Potential Therapeutic Target For Gastric Cancer Research Articles

Abstract C074: Preclinical study of antitumor effect of alpelisib combined with paclitaxel in gastric caner

Abstract Background: PIK3CA, encoding p110α subunit of PI3K, is the third most frequently mutated gene in gastric cancer (GC): 9–13% of non-hypermutated tumors and 32% of hypermutated tumors. Thus, PI3K pathway could be a potential therapeutic target in GC. Nevertheless, only few studies have been conducted so far. Materials and Methods: This preclinical study aimed to investigate the anti-tumor effects of alpelisib (Novartis, Basel, Switzerland), a PI3K p110α-specific inhibitor, using in vitro and in vivo models of GC. The combinatorial effects of alpelisib and paclitaxel, a commonly used agent for GC, were also evaluated. Results: Among eight GC cell lines (SNU1, SNU16, SNU484, SNU601, SNU638, SNU668, AGS, and MKN1), three harbored PIK3CA hotspot mutations: E542K in SNU601, E545A and E453K in AGS, and E545K in MKN1, while the others were PIK3CA wild-type. These three PIK3CA mutant cells were predominantly sensitive to alpelisib. Alpelisib monotherapy decreased AKT and S6K1 phosphorylation and induced G0/G1 cell cycle arrest regardless of PIK3CA mutational status. Moreover, in PIK3CA mutant cells, GSK3β and BAD phosphorylation was potently abrogated by alpelisib alone, which was not evident in PIK3CA wild-type cells. Alpelisib in combination with paclitaxel demonstrated synergistic anti-proliferative effects, preferentially in PIK3CA mutant cells, by increasing caspase 3/7 activity and resulting in increased apoptosis. Moreover, the alpelisib and paclitaxel combination more potently inhibited the cell migration in wound healing assays and the expression of epithelial-mesenchymal transition (EMT)-associated proteins including Snail and Twist in PIK3CA mutant cells, compared with PIK3CA wild-type cells. In a mouse xenograft model of MKN1 cells, alpelisib combined with paclitaxel significantly enhanced the anti-tumor activity by decreasing Ki-67 expression and increasing TUNEL expression. Moreover, this combination tended to prolong survival of tumor-bearing mice for 4 weeks of treatment period without resulting in significant change in body weight. Conclusions: Alpelisib alone or in combination with paclitaxel demonstrated promising anti-tumor activity and tolerability in in vitro and in vivo models of PIK3CA mutant GC via inactivating PI3K down-stream molecules, decreasing cell migration, and increasing apoptosis. Our data suggest that this novel combination is worthy of further clinical investigations in patients with PIK3CA mutant GC. Keywords: alpelisib; BYL719; paclitaxel; PIK3CA; gastric cancer; combination; chemotherapy Citation Format: Ji-Won Kim, Kui-Jin Kim, Ji Hea Sung, Koung Jin Suh, Ji Yun Lee, Se Hyun Kim, Jin Won Kim, Jeong-Ok Lee, Yu Jung Kim, Soo-Mee Bang, Jee Hyun Kim, Jong Seok Lee, Keun-Wook Lee. Preclinical study of antitumor effect of alpelisib combined with paclitaxel in gastric caner [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C074. doi:10.1158/1535-7163.TARG-19-C074

Downregulation of NUSAP1 suppresses cell proliferation, migration, and invasion via inhibiting mTORC1 signalling pathway in gastric cancer.

Gastric cancer (GC) is one of the most common causes of cancer-related death worldwide, and outstanding biomarkers for therapeutic targets or predicting GC survival are still lacking. Increasing evidence indicated that nucleolar and spindle associated protein 1 (NUSAP1) involved in regulating the progression of various cancers; however, its specific role in GC remained unclear. In this study, we found that NUSAP1 was upregulated in the GC tissues and cell lines via analysing data from The Cancer Genome Atlas (TCGA), gene expression omnibus (GEO), qRT-PCR, and western blot assays. Patients with high NUSAP1 expression levels showed shorter free-progression survival (FPS), larger tumour size, and higher lymphatic metastasis rate compared with those with low NUSAP1 expression. Further functional experiments revealed knockdown of NUSAP1 could inhibit the growth, migration, and invasion of GC cells in vitro and vivo. Additionally, silencing NUSAP1 induced G0/G1 phase arrest, apoptosis, and suppressed the epithelial-mesenchymal transition (EMT) process. Finally, we performed gene set enrichment analysis (GSEA) and observed NUSAP1 was positive with mTORC1 signalling pathway, which was verified by the subsequent immunoblotting. In conclusion, our findings suggested that NUSAP1 contributed to GC progression and may act as a potential therapeutic target for GC. SIGNIFICANCE OF THE STUDY: Our results firstly illuminated that NUSAP1 expression was significantly upregulated in GC tissues and predicted poor FPS. Silencing it could attenuate GC progression via inhibiting mTORC1 signalling pathway. Hence, NUSAP1 may act as a promising therapy target for GC.

MST4 Predicts Poor Prognosis And Promotes Metastasis By Facilitating Epithelial-Mesenchymal Transition In Gastric Cancer.

BackgroundMetastasis is the main cause for gastric cancer (GC)-related deaths. Better understanding of GC metastatic mechanism would provide novel diagnostic markers and therapeutic targets. Though it has been reported that mammalian sterile-20-like kinase 4 (MST4) exerts the oncogenic role in other tumors, the prognostic value and biological role of MST4 in GC are still unknown.MethodsThe expression level of MST4 in GC was analyzed by using TCGA database. Then, Western blot and polymerase chain reaction (PCR) were used to determine the MST4 expression in GC tissues and cell lines. Immunohistochemistry was performed to investigate the expression of proteins in human GC tissues, and its correlation with clinicopathologic parameters as well as the prognosis for patients with GC was analyzed. In addition, the biological function and its molecular mechanism of MST4 in GC were investigated by in vitro and in vivo assays.ResultsIt demonstrated that MST4 expression was significantly upregulated in GC tissues and cell lines. High expression of MST4 was correlated with aggressive clinicopathological parameters such as lymph node metastasis, lymphovascular invasion (all P < 0.05). GC patients with high MST4 expression had both shorter overall survival (OS) and disease-free survival (DFS) than those with low MST4 expression (all P < 0.05). MST4 expression was an independent and significant risk factor for OS and DFS of GC patients (all P < 0.05). Results of functional experiments showed that MST4 could promote GC cells migration, invasion in vitro and metastasis in vivo. In terms of mechanism, MST4 promoted metastasis by facilitating epithelial–mesenchymal transition (EMT) through activating Ezrin pathway in GC. Further studies indicate that down-regulated miR-124-3p expression contributes to upregulated MST4 expression in GC.ConclusionOur data showed that MST4 predicts poor prognosis and promotes metastasis by facilitating epithelial–mesenchymal transition in GC. Therefore, our study suggests that MST4 can be used as a valuable prognostic biomarker and a potential therapeutic target in GC.