexpressing SNRPA exhibited opposite results. Moreover, we identified NGF (Nerve growth factor) as a downstream effector of SNRPA and further proved that NGF was crucial for SNRPA-mediated GC cell growth. These findings suggested that SNRPA may contribute to GC progression via NGF and could be a prognostic biomarker for GC.

LMO3 promotes gastric cancer cell invasion and proliferation through Akt-mTOR and Akt-GSK3β signaling

The present study assessed the biological functions of LIM domain only 3 (LMO3) in gastric cancer (GC) investigated and the underlying molecular mechanisms. It was revealed that the expression of LMO3 was significantly upregulated in GC tissues. A GC tissue microarray (n=164) indicated that LMO3 expression was closely associated with clinicopathological factors, as well as overall survival and disease-free survival of patients. After knockdown of LMO3 in MGC-803 and SGC-7901 cells, the invasion and proliferation were obviously suppressed. Furthermore, LMO3 knockdown suppressed the phosphorylation of Akt, mammalian target of rapamycin (mTOR) and glycogen synthase kinase (GSK)3β signaling. An inhibitor of mTOR, dactolisib, abrogated recombinant LMO3 protein-induced GC cell invasion and proliferation, while an inhibitor of GSK3β, CHIR-98014, only abrogated rLMO3 protein-induced proliferation. These results suggested that LMO3 promotes GC cell invasion and proliferation mainly through Akt/mTOR and Akt/GSK3β signaling. LMO3 may serve as a potential therapeutic target for GC in the future.

FGFR2 Promotes Gastric Cancer Progression by Inhibiting the Expression of Thrombospondin4 via PI3K-Akt-Mtor Pathway

Background/Aims: Fibroblast growth factor receptor 2 (FGFR2) has attracted considerable interest as a therapeutic target in gastric cancer (GC). There is growing evidence to suggest that the bioavailability of the potent pro-tumor function of FGFR2 is associated with thrombospondins (TSPs). As a follow-on from our previous study, here we evaluated the potential clinical significance and mechanism of the relationship between FGFR2 and TSP4 in GC. Methods: Expression levels of FGFR2 and TSP4 were detected by immunohistochemistry in GC tissue microarray slides. SGC7901 and MKN28 cell lines were used to confirm the relationship between FGFR2 and TSP4. In vitro cell viability, colony formation, and invasion and migration assays were performed to evaluate the effect of FGFR2-TSP4 axis on tumor cell activities. The mechanism of TSP4 regulated by FGFG2 was explored via small molecular inhibitors in vitro and a xenograft model. Results: FGFR2 was shown to be markedly overexpressed in GC tissues and was correlated with a high risk of lymph node metastasis, late clinical stage, and poor prognosis. Low TSP4 expression was associated with shorter overall survival (OS) and advanced stage in GC patients. Interestingly, correlation analysis indicated that FGFR2 was negatively associated with TSP4. Indeed, in vitro and in vivo experiments suggested FGFR2 activation could downregulate TSP4 expression, which played an important role in the proliferation, invasion and migration of GC cells. We also found involvement of the PI3K-AKT-mTOR pathway in the FGFR2-TSP4 axis. Conclusion: The FGFR2 signal promotes human GC progression through the downregulation of TSP4 via PI3K-AKT-mTOR pathway. Our findings provide a foundation for further investigating promising therapeutic strategies for GC overexpressing FGFR2.