Primary Gastric Cancer Samples Research Articles

Exploration of different immunogenomic TMEs in gastric cancer based on HER2 and PD-L1 expression.

e16073 Background: Recent results from the KEYNOTE-811 study showed that first-line pembrolizumab was superior to placebo with respect to progression-free survival (PFS) in combination with trastuzumab and chemotherapy in patients with HER2 positive metastatic gastric cancer (GC). Interestingly, In the subgroup analysis, the PFS benefit with pembrolizumab was significant in patients with tumors with PD-L1 CPS ≥1, whereas there was no benefit in the small subgroup of patients with tumors with CPS < 1. Therefore, this study is to investigate immune-genomic differences in the tumor microenvironment according to the composition of surrounding cells and the expression status of HER2 and PD-L1. Methods: Utilizing single cell RNA sequencing (scRNA seq) and TCR sequencing, we examined 14 HER2 positive and 18 negative primary GC samples with CPS score based on PD-L1 IHC 22C3 pharmDx. Among the HER2-positive group, there were 4 samples in the CPS < 1 (negative) and 10 samples in the CPS ≥1 (positive) group. In the HER2 negative group, there were 6 samples in the CPS < 1 and 12 samples in the CPS ≥1. Results: First, when analyzing the distribution of total cells according to HER2 status, in the HER2 positive group, more endothelial cells ( p= 0.034) and fibroblasts ( p= 0.054) appeared in the PD-L1 negative group compared to the PD-L1 positive group, while there was no difference in the HER2 negative group. Furthermore, Through TCR analysis, the exhausted and cytotoxic score exhibit contrasting trends between HER2 negative and positive groups. In HER2-positive patients, the exhausted and cytotoxic score appear significantly higher in the CPS negative group. Next, we evaluated the proportion of immune cells according to HER2 status. The observed of CD4 Treg ( p= 0.084) and CD8 exhausted T (Tex), ( p= 0.036) cells was increased in the HER2 positive compared to the negative. When regrouped according to PD-L1 expression, only the HER2 positive group showed a tendency to increase the observation of CD4 Treg and CD8 Tex cells compared to positive in PD-L1 negative. In addition, we investigated whether the expression pattern of other immune checkpoint molecules differed depending on the PD-L1 expression pattern in both HER2 positive and negative patients. In the HER2 negative group, the expression of immune checkpoint proteins was similar regardless of the CPS expression group, or the expression of some proteins was higher in the CPS positive group. In other hands, in HER2 positive, CPS < 1 group had higher expression of immune checkpoint proteins, such as PDCD1, LAG2, BTLA, VISTA and TIGHT. Conclusions: The high proportion of CD4 Tregs, CD8 Tex and the elevated exhausted score in the subgroup with CPS < 1 in HER2 positive GCs suggesting immune exclusive environment compared to her-2 and PD-L1 coexpressing group. Considering increased expression of immune checkpoint proteins other than PD-1/PD-L1, for patients with HER2-positive and CPS < 1, combination therapy with other immune checkpoint inhibitors rather than anti-PD-1/PD-L1 may be suggested.

Single cell and bulk RNA sequencing identifies tumor microenvironment subtypes and chemoresistance-related IGF1+ cancer-associated fibroblast in gastric cancer

BackgroundThe tumor microenvironment (TME) significantly influences prognosis and drug resistance in various tumors, yet its heterogeneity and the mechanisms affecting therapeutic response remain unclear in gastric cancer (GC). MethodsThe heterogenous TME were explored with single-cell RNA-sequencing (scRNA-seq) data of 50 primary GC samples. We then identified four GC TME subtypes with nonnegative matrix factorization (NMF) and constructed a pearson nearest-centroid classifier based on subtype-specific upregulated genes. Genomic features and clinical significance of four subtypes were comprehensively evaluated. We reclustered fibroblasts to identify cancer-associated fibroblast (CAF) subtype associated with poor clinical outcomes. RT-qPCR and double immunofluorescence staining were applied to validate the findings. Cellchat analysis elucidated potential molecular mechanisms of the CAF subtype in GC disease progression and chemotherapy resistance. FindingsThe GC TME exhibited high heterogeneity, influencing chemo-sensitivity. Four TME-based subtypes predicting response to immunotherapy and chemotherapy were identified and validated in 1406 GC patients. Among which, ISG1 subtype displayed higher fibroblasts infiltration and heightened oncogenic pathways, and inferior response to chemotherapy with unfavorable prognosis. Microsatellite instability-high (MSI-H) GCs within four TME subtypes showed immunological heterogeneity. We then reported an IGF1-overexpressing CAF was associated with chemo-resistance and GC recurrence. Cell communication analysis revealed IGF1+ CAF may induce drug-resistant phenotypes in tumor cells through IGF1-α6β4 integrin ligand-receptor binding and activation of EMT biological process. InterpretationWe identified four TME-based subtypes with different clinical outcomes and IGF1+ CAFs contributing to poor clinical outcomes in GC, which might provide guidance for individualized treatment and facilitate the development of novel therapeutic targets.

