Human Gastric Cancer Samples Research Articles

ISL1 and AQP5 complement each other to enhance gastric cancer cell stemness by regulating CD44 expression.

Gastric cancer, a prevalent and life-threatening malignancy, is believed to involve cancer stem cells (CSCs) as a contributing factor to tumor progression. Insulin gene enhancer binding protein-1 (ISL1) is a transcription factor, and it has not been elucidated how ISL1 regulates gastric carcinogenesis. The aim of this paper is to investigate the role of ISL1 in gastric cancer development. In this study, we investigated the effects of ISL1 on the stem-like properties of human gastric cancer cells by applying transcriptional, flow, and immunofluorescence techniques. In human gastric cancer samples, there is an observed elevation in ISL1 expression, which correlates with the expression of stem cell markers, notably LGR5. Functionally, ISL1 fosters the self-renewal, cell proliferation, migration, and the clonogenic potential of gastric cancer cells in vitro. Furthermore, it enhances the ability of these cells to form tumors and metastasize in vivo. Additionally, ISL1 collaborates with AQP5, collectively intensifying the tumorigenicity of gastric cancer cells. Mechanistically, transcriptomic analysis of cells overexpressing ISL1 unveils a notable activation of the forkhead box O (FOXO) pathway. This activation leads to increased nuclear expression of forkhead box O3 (FOXO3), subsequently resulting in elevated expression of the stemness-associated gene CD44 in gastric cancer cells. These findings shed light on the role of ISL1 in promoting the stem-like characteristics of gastric cancer cells and emphasize the connection between ISL1 and AQP5 as a novel therapeutic target for individuals with gastric cancer.

Abstract 5583: Helicobacter pylori induces the wee1, promoting immune suppressive in human gastric adenocarcinoma

Abstract Background: Gastric cancer (GC) is the fourth most common cause of cancer-related death worldwide. GC has a dismal 5-year survival rate of about 32%. Helicobacter pylori (H. pylori) infection is the strongest risk factor for gastric carcinogenesis. 75-90% of gastric cancer patients exhibit positive H. pylori serology. WEE1 is a nuclear kinase that regulates cell cycle progression by inhibiting cyclin-dependent kinases. Methods & Results: Analysis of non-cancer normal stomach tissue (n=360) and human GC samples (n=1221) identified that WEE1 mRNA was significantly overexpressed in GC. Using immunohistochemistry (IHC) staining in 41 normal and 44 GC human samples, the data indicated that WEE1 protein was significantly overexpressed in GC. Western blot and immunofluorescence (IF) staining confirmed that H. pylori infection induces WEE1 overexpression in GC cells with an unexpected cytosolic localization of WEE1 in GC cells. Tff1 knockout GC mouse model (Tff1-/-) was infected with mouse-adapted H. pylori strain PMSS1. Western blot and immunohistochemistry data demonstrated that H. pylori infection induced WEE1 expression in Tff1 -/- HGD/cancer mouse gastric tissues compared to control Tff1 -/- mouse stomach. To study how H. pylori infection induces WEE1 in GC, analysis from miRTAR, Target Scan, and miRDB demonstrated that WEE1 is predicted to be targeted by miR-497-5p. Using qRT-PCR analysis, we assessed 30 normal and 30 GC human tissue samples, revealing a pronounced downregulation of miR-497-5p in GC specimens. Western blot analysis confirmed that the restoration of miR-497-5p in GC cells markedly reduced WEE1 protein expression. Interestingly, H. pylori infection further decreased miR-497-5p levels in GC cells, upregulating WEE1 protein expression. Since the miR-497-5P host gene promoter region contains CpG islands, we confirmed that treatment with 5 Azacytidine (demethylating agent) or DNMT1 knockdown induced expression of miR-497-5p with decreased WEE1 level in GC cells. Furthermore, our preliminary results indicate that aberrant WEE1 overexpression in GC demonstrates a significant positive correlation with immune-suppression gene signatures, underscoring its predictive value for unfavorable patient survival outcomes in GC. Using the TFF1 knockout mouse GC model, we found that WEE1 inhibition notably decreases the number and activation of CD4+ and CD8+ T cells in neoplastic gastric tissues. These findings also indicate a crucial relationship between WEE1 and the immunosuppressive microenvironment in GC, establishing WEE1 as a promising therapeutic target to enhance therapeutic efficacy and overcome immunotherapy resistance in GC. Conclusions: Our findings demonstrated a novel mechanism in which H. pylori activates the WEE1 in GC, promoting an immune suppressive microenvironment. WEE1 inhibition with immunotherapy could be a promising therapeutic method for GC patients. Citation Format: Md Obaidul Islam, Krishnapriya Thangaretnam, Heng Lu, Dunfa Peng, Nadeem Sidiq Bhat, Mohammed Soutto, Wael El-Rifai, Zheng Chen. Helicobacter pylori induces the wee1, promoting immune suppressive in human gastric adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5583.