Gastric cancer metastasis-related NT5DC2 indicates unfavorable prognosis of patients.

Approximately 80 to 90% of patients with gastric cancer (GC) eventually develop into metastatic GC nowadays,because GC is difficult to be diagnosed at an early stage. GC patients with metastases typically have a poor prognosis. It is necessary to explore a potential prognostic marker in metastatic GC. All GC data were obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases. The metastasis-related candidate gene and its role in GC were analyzed by comprehensive analysis. Totally 1049 metastasis-related genes were identified in GC. Univariate Cox regression analysis screened the top 10 genes (PDHX, SLC43A1, CSAG2, NT5DC2, CSAG1, FMN1, MED1, HIVEP2, FNDC3A, and PPP1R2) that were closely correlated with prognosis of GC patients. Among which, NT5DC2 was screened as the target gene for subsequent study. The NT5DC2 expression were increased in primary GC and metastatic GC samples. Moreover, GC patients with high NT5DC2 expression exhibited shorter overall survival and post progression survival, and the NT5DC2 was metastatic GC patients' independent prognostic factor. Totally 29 pathways were activated in metastatic GC samples with high NT5DC2 expression. Four immune cells' infiltration were significantly different between NT5DC2 high and low expressed metastatic GC patients. NT5DC2 showed significantly negative correlations with 6 types of immune cells' critical marker genes and 5 types of immune cell infiltration. The 10 immune checkpoint expressions were decreased in high NTDC2 expression metastatic GC patients. NT5DC2 plays a prognostic role in metastatic GC. GC patients with high NT5DC2 expression indicates unfavorable prognosis.

Proteomic profiling of gastric cancer with peritoneal metastasis identifies a protein signature associated with immune microenvironment and patient outcome

BackgroundPeritoneal metastasis (PM) frequently occurs in patients with gastric cancer (GC) and is a major cause of mortality. Risk stratification for PM can optimize decision making in GC treatment.MethodsA total of 25 GC patients (13 with synchronous, 6 with metachronous PM and 6 PM-free) were included in this study. Quantitative proteomics by high-depth tandem mass tags labeling and whole-exome sequencing were conducted in primary GC and PM samples. Proteomic signature and prognostic model were established by machine learning algorithms in PM and PM-free GC, then validated in two external cohorts. Tumor-infiltrating immune cells in GC were analyzed by CIBERSORT.ResultsHeterogeneity between paired primary and PM samples was observed at both genomic and proteomic levels. Compared to primary GC, proteome of PM samples was enriched in RNA binding and extracellular exosomes. 641 differently expressed proteins (DEPs) between primary GC of PM group and PM-free group were screened, which were enriched in extracellular exosome and cell adhesion pathways. Subsequently, a ten-protein signature was derived based on DEPs by machine learning. This signature was significantly associated with patient prognosis in internal cohort and two external proteomic datasets of diffuse and mixed type GC. Tumor-infiltrating immune cell analysis showed that the signature was associated with immune microenvironment of GC.ConclusionsWe characterized proteomic features that were informative for PM progression of GC. A protein signature associated with immune microenvironment and patient outcome was derived, and it could guide risk stratification and individualized treatment.

HRCT1, negatively regulated by miR-124-3p, promotes tumor metastasis and the growth of gastric cancer by activating the ERBB2-MAPK pathway.

Gastric cancer is the fourth leading cause of cancer-related deaths worldwide. And patient outcomes are poor due to tumor relapse and metastasis. To develop new therapeutic strategies, it is of great importance to explore the mechanism underlying the progression of gastric cancer. Primary gastric cancer samples with lymph node metastases (LNM) and without LNM were subjected to mRNA microarray assay. The differentially expressed genes were confirmed by RT-qPCR. HRCT1 protein expression was further detected using an immunohistochemistry (IHC) assay. In vitro and in vivo assays were performed to investigate the role of HRCT1 in tumor invasion, metastasis, and proliferation. The expressions of the downstream target genes of HRCT1 were detected by microarray, RT-qPCR and Western blot assays. Dual-luciferase reporter and Western blot assays were carried out to identify miRNAs target to HRCT1. HRCT1 was upregulated in gastric cancer, and high expression of HRCT1 was associated with poor overall survival (OS) and disease-free survival (DFS). Moreover, HRCT1protein expression was an independent predictor for poor OS and DFS. HRCT1 could promote gastric cancer cells' migration, invasion, and proliferation in vitro as well as tumor metastasis and growth in vivo. Notably, our data showed that HRCT1 promoted gastric cancer progression by activating the ERBB2-MAPK signaling pathway. At least partially, the expression of HRCT1 could be negatively regulated by miR-124-3p. The upregulated expression of HRCT1 predicts poor survival for patients with gastric cancer. HRCT1 promotes tumor progression by activating the ERBB2-MAPK pathway. HRCT1, negatively regulated by miR-124-3p, may be a potential therapeutic target for patients with gastric cancer.

Association of peripheral basophils with tumor M2 macrophage infiltration and outcomes of the anti-PD-1 inhibitor plus chemotherapy combination in advanced gastric cancer

BackgroundAlthough the anti-programmed death-1 (PD-1) inhibitor plus chemotherapy combination has been approved as the standard first-line treatment for advanced gastric cancer, a proportion of patients do not significantly benefit from this therapy. Who would respond poorly to this treatment and the underlying mechanisms of treatment failure are far from clear.MethodsWe retrospectively analyzed the associations between the peripheral basophils at baseline and clinical outcomes in 63 advanced gastric cancer patients treated with anti-PD-1 plus chemotherapy and 54 patients treated with chemotherapy alone. Immunohistochemistry and immunofluorescence staining in gastric cancer samples were utilized to investigate the basophil-related immunophenotype.ResultsThe optimal cutoff of basophil count to distinguish responders to anti-PD-1 plus chemotherapy from non-responders was 20.0/μL. Compared with the low basophil group (≤ 20.0/μL, n = 40), the high basophil group (> 20.0/μL, n = 23) had a significantly lower objective response rate (ORR 17.4% vs. 67.5%, p = 0.0001), worse progression-free survival (median PFS 4.0 vs. 15.0 months, p = 0.0003), and worse overall survival (median OS not reached, p = 0.027). Multivariate analyses identified a basophil count of > 20.0/μL as an independent risk factor for a worse ORR (OR 0.040, 95% CI 0.007–0.241, p = 0.0004), worse PFS (HR 3.720, 95% CI 1.823–7.594, p = 0.0003) and worse OS (HR 3.427, 95% CI 1.698–6.917, p = 0.001). In contrast, there was no significant association between peripheral basophil counts and tumor response or survival in the chemotherapy-alone group (p > 0.05). In primary gastric cancer samples, we observed a correlation between higher peripheral basophil counts and the accumulation of tumor-infiltrating basophils (r = 0.6833, p = 0.005). Tumor-infiltrating basophils were found to be spatially proximate to M2 macrophages within TME and positively correlated with tumor M2 macrophage infiltration (r = 0.7234, p = 0.0023). The peripheral basophil counts also had a significant positive correlation with tumor-infiltrating M2 macrophage counts (r = 0.6584, p = 0.003). Further validation in tumor samples treated with the neoadjuvant anti-PD-1 inhibitor plus chemotherapy combination suggests that the peripheral basophils, tumor infiltration of basophils, and M2 macrophages were significantly more abundant in non-responders than in responders (p = 0.0333, p = 0.0007, and p = 0.0066, respectively).ConclusionsThe peripheral basophil count was observed to be a potential biomarker of anti-PD-1 efficacy for advanced gastric cancer. Moreover, basophils may induce an immune-evasive tumor microenvironment by increasing M2 macrophage infiltration, which could be a potential immunotherapeutic target for advanced gastric cancer.

Co-expression of VEGF-B and FLT-1 correlates with malignancy and prognosis of gastric cancer.

Background: This study aims to investigate the correlation of VEGF-B and FLT-1 co-expression with the prognosis of gastric cancer (GC). Materials & methods: Primary GC samples and adjacent tissues were obtained from 96 patients. Results: Both VEGF-B and FLT-1 were testified to be upregulated in the human GC compared with adjacent tissues. Spearman's rank correlation analysis indicated that VEGF-B and FLT-1 expression were correlated (r=0.321, p = 0.0015). High VEGF-B and FLT-1 co-expression patients showed poor prognosis when compared with low VEGF-B and FLT-1 co-expression patients (p = 0.0169). Conclusion: The high co-expression of VEGF-B and FLT-1 in GC shows a poor prognosis of overall survival, and targeted therapy against the interaction between VEGF-B and FLT-1 is worth further detailed analysis.