Abstract 4744: Overcoming MK1775 resistance in upper gastrointestinal cancers with combined WEE1 and CHK1 inhibition

Abstract Background: Upper gastrointestinal cancers (UGC), encompassing esophageal and gastric cancers, rank among the top causes of cancer-related deaths globally, with esophageal cancer being the sixth and gastric cancer the fourth leading cause. Esophageal adenocarcinoma (EAC), predominant in the western world, shares histopathological and molecular characteristics with gastric cancer (GC). Despite these similarities, treatment challenges persist, exemplified by the limited efficacy of the WEE1 inhibitor MK1775 in clinical trials due to drug resistance. Our study investigates novel mechanisms underlying this resistance in UGC. Methods and results: RNA sequencing and immunohistochemistry staining demonstrated notable overexpression of WEE1 in over two hundred human EAC and GC samples compared to normal tissues. Concurrently, Western blot analysis revealed a positive correlation between WEE1 and CHK1 overexpression across 16 UGC cell lines. To categorize UGC cells based on their sensitivity to MK1775, we conducted ATP-glo cell viability assays, determining their half-maximal inhibitory concentration (IC50) values. Utilizing nuclear-cytosol separation, our data intriguingly uncovered, for the first time, that in MK1775-resistant cells, MK1775 treatment induces the abnormal translocation of phosphorylated CHK1 (p-CHK1) from the nucleus to the cytoplasm, a shift not observed in MK1775-sensitive cells. Further analysis through Western blot suggested that the cytoplasmic presence of p-CHK1 might trigger the activation of oncogenic STAT3 following MK1775 treatment. This activation appears to stimulate the expression of genes that promote cancer cell survival and drug resistance by enhancing cell survival and inhibiting apoptosis. Notably, combining the WEE1 inhibitor with the CHK1 inhibitor CHIR124 effectively inhibited CHK1 kinase activity and abrogated STAT3 phosphorylation, DNA binding, and transcriptional activity in UGC cells. Our findings strongly indicate that a combined therapy of WEE1 and CHK1 inhibitors could be a promising strategy to overcome MK1775 resistance in UGC cells. Our ongoing research will employ human-derived UGC organoids, patient-derived xenografts (PDXs), and human tissue samples to further validate these findings. Conclusions: Our study underscores the potential of combining WEE1 and CHK1 inhibitors to effectively combat MK1775 resistance in UGC, offering a promising direction for improved treatment strategies and patient outcomes in these challenging malignancies. Citation Format: Krishnapriya Thangaretnam, MD Obaidul Islam, Heng Lu, Dunfa Peng, Nadeem Sidiq Bhat, Mohammed Soutto, EL-Rifai Wael, Zheng Chen. Overcoming MK1775 resistance in upper gastrointestinal cancers with combined WEE1 and CHK1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4744.

CLEC4E upregulation in gastric cancer: A potential therapeutic target correlating with tumor-associated macrophages

BackgroundCLEC4E has been reported to promote lung cancer progression. Tumor-associated macrophages (TAMs) play an important role in tumorigenesis. Whether the expression of CLEC4E in TAMs is associated with gastric carcinogenesis remains unclear. MethodsThe TIMER, UALCAN, UCSC Xena, and KM plotter databases are used to examine the expression of CLEC4E and its prognostic significance in gastric cancer (GC). Additionally, GO, KEGG, and GSEA analysis were conducted, and single-cell RNA-seq (scRNA-seq) datasets were utilized. The Coremine medical database was used to predict therapeutic drugs, and molecular docking was performed. Human GC samples were obtained, and co-culture models were constructed to evaluate the effects of CLEC4E in TAMs on tumor growth, migration, and invasion in vitro. ResultsCLEC4E was significantly upregulated in GC, and high CLEC4E expression was associated with poor prognosis. Western blotting and immunostaining showed increased protein levels of CLEC4E in GC. GO, KEGG, and GSEA results indicated that CLEC4E is involved in immune response. Immune infiltration analysis demonstrated that CLEC4E expression positively correlated with multiple immune cell types. scRNA-seq analyses revealed that CLEC4E was predominantly expressed in myeloid cells specifically TAMs, in GC. In vitro experiments confirmed that MFC induced CLEC4E expression in TAMs to mediate tumor progression. Specifically targeting CLEC4E by si-CLEC4E or stigmasterol inhibited cancer cell migration and invasion. ConclusionCLEC4E is a potential prognostic biomarker and new therapeutic target for GC that can be specifically targeted by stigmasterol.