Distinct tumor immune microenvironments in primary and metastatic lesions in gastric cancer patients

This study compared the tumor immune microenvironments (TIMEs) of primary gastric cancer (PGC) and paired metastatic gastric cancer (MGC). CD4+ and CD8+ T-cell density and PD-L1 expression were evaluated by multiplex immunohistochemistry, DNA mismatch repair (MMR) by immunohistochemistry, and immune-related genes by RNA sequencing. Twenty-three patients who underwent surgical treatment for PGC and MGC were enrolled in this study. CD8+ T-cell, PD-L1+ cell, and PD-L1+CK+ cell densities were significantly lower in MGC than PGC. PD-L1 positivity using a combined positive score (≥ 1%) and deficient MMR were observed in 52.2% and 8.7% of PGC samples, respectively, whereas both occurred in only 4.3% of MGC samples. The most frequent TIME types were inflamed (34.8%) and adaptive immune resistance (34.8%) in PGC, and immune desert (65.2%) and immunological ignorance (73.9%) in MGC. In transcriptome analysis, the expression of the T-cell inflamed gene set and co-stimulatory gene module was down-regulated in MGC compared to PGC. The total CD8+ T-cell density was an independent prognostic marker in both PGC and MGC (univariate P = 0.002, multivariate P = 0.006). Our result suggest that the TIME of metastatic tumors was less immunologically active compared to that of primary tumors in gastric cancer patients.

RETRACTED ARTICLE: The POU2F1/miR-4490/USP22 axis regulates cell proliferation and metastasis in gastric cancer

PurposeGrowing evidence indicates that aberrant expression of microRNAs contributes to tumor development. However, the biological role of microRNA-4490 (miR-4490) in gastric cancer (GC) remains to be clarified.MethodsTo explore the function of miR-4490 in GC, we performed colony formation, EdU incorporation, qRT-PCR, Western blotting, in situ hybridization (ISH), immunohistochemistry (IHC), flow cytometry, ChIP and dual-luciferase reporter assays. In addition, the growth, migration and invasion capacities of GC cells were evaluated.ResultsWe found that miR-4490 was significantly downregulated in primary GC samples and in GC-derived cell lines compared with normal controls, and that this expression level was negatively correlated with GC malignancy. Exogenous miR-4490 expression not only reduced cell cycle progression and proliferation, but also significantly inhibited GC cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro. Mechanistically, we found that miR-4490 directly targets USP22, which mediates inhibition of GC cell proliferation and EMT-induced metastasis in vitro and in vivo. Moreover, we found through luciferase and ChIP assays that transcription factor POU2F1 can directly bind to POU2F1 binding sites within the miR-4490 and USP22 promoters and, by doing so, modulate their transcription. Spearman’s correlation analysis revealed a positive correlation between USP22 and POU2F1 expression and negative correlations between miR-4490 and USP22 as well as miR-4490 and POU2F1 expression in primary GC tissues.ConclusionBased on our results we conclude that miR-4490 acts as a tumor suppressor, and that the POU2F1/miR-4490/USP22 axis plays an important role in the regulation of growth, invasion and EMT of GC cells.

Role of LATS1/2 in Prognosis of Advanced Gastric Cancer and Its Relationship With the Tumor Immune Microenvironment.

Background: Gastric cancer (GC) remains a refractory cancer particularly in Eastern Asia. Large tumor suppressor kinases 1/2 (LATS1/2) are core members of the Hippo pathway. The role of LATS1/2 in the prognosis of different subtypes of advanced gastric cancer and its relationship with the tumor immune microenvironment in GC remain unknown. Exploring the role of LATS1/2 in GC might provide potential immunotherapeutic approaches for treating GC.Methods: Four hundred and ninety surgically resected primary GC samples were assessed for LATS1/2, CD8, FOXP3, and CD163. Correlations between LATS1/2 expression and immune-related markers were investigated and the prognoses of patients with different GC subtypes were analyzed.Results: CD8 and CD163 appeared to be favorable and adverse prognostic factors, respectively. LATS1/2 and FOXP3 did not predict patients' overall survival. However, in microsatellite-stable GC patients, high LATS1/2 and FOXP3 expression and low CD8 expression predicted poor prognoses. Furthermore, high LATS1/2 expression was significantly correlated with decreased CD8 and increased FOXP3. Combined analysis of LATS1/2, CD8, and FOXP3 had better prognostic accuracy than did each marker individually.Conclusions: Different biological molecules can predict the prognoses of different types of GC patients. LATS1/2, core kinases in the Hippo pathway, are closely related to CD8 and FOXP3. Further understanding the mechanisms of LATS1/2 in CD8+ T cells and FOXP3+ Treg cells provides further theoretical basis and potential targets for GC immunotherapy.