Overexpression of FERM Domain Containing Kindlin 2 (FERMT2) in Fibroblasts Correlates with EMT and Immunosuppression in Gastric Cancer.

The mesenchymal feature, dominated by epithelial mesenchymal transition (EMT) and stromal cell activation, is one of the main reasons for the aggressive nature of tumors, yet it remains poorly understood. In gastric cancer (GC), the fermitin family homolog-2 (FERMT2) is involved in macrophage signaling, promoting migration and invasion. However, the function of FERMT2 in fibroblasts remains unclear. Here, we demonstrated that downregulation of FERMT2 expression can block EMT in GC cells by inhibiting fibroblast activation in vitro. Furthermore, we found that, in addition to the known pathways, fibroblast-derived FERMT2 promotes M2-like macrophage growth and that in human GC samples, there is a strong positive correlation between FERMT2 and CD163 and CD206 levels. Notably, high FERMT2 expression was significantly associated with poor clinical outcomes and was upregulated in patients with advanced disease. Taken together, our results provide evidence that the fibroblast-FERMT2-EMT-M2 macrophage axis plays a critical role in the GC mesenchymal phenotype and may be a promising target for the treatment of advanced GC.

PSMA1 mediates tumor progression and poor prognosis of gastric carcinoma by deubiquitinating and stabilizing TAZ

The deubiquitinating enzyme family in tumor progression play important role in intracellular protein degradation. The proteasome subunit alpha type 1 (PSMA1) has been reported to act as an oncogene in several human cancers. The present study aimed to reveal the functional significance of PSMA1 in gastric cancer (GC) progression and the underlying mechanisms. The expression of PSMA1 in human GC samples and GC cell lines was examined by western blot analysis, real-time PCR, immunohistochemistry (IHC), and in vitro ubiquitination assays and established a xenograft mouse model. We found that PSMA1 was upregulated in GC and promoted proliferation, migration and invasion in GC cells. Herein, we report transcriptional co-activator with PDZ-binding motif (TAZ) was a downstream gene of PSMA1. Mechanistically, PSMA1 directly interacted with and stabilized TAZ via deubiquitination in GC. Furthermore, we found that TAZ was the essential mediator of PSMA1-modulated oncogenic activity in vitro and in vivo. Examination of clinical samples confirmed that elevated mediators of PSMA1, concomitant with increased TAZ abundance, correlate with human GC progression. These data suggested that PSMA1 promotes GC progression and proliferation by deubiquitinating TAZ. PSMA1 promotes GC progression and proliferation regarding PSMA1-mediated deubiquitinating enzyme activity and suggest potential therapeutic targets for GC management.

Obesity promotes lipid accumulation in lymph node metastasis of gastric cancer: a retrospective case‒control study

BackgroundThe connection between obesity, lipid accumulation, and lymph node metastasis (LNM) in gastric cancer (GC) is unclear.MethodsThe association of body mass index (BMI) and serum lipid levels with LNM was measured by calculating the odds ratio (OR) and 95% confidence interval (CI) in 1,058 eligible GC patients with a mean age of 61.4 years. Meanwhile, differentially expressed genes (DEGs) were identified between lymph node metastasis-positive (N +) and -negative (N0) groups using public RNA-seq data. Neutral lipids in human GC samples were detected by Oil red O staining. The expression of cluster of differentiation 36 (CD36), fatty acid synthase (FASN), and lipoprotein lipase (LPL) was detected by immunohistochemistry (IHC) and quantitative real-time PCR.ResultsCompared with normal-weight patients, overweight (OR = 2.02, 95% CI = 1.26–3.23) and obese (OR = 1.83, 95% CI = 1.15–2.91) patients showed increased ORs for LNM. However, no significant results were obtained for serum lipids in the multivariable-adjusted model (P > 0.05). Subgroup analysis suggested that increased low-density lipoprotein cholesterol was a risk factor in females (OR = 1.27, 95% CI = 1.02–1.59). Functional enrichment analysis of DEGs revealed a connection between lipid metabolism and LNM. Meanwhile, lipid staining showed a mass of lipids in obese N + tumor samples, and IHC analysis indicated an increase in LPL and CD36 expression in N + cases, implying a crucial role for exogenous lipid supply in LNM.ConclusionsHigh BMI significantly increases the risk of LNM in GC and promotes lipid accumulation in GC cells in LNM.

Dual roles of β-arrestin 1 in mediating cell metabolism and proliferation in gastric cancer

β-Arrestin 1 (ARRB1) has been recognized as a multifunctional adaptor protein in the last decade, beyond its original role in desensitizing G protein-coupled receptor signaling. Here, we identify that ARRB1 plays essential roles in mediating gastric cancer (GC) cell metabolism and proliferation, by combining cohort analysis and functional investigation using patient-derived preclinical models. Overexpression of ARRB1 was associated with poor outcome of GC patients and knockdown of ARRB1 impaired cell proliferation both ex vivo and invivo. Intriguingly, ARRB1 depicted diverse subcellular localizations during a passage of organoid cultures (7 d) to exert dual functions. Further analysis revealed that nuclear ARRB1 binds with transcription factor E2F1 triggering up-regulation of proliferative genes, while cytoplasmic ARRB1 modulates metabolic flux by binding with the pyruvate kinase M2 isoform (PKM2) and hindering PKM2 tetramerization, which reduces pyruvate kinase activity and leads to cellular metabolism shifts from oxidative phosphorylation to aerobic glycolysis. As ARRB1 localization was shown mostly in the cytoplasm in human GC samples, therapeutic potential of the ARRB1-PKM2 axis was tested, and we found tumor proliferation could be attenuated by the PKM2 activator DASA-58, especially in ARRB1high organoids. Together, the data in our study highlight a spatiotemporally dependent role of ARRB1 in mediating GC cell metabolism and proliferation and implies reactivating PKM2 may be a promising therapeutic strategy in a subset of GC patients.

Gasdermin B over-expression modulates HER2-targeted therapy resistance by inducing protective autophagy through Rab7 activation.

BackgroundGasdermin B (GSDMB) over-expression promotes poor prognosis and aggressive behavior in HER2 breast cancer by increasing resistance to therapy. Decoding the molecular mechanism of GSDMB-mediated drug resistance is crucial to identify novel effective targeted treatments for HER2/GSDMB aggressive tumors.MethodsDifferent in vitro approaches (immunoblot, qRT-PCR, flow cytometry, proteomic analysis, immunoprecipitation, and confocal/electron microscopy) were performed in HER2 breast and gastroesophageal carcinoma cell models. Results were then validated using in vivo preclinical animal models and analyzing human breast and gastric cancer samples.ResultsGSDMB up-regulation renders HER2 cancer cells more resistant to anti-HER2 agents by promoting protective autophagy. Accordingly, the combination of lapatinib with the autophagy inhibitor chloroquine increases the therapeutic response of GSDMB-positive cancers in vitro and in zebrafish and mice tumor xenograft in vivo models. Mechanistically, GSDMB N-terminal domain interacts with the key components of the autophagy machinery LC3B and Rab7, facilitating the Rab7 activation during pro-survival autophagy in response to anti-HER2 therapies. Finally, we validated these results in clinical samples where GSDMB/Rab7/LC3B co-expression associates significantly with relapse in HER2 breast and gastric cancers.ConclusionOur findings uncover for the first time a functional link between GSDMB over-expression and protective autophagy in response to HER2-targeted therapies. GSDMB behaves like an autophagy adaptor and plays a pivotal role in modulating autophagosome maturation through Rab7 activation. Finally, our results provide a new and accessible therapeutic approach for HER2/GSDMB + cancers with adverse clinical outcome.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13046-022-02497-w.