LncRNA PVT1 induces aggressive vasculogenic mimicry formation through activating the STAT3/Slug axis and epithelial-to-mesenchymal transition in gastric cancer.

Vasculogenic mimicry (VM), a vessel-like network formed by highly aggressive tumor cells, plays an important role in accelerating cancer progression. This special vascularization pattern is closely associated with a poor prognosis in various cancers. As yet, however, the regulatory mechanism of VM formation is largely unknown. In this study, we assess whether the long noncoding RNA PVT1 is involved in VM generation in gastric cancer. VM formation was determined by immunohistochemistry using PAS/CD31 double staining in gastric cancers and Matrigel tube formation in vitro. qRT-PCR and Western blotting were used to assess mRNA and protein expression. Interaction between PVT1 and STAT3 was determined using a RNA pull-down assay. Luciferase reporter and chromatin immunoprecipitation assays were performed to evaluate transcriptional activity of STAT3 on the Slug gene promoter. We found that PVT1 can induce VM generation both in vitro and in vivo. Mechanistically, we found that PVT1 interacted with and activated STAT3 through a 850-1770nt fragment. PVT1 facilitated STAT3 recruitment to the Slug promoter and transcriptionally enhanced Slug expression, thereby triggering epithelial-to-mesenchymal transition (EMT) and VM capillary formation. STAT3 inhibition effectively blocked PVT1-mediated VM. In primary gastric cancer samples, a positive correlation was found between PVT1 and Slug upregulation, and patients with a high PVT1 and Slug expression exhibited markedly shorter survival times. Our results shed light on the role of PVT1 in gastric cancer cell-dependent VM formation. Our findings provide valuable clues for the design of new anti-angiogenic therapeutic strategies. The PVT1/STAT3 axis may serve as a potential target in gastric cancer treatment.

Abstract 4028: Tenascin C in gastric cancer stroma is a predictive marker for postoperative prognosis

Abstract Background: Tenascin C, a glycoprotein in the extracellular matrix is reportedly associated with poor clinical outcome in several malignancies. We have previously reported that tenascin C in colorectal cancer stroma is a predictive marker for liver metastasis (Br J Cancer 117:1360-70, 2017). In this study, we evaluated the clinicopathological and prognostic significance of tenascin C expression in gastric cancer stroma. Materials and methods: Expression levels of tenascin C protein were immunohistochemically analyzed in 176 primary gastric cancer samples. Tenascin C staining intensity and relativity were classified into four scores and two groups, respectively. The correlation of the tenascin C staining intensity and relativity with clinicopathological factors, postoperative overall survival (OS) and disease-free survival (DFS) were analyzed. Results: There were positive correlations between the tenascin C staining intensity or relativity and tumor depth, lymphatic invasion, venous invasion, lymph node metastasis, pathological stage, high preoperative carcinoembryonic antigen (CEA)>5ng/ml, and high preoperative carbohydrate antigen (CA19-9) >37U/ml. Multivariate analysis revealed that high tenascin C staining intensity significantly correlated with tumor invasion into the muscularis propria (p=0.015) and venous invasion (p=0.042), and that high tenascin C staining relativity correlated with venous invasion (p=0.013). Gastric cancer patients with high tenascin C staining intensity had significantly shorter postoperative OS (p<0.001), whereas tenascin C staining relativity did not correlate with postoperative OS (p=0.05). High tenascin C staining intensity and relativity significantly correlated with shorter postoperative DFS (p<0.001 and 0.005, respectively). Cox proportional hazard model identified high tenascin C staining intensity as an independent prognostic factor for poor postoperative OS (hazard ratio (HR): 13.16, 95% confidence interval (CI): 1.362-125.000, p=0.026) and DFS (HR: 6.02, 95%CI: 1.538-23.810, p=0.010). Conclusions: Tenascin C protein expression in primary tumor stroma was identified as a predictive marker for postoperative prognosis in patients with gastric cancer. Citation Format: Hirotoshi Kikuchi, Tomohiro Murakami, Sanshiro Kawata, Amane Hirotsu, Tomohiro Matsumoto, Yusuke Ozaki, Yoshihiro Hiramatsu, Kinji Kamiya, Yoshifumi Morita, Takanori Sakaguchi, Hiroyuki Konno, Hiroya Takeuchi. Tenascin C in gastric cancer stroma is a predictive marker for postoperative prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4028.

Regulatory T cells and M2 macrophages present diverse prognostic value in gastric cancer patients with different clinicopathologic characteristics and chemotherapy strategies

BackgroundGastric cancer (GC) remains a refractory cancer worldwide. Currently, exploring the differences of the immune status in GC patients with different subgroups might provide promising immunotherapeutic approaches for the treatment of GC.MethodsIn this study, a total of 598 surgically resected FFPE primary gastric cancer samples were assessed for FOXP3, CD163, CD3, CD8, and PD-L1 markers. The correlations between the immune markers expression and clinicopathological features and prognosis were investigated retrospectively.ResultsIn general, PD-L1, CD3, and CD8 could be regarded as favorable prognostic factors. Our data demonstrated that high infiltration of FOXP3+ Treg indicates better prognosis in stage I–II patients, while the converse outcome was noted in stage III–IV patients. Our data also confirmed different prognostic value in different pathological classifications, chemotherapy strategies, and locations, with or without lymph node metastasis. Also, M2 macrophages indicated poor prognosis in general. However, high M2 macrophage infiltration suggests a favorable prognosis in signet ring cell carcinoma and mucinous adenocarcinoma. Moreover, the prognostic value of the two indices when they are combined is reported.ConclusionsThese results suggested that different immune statuses are exhibited in different subgroups of GC, which may direct further understanding of the immune status of GC as well as provide a further theoretical basis and potential targets for GC immunotherapy.

RPS15A promotes gastric cancer progression via activation of the Akt/IKK-β/NF-κB signalling pathway.

This study aimed to investigate the clinical significance, potential biological function and underlying mechanism of RPS15A in gastric cancer (GC) progression. RPS15A expression was detected in 40 pairs of GC tissues and matched normal gastric mucosae (MNGM) using qRT‐PCR analysis. Immunohistochemistry assay was conducted using a tissue microarray including 186 primary GC samples to characterize the clinical significance of RPS15A. A series of in vitro and in vivo assays were performed to elucidate the biological function of RPS15A in GC development and underlying molecular mechanisms. The expression of RPS15A was significantly up‐regulated in GC samples compared to MNGM, and its expression was closely related to TNM stage, tumour size, differentiation, lymph node metastasis and poor patient survival. Ectopic expression of RPS15A markedly enhanced the proliferation and metastasis of GC cells both in vitro and in vivo. RPS15A overexpression also promoted the epithelial‐mesenchymal transition (EMT) phenotype formation of GC cells. Investigations of underlying mechanisms found that RPS15A activated the NF‐κB signalling pathway by inducing the nuclear translocation and phosphorylation of the p65 NF‐κB subunit, transactivation of NF‐κB reporter and up‐regulating target genes of this pathway. In addition, RPS15A overexpression activated, while RPS15A knockdown inhibited the Akt/IKK‐β signalling axis in GC cells. And both Akt inhibitor LY294002 and IKK inhibitor Bay117082 neutralized the p65 and p‐p65 nuclear translocation induced by RPS15A overexpression. Collectively, our findings suggest that RPS15A activates the NF‐κB pathway through Akt/IKK‐β signalling axis, and consequently promotes EMT and GC metastasis. This newly identified RPS15A/Akt/IKK‐β/NF‐κB signalling pathway may be a potential therapeutic target to prevent GC progression.

FGF18, a prominent player in FGF signaling, promotes gastric tumorigenesis through autocrine manner and is negatively regulated by miR-590-5p

Fibroblast growth factors (FGFs) and their receptors are significant components during fundamental cellular processes. FGF18 plays a distinctive role in modulating the activity of both tumor cells and tumor microenvironment. This study aims to comprehensively investigate the expression and functional role of FGF18 in gastric cancer (GC) and elucidate its regulatory mechanisms. The upregulation of FGF18 was detected in seven out of eleven (63.6%) GC cell lines. In primary GC samples, FGF18 was overexpressed in genomically stable and chromosomal instability subtypes of GC and its overexpression was associated with poor survival. Knocking down FGF18 inhibited tumor formation abilities, induced G1 phase cell cycle arrest and enhanced anti-cancer drug sensitivity. Expression microarray profiling revealed that silencing of FGF18 activated ATM pathway but quenched TGF-β pathway. The key factors that altered in the related signaling were validated by western blot and immunofluorescence. Meanwhile, treating GC cells with human recombinant FGF18 or FGF18-conditioned medium accelerated tumor growth through activation of ERK-MAPK signaling. FGF18 was further confirmed to be a direct target of tumor suppressor, miR-590-5p. Their expressions showed a negative correlation in primary GC samples and more importantly, re-overexpression of FGF18 partly abolished the tumor-suppressive effect of miR-590-5p. Our study not only identified that FGF18 serves as a novel prognostic marker and a therapeutic target in GC but also enriched the knowledge of FGF-FGFR signaling during gastric tumorigenesis.

SRGAP1, a crucial target of miR-340 and miR-124, functions as a potential oncogene in gastric tumorigenesis

Slit-Robo GTPase-activating protein 1 (SRGAP1) functions as a GAP for Rho-family GTPases and downstream of Slit-Robo signaling. We aim to investigate the biological function of SRGAP1 and reveal its regulation by deregulated microRNAs (miRNAs) in gastric cancer (GC). mRNA and protein expression of SRGAP1 were examined by quantitative reverse transcription PCR (qRT-PCR) and western blot. The biological role of SRGAP1 was demonstrated through siRNA-mediated knockdown experiments. The regulation of SRGAP1 by miR-340 and miR-124 was confirmed by western blot, dual luciferase activity assays and rescue experiments. SRGAP1 is overexpressed in 9 out of 12 (75.0%) GC cell lines. In primary GC samples from TCGA cohort, SRGAP1 shows gene amplification in 5/258 (1.9%) of cases and its mRNA expression demonstrates a positive correlation with copy number gain. Knockdown of SRGAP1 in GC cells suppressed cell proliferation, reduced colony formation, and significantly inhibited cell invasion and migration. Luciferase reporter assays revealed that SRGAP1 knockdown significantly inhibited Wnt/β-catenin pathway. In addition, SRGAP1 was found to be a direct target of two tumor-suppressive miRNAs, miR-340 and miR-124. Concordantly, these two miRNAs were downregulated in primary gastric tumors and these decreasing levels w5ere associated with poor outcomes. Expression of miR-340 and SRGAP1 displayed a reverse relationship in primary samples and re-expressed SRGAP1, rescued the anti-cancer effects of miR-340. Taken together, these data strongly suggest that, apart from gene amplification and mutation, the activation of SRGAP1 in GC is partly due to the downregulation of tumor-suppressive miRNAs, miR-340 and miR-124. Thus SRGAP1 is overexpressed in gastric carcinogenesis and plays an oncogenic role through activating Wnt/β-catenin pathway.

SLC34A2 regulates miR-25-Gsk3β signaling pathway to affect tumor progression in gastric cancer stem cell-like cells.

A novel paradigm in tumor biology suggests that gastric cancer progression is driven by gastric cancer stem cell-like cells (GCSCs), but molecular mechanisms regulating tumorigenic and self-renewal potential of GCSCs are still unclear. Here, we aim to investigate biological function of SLC34A2 in regulating sphere formation and tumorigenicity (both are the hallmark of CSCs) of GCSCs and its underlying mechanisms. Our findings testified that CD44+ cells which were derived from fresh primary gastric cancer samples and cell lines displayed stem cell-like features. Significantly, SLC34A2 is increased in CD44+ GCSCs compared with those in adherent counterpart from CD44+ GCSCs. On clinic, SLC34A2 is overexpressed in primary tumor tissues compared with adjacent counterparts. We showed that SLC34A2 regulated sphere formation and self-renewal properties of CD44+ GCSCs in vitro and in vivo. Mechanistic investigations revealed that Gsk3β was the most strikingly up-regulated gene in response to SLC34A2 knockdown in GCSCs and Wnt/β-cantenin signaling was required for SLC34A2-mediated sphere formation. Furthermore, SLC34A2 directly binds specific sites in the miR-25 promoter region and that the promoter activity is decreased after the mutation of putative SLC34A2-binding sites, indicating that SLC34A2 is required for the transcriptional induction of miR-25. Meanwhile, luciferase assays showed that miR-25 directly targeted Gsk3β in CD44+ GCSCs. Overall, our findings define a SLC34A2-miR-25-Gsk3β pathway in the regulation of GCSCs features and gastric cancer progression, with potential therapeutic applications in blocking their progression.

PKM2 promotes cell migration and inhibits autophagy by mediating PI3K/AKT activation and contributes to the malignant development of gastric cancer

Pyruvate kinase M2 (PKM2) is a key kinase of glycolysis and is characteristic of all proliferating cells. The role of PKM2 in gastric cancer (GC) is still ambiguous and yet to be determined. To better understand the role of PKM2 in both the migration and invasion of GC, we measured the expression of PKM2 in GC cell lines using qRT-PCR and western blot. The prognostic value of PKM2 was analyzed by Immunohistochemistry in a cohort containing 88 GC patients. PKM2 was knocked down by the short hairpin RNA plasmid vector in NCI-N87 and BGC-823 cells, and the biological behavior and downstream signaling pathways were also investigated in vitro. Subcutaneous xenografts and pulmonary metastases models were constructed in nude mice to compare the differences in tumorgenesis and metastasis after Knockdown of PKM2. Our results obtained from in vitro cell biological behavior, in vivo tumorigenicity studies, and primary GC samples revealed an oncogenic role for PKM2 in GC. Furthermore, for those GC patients who received radical resection, PKM2 might serve as a novel prognostic biomarker and target which would allow for a brand new treatment strategy for GC in the clinical settings.

MicroRNA-1 acts as a tumor suppressor microRNA by inhibiting angiogenesis-related growth factors in human gastric cancer

BackgroundWe recently reported that miR-1 was one of the most significantly downregulated microRNAs in gastric cancer (GC) patients from The Cancer Genome Atlas microRNA sequencing data. Here we aim to elucidate the role of miR-1 in gastric carcinogenesis.MethodsWe measured miR-1 expression in human GC cell lines and 90 paired primary GC samples, and analyzed the association of its status with clinicopathological features. The effect of miR-1 on GC cells was evaluated by proliferation and migration assay. To identify the target genes of miR-1, bioinformatic analysis and protein array analysis were performed. Moreover, the regulation mechanism of miR-1 with regard to these predicted targets was investigated by quantitative PCR (qPCR), Western blot, ELISA, and endothelial cell tube formation. The putative binding site of miR-1 on target genes was assessed by a reporter assay.ResultsExpression of miR-1 was obviously decreased in GC cell lines and primary tissues. Patients with low miR-1 expression had significantly shorter overall survival compared with those with high miR-1 expression (P = 0.0027). Overexpression of miR-1 in GC cells inhibited proliferation, migration, and tube formation of endothelial cells by suppressing expression of vascular endothelial growth factor A (VEGF-A) and endothelin 1 (EDN1). Conversely, inhibition of miR-1 with use of antago-miR-1 caused an increase in expression of VEGF-A and EDN1 in nonmalignant GC cells or low-malignancy GC cells.ConclusionsMiR-1 acts as a tumor suppressor by inhibiting angiogenesis-related growth factors in human gastric cancer. Downregulated miR-1 not only promotes cellular proliferation and migration of GC cells, but may activates proangiogenesis signaling and stimulates the proliferation and migration of endothelial cells, indicating the possibility of new strategies for GC therapy.

Acidified bile acids enhance tumor progression and telomerase activity of gastric cancer in mice dependent on c-Myc expression.

c‐Myc overexpression has been implicated in several malignancies including gastric cancer. Here, we report that acidified bile acids enhance tumor progression and telomerase activity in gastric cancer via c‐Myc activation both in vivo and in vitro. c‐Myc mRNA and protein levels were assessed in ten primary and five local recurrent gastric cancer samples by quantitative real‐time polymerase chain reaction and western blotting analysis. The gastric cancer cell line MGC803 was exposed to bile salts (100 μmol/L glycochenodeoxycholic acid and deoxycholic acid) in an acid medium (pH 5.5) for 10 min daily for 60 weeks to develop an MGC803‐resistant cell line. Control MGC803 cells were grown without acids or bile salts for 60 weeks as a control. Cell morphology, proliferation, colony formation and apoptosis of MGC803‐resistant cells were analyzed after 60 weeks. To determine the involvement of c‐Myc in tumor progression and telomere aging in MGC803‐resistant cells, we generated xenografts in nude mice and measured xenograft volume and in vivo telomerase activity. The c‐Myc and hTERT protein and mRNA levels were significantly higher in local recurrent gastric cancer samples than in primary gastric cancer samples. MGC803‐resistant cells showed a marked phenotypic change under normal growth conditions with more clusters and acini, and exhibited increased cell viability and colony formation and decreased apoptosis in vitro. These phenotypic changes were found to be dependent on c‐Myc activation using the c‐Myc inhibitor 10058‐F4. MGC803‐resistant cells also showed a c‐Myc‐dependent increase in xenograft growth and telomerase activity in vivo. In conclusion, these observations support the hypothesis that acidified bile acids enhance tumor progression and telomerase activity in gastric cancer and that these effects are dependent on c‐Myc activity. These findings suggest that acidified bile acids play an important role in the malignant progression of local recurrent gastric cancer.