Abstract 5810: LPAR2 depletion suppresses gastric cancer via alteration of mitochondrial energy metabolism

Abstract Introduction: Gastric cancer is the second most common cause of cancer-related death worldwide. Altered metabolism is considered a primary hallmark of tumorigenesis, as it can regulate important processes associated with proliferation, migration, and invasion. Lysophosphatidic acid (LPA), a multifunctional endogenous phospholipid, plays a vital role in cellular homeostasis and malignant behavior of the cancer cells through G-protein coupled receptors. Although several signaling pathways have been reported as a critical mediator of gastric cancer, LPA-LPAR2 axis-mediated alteration of energy metabolism in gastric cancer progression has not been established yet. Therefore, our study focused on the molecular mechanism of LPA-LPAR2 axis-mediated alteration of mitochondrial bioenergetics during gastric cancer progression. Method: TCGA database analysis was carried out to check the mRNA levels of the LPA receptors. Expression of LPA receptors was investigated in human gastric cancer samples by immunoblotting, RT-PCR, and immunohistochemical analysis. We elucidated the functional effects of LPA on gastric cancer by performing ECIS proliferation, migration assay, scratch assay, and transwell invasion assay in gastric cancer cells. To confirm the involvement of the specific LPA receptor on gastric cancer progression, we treated the cells with LPA1-3 receptor blocker (Ki16425) and LPA2 receptor-specific antagonist. Following LPA receptor blockade, we analyzed the glycolytic and mitochondrial functions by measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using a Seahorse XF24 analyzer. Result: Our TCGA dataset analysis, LPA ELISA, and the Western blotting result showed the presence of LPA receptors in human gastric cancer tissues, specifically LPAR2 was robustly increased (P<0.001) in human gastric cancer tissue samples, and LPA level was significantly higher (P<0.001), suggesting a possible role of LPA in gastric cancer. LPA treatment increased proliferation, migration, and invasion activity in AGS and NCI-N87 gastric cancer cell lines. In contrast, LPA receptor antagonist Ki16425 abrogated the LPA-induced effect. Our seahorse analysis results showed that LPA treatment increased the OCR (P<0.001) and the ECAR (P<0.001), whereas LPAR2 specific receptor antagonists abrogated this effect (P<0.001). Conclusion: Together, our results suggested that the LPA-LPAR2 axis mediates gastric cancer initiation and progression by increasing energy metabolism via oxidative phosphorylation and glycolysis. Thus, targeting the LPAR2 receptor may give us a novel therapeutic approach to treat gastric cancer. Citation Format: Hosne Ara, Susmita Bhattarai, Sudha Sharma, Utsab Subedi, Xiuping Yu, Md. Shenuarin Bhuiyan, Sumitra Miriyala, Manikandan Panchatcharam. LPAR2 depletion suppresses gastric cancer via alteration of mitochondrial energy metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5810.

Abstract 5849: Activation of STAT3 is mediated by FGFR4 induction via SRC signaling after H. pylori infection

Abstract Background: Gastric cancer is considered the 5th most common form of cancer and 4th leading cause of cancer-related deaths worldwide. Due to the late stage of diagnosis, gastric cancer carries poor prognosis and poor overall survival. Infection with H. pylori is the strongest known risk factor. Fibroblast Growth Factor Receptor 4 (FGFR4) belongs to a family of highly conserved tyrosine kinases. This study investigated the molecular mechanism of FGFR4 regulation in response to H. pylori infection. Methods and Results: Among all the FGFRs, FGFR4 mRNA expression level was the most significantly elevated in human gastric cancer samples as compared to normal samples, using Real-time qPCR. These results were confirmed using the TCGA and Geo database analysis, confirming that FGFR4 is highly expressed in gastric cancer. Using western blot, we found that FGFR4 is significantly expressed in dysplasia and adenocarcinoma lesions in the TFF1-KO gastric cancer mouse model. Following infection with H. pylori in in-vitro (J166, 7.13) and in-vivo (PMSS1) models, we detected a significant increase in the expression of FGFR4 at the protein and mRNA levels. We also detected activation of STAT3 in human gastric cell lines and mice gastric tissues. Furthermore, analysis of FGFR4 promoter revealed several putative binding sites for STAT3. To confirm that FGFR4 expression is dependent upon STAT3 activation, we performed ChIP assay and confirmed direct functional binding of STAT3 on FGFR4 promoter. Using the FGF19, the ligand for FGFR4, we found that FGF19-FGFR4 axis played a vital role in activating STAT3 through an SRC-dependent mechanism and discovered that there is a feedforward activation loop between FGFR4 and STAT3. Functionally, we found that FGFR4 protected against H. pylori-induced DNA damage and cell death. Conclusion: Our study established a feedforward activation loop between FGFR4 and STAT3 in response to H. pylori infection and FGFR4 could be a possible druggable moiety for future therapies in gastric cancer. Citation Format: Nadeem S. Bhat, Mohammed Soutto, Xing Zhang, Zheng Chen, Shoumin Zhu, Heng Lu, Dunfa Peng, Zekuan Xu, Wael El-Rifai. Activation of STAT3 is mediated by FGFR4 induction via SRC signaling after H. pylori infection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5849.

Abrogation of ARF6 in promoting erastin-induced ferroptosis and mitigating capecitabine resistance in gastric cancer cells.

ADP ribosylation factor 6 (ARF6) is a member of the Rat sarcoma virus (RAS) superfamily that is involved in the regulation of vesicular trafficking, membrane lipid remodeling, and signaling pathways. Our earlier work discovered that ARF6, as a downstream effector of the Kirsten rat sarcoma viral oncogene (Kras)/extracellular signal-regulated kinases (ERK) signaling pathway, may increase proliferation and induce the Warburg effect in gastric cancer (GC) cells. Additionally, ARF6 appears to be a potential biomarker for predicting the prognosis of GC. Ferroptosis has recently been described as a type of nonapoptotic iron-dependent cell death that is strongly associated with the Kras mutation. Therefore, it is critical to continue investigating the link between ARF6 and ferroptosis. We first created ARF6 silenced cancer cell lines with lentivirus transfection. The knockdown efficiency was confirmed through quantitative polymerase chain reaction (qPCR) and western blotting. Subsequently, we used Cell Counting Kit-8 (CCK-8) and malondialdehyde (MDA) assay for lipid peroxidation measurement. Following this, qPCR and western blotting were conducted to clarify the mechanism involved. Finally, immunohistochemistry was used to stain human GC samples. Our findings established that, whereas ARF6 did not directly regulate lipid peroxidation, it did render GC cells susceptible to oxidative stress, particularly erastin-induced lipid peroxidation. Additionally, our research demonstrated that ARF6 may control capecitabine resistance via several routes. ARF6 may play a critical role in the development of GC.

SOCS3 gene silencing does not occur through methylation and mutations in gastric cancer.

Gastric cancer (GC) is ranked the third leading cause of cancer-related deaths worldwide. Mutations and epigenetic alterations in several essential genes, including p53, KRAS, PIK3CA, FAT4 and ARID1A, are often reported. Furthermore, loss of SOCS3 expression was reported in GC, suggesting its tumor suppressor role. To assess the mutational and methylation status of SOCS3, we performed gene panel exome sequencing on 47 human GC samples. The SOCS3 gene was rarely mutated, suggesting alternative regulation mechanisms, such as promoter hypermethylation and/or long non-coding RNAs (lncRNAs). We first explored SOCS3 promoter methylation status in 44 human GC samples by methylation-specific PCR (MS-PCR). Thirteen out of forty-four patients (29.5%) displayed a methylation pattern. Then, to see whether SOCS3 expression is silenced by CpG methylation, we examined publicly available databases (cbioportal and The Cancer Genome Atlas (TCGA)). The analysis revealed β values lower than 0.1, indicating hypo-methylation in healthy and GC samples. Moreover, moderate methylation (β < 0.4) and high methylation (β > 0.4) did not affect the free survival, suggesting that methylation is unlikely to be the mechanism rulingSOCS3silencing in GC. Next, to assess the regulatory effects of lncRNAs on SOCS3, we silenced the AC125807.2-lncRNA and quantified the SOCS3 gene expression in AGS and NCI-N87 gastric cancer cell line. SOCS3 was found to be downregulated following AC125807.2-lncRNA silencing in AGS cells, suggesting the potential implication of lncRNA AC125807.2 in SOCS3 regulation. However, in NCI-N87 cells, there was no significant change in SOCS3 expression. In conclusion, neither mutations nor hypermethylation was associated with the SOCS3downregulation in GC, and alternative mechanisms, including non-coding RNAs-mediated gene silencing, may be proposed.

MiR-449b-5p modifies glycolysis by lactate dehydrogenase A in gastric cancer

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To investigate the role of miR-449b-5p in gastric cancer (GC) metabolism.&#x0D; Methods: Human GC samples and their corresponding normal tissues were used in this study. Cell survival ability was evaluated using a commercial MTT kit. Glucose uptake, lactate production and ATP content were assessed using commercial kits. The expression levels of miRNAs were assessed by stem-loop reverse transcription-polymerase chain reaction (RT-PCR).&#x0D; Results: MiR-449b-5p expression decreased in both GC tissues and cells. When the cells with low level of miR-449b-5p were transfected with miR-449b-5p mimics, glycolysis was inhibited (p &lt; 0.05). Lactate dehydrogenase A (LDHA) was predicted as a target gene of miR-449b-5p and verified using luciferase reporter assay. MiR-449b-5p expression was up-regulated in GC cells (p &lt; 0.05). Furthermore, miR- 449b-5p expression was related to long overall survival time in patients with GC.&#x0D; Conclusion: The findings demonstrate that miR-449b-5p reverses the glycolytic state of GC cells by targeting LDHA expression, and thus, it can potentially be developed for the treatment of gastric cancer.&#x0D; &#x0D; &#x0D; &#x0D;

PRMT5 acts as a tumor suppressor by inhibiting Wnt/β-catenin signaling in murine gastric tumorigenesis.

Previous studies have demonstrated the in vitro oncogenic role of protein arginine methyltransferase 5 (PRMT5) in gastric cancer cell lines. The in vivo function of PRMT5 in gastric tumorigenesis, however, is still unexplored. Here, we showed that Prmt5 deletion in mouse gastric epithelium resulted in spontaneous tumorigenesis in gastric antrum. All Prmt5-deficient mice displayed intestinal-type gastric cancer within 4 months of age. Of note, 20% (2/10) of Prmt5 mutants finally developed into invasive gastric cancer by 8 months of age. Gastric cancer caused by PRMT5 loss exhibited the increase in Lgr5+ stem cells, which are proposed to contribute to both the gastric tumorigenesis and progression in mouse models. Consistent with the notion that Lgr5 is the target of Wnt/β-catenin signaling, whose activation is the most predominant driver for gastric tumorigenesis, Prmt5 mutant gastric cancer showed the activation of Wnt/β-Catenin signaling. Furthermore, in human gastric cancer samples, PRMT5 deletion and downregulation were frequently observed and associated with the poor prognosis. We propose that as opposed to the tumor-promoting role of PRMT5 well-established in the progression of various cancer types, PRMT5 functions as a tumor suppressor in vivo, at least during gastric tumor formation.

Histidine-rich calcium binding protein promotes gastric cancer cell proliferation, migration, invasion and epithelial-mesenchymal transition through Raf/MEK/ERK signaling.

Histidine-rich calcium binding protein (HRC) is a new type of Ca2+ homeostasis regulator, which acts as a nonnegligible role in regulating intracellular calcium homeostasis. Here, we demonstrated that HRC expression was upregulated in human gastric cancer (GC) samples, and its expression level was closely correlated with the overall survival (OS) rate of GC patients and the malignant potential of GC cell lines. Knockdown of HRC inhibited migration, invasion, and proliferation of GC cell lines in vitro, while HRC overexpression promoted GC cell migration, invasion, and proliferation in vitro, as well as the growth of subcutaneous tumors and peritoneal tumors in vivo. In terms of the mechanism, knockdown of HRC reduced the intracellular calcium ion level and the CaM protein level. Through cell function experiments, we found that HRC regulated the Raf/MEK/ERK pathway through Ca2+/CaM signaling and ultimately affected the epithelial‑mesenchyme transition (EMT) of GC. In summary, we revealed that HRC represents a potential target for GC treatment.

Long Noncoding RNA RP11-357H14.17 Plays an Oncogene Role in Gastric Cancer by Activating ATF2 Signaling and Enhancing Treg Cells.

Background Gastric cancer (GC) is one of the most common malignant tumors in the world. The potential functions and mechanisms of long noncoding RNAs (lncRNAs) in GC development are still unclear. It is of great significance to explore the prognostic value of LncRNA signatures for GC. Methods LncRNAs differently expressed in GC and their prognostic value were studied based on The Cancer Genome Atlas (TCGA) database. The functional regulatory network and immune infiltration of RP11-357H14.17 were further studied using a variety of bioinformatics tools and databases. Results We found that the high expression of RP11-357H14.17 was closely associated with shortened overall survival (OS) and poor prognosis in gastric cancer patients. We also found that its expression was related to clinical features including tumor volume, metastasis, and differentiation. Functional enrichment analysis revealed that RP11-357H14.17 is closely related to enhanced DNA replication and metabolism; ssGSEA analysis implied the oncogenic roles of RP11-357H14.17 was related to ATF2 signaling and Treg cell differentiation. Furthermore, we verified such link by using real-time PCR and IHC staining in human GC samples. Conclusion We demonstrate that RP11-357H14.17 may play a crucial role in the occurrence, development, and malignant biological behavior of gastric cancer as a potential prognostic marker for gastric cancer.

\u03b2-Galactosidase is a target enzyme for detecting peritoneal metastasis of gastric cancer

Diagnosis of peritoneal metastasis in gastric cancer (GC) is essential for determining appropriate therapeutic strategies and avoiding non-essential laparotomy or gastrectomy. Recently, a variety of activatable fluorescence probes that can detect enzyme activities have been developed for cancer imaging. The aim of this study was to identify the key enzyme involved in peritoneal metastasis in GC. The enzymatic activity of gamma-glutamyl transpeptidase, dipeptidyl peptidase IV, and β-galactosidase (β-Gal) was assessed in lysates prepared from preserved human GC (n = 89) and normal peritoneal (NP; n = 20) samples. β-Gal activity was significantly higher in the human GC samples than in NP samples, whereas no differences were observed in the activities of the other enzymes. Therefore, we used SPiDER-βGal, a fluorescent probe that can be activated by β-Gal, for imaging GC cell lines, peritoneal metastasis in a mouse model, and fresh human resected GC samples (n = 13). All cell lines showed fluorescence after applying SPiDER-βGal, and metastatic nodules in the mice gradually developed high fluorescence that could be visualized with SPiDER-βGal. The human GC samples showed significantly higher fluorescence than NP samples. β-Gal is a useful target enzyme for fluorescence imaging of peritoneal metastasis in GC.

Tumor-specific overexpression of histone gene, H3C14 in gastric cancer is mediated through EGFR-FOXC1 axis

Incorporation of different H3 histone isoforms/variants have been reported to differentially regulate gene expression via alteration in chromatin organization during diverse cellular processes. However, the differential expression of highly conserved histone H3.2 genes, H3C14 and H3C13 in human cancer has not been delineated. In this study, we investigated the expression of H3.2 genes in primary human gastric, brain, breast, colon, liver, and head and neck cancer tissues and tumor cell lines. The data showed overexpression of H3.2 transcripts in tumor samples and cell lines with respect to normal counterparts. Furthermore, TCGA data of individual and TCGA PANCAN cohort also showed significant up-regulation of H3.2 genes. Further, overexpressed H3C14 gene coding for H3.2 protein was regulated by FOXC1 transcription factor and G4-cassette in gastric cancer cell lines. Elevated expression of FOXC1 protein and transcripts were also observed in human gastric cancer samples and cell lines. Further, FOXC1 protein was predominantly localized in the nuclei of neoplastic gastric cells compared to normal counterpart. In continuation, studies with EGF induction, FOXC1 knockdown, and ChIP-qPCR for the first time identified a novel axis, EGFR-FOXC1-H3C14 for regulation of H3C14 gene overexpression in gastric cancer. Therefore, the changes the epigenomic landscape due to incorporation of differential expression H3 variant contributes to change in gene expression pattern and thereby contributing to pathogenesis of cancer.

The LPS-Treated Human Gastric Cancer Cells (AGS) Show a Significant Higher Tendency to Proliferation, Inflammation and Cannabinoid Receptor 1 Expression

Background: Cannabinoid receptor 1 (CB1R) that located throughout the central, peripheral, and enteric nervous system can be found all over the gastrointestinal tract. Cannabinoid receptors (CBRs) play important roles in pathophysiological processes and have been identified as a therapeutic target for developing novel anticancer agents. Objectives: The purposes of this study were to evaluate the CB1R expression in human gastric cancer, mRNA and protein expression of CB1R under lipopolysaccharide (LPS)-mediated inflammation condition in human gastric cancer cells (AGS), and the effects of inflammation on cell proliferation in LPS-stimulated AGS cells. Methods: CB1R mRNA expression in human gastric cancer samples and AGS cells were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The expression level of CB1R, after inflammation induction using LPS, was evaluated by qRT-PCR and western blot techniques. Cell proliferation was evaluated using 5-bromo-2-deoxyuridine (BrdU) labeling assay. Results: CB1R mRNA was significantly higher in human gastric cancer samples compared to adjacent normal tissues. LPS induced CB1R mRNA and protein expression in stimulated cells and promoted the proliferation of AGS cells. Conclusions: Our results show the increased expression of CB1R in gastric cancer samples and reveal that LPS induction increases the expression of CB1R and promotes cell proliferation in AGS cells. Accordingly, CB1R may be suggested as a potential molecular target for diagnostic and therapeutic aims in patients with gastric cancer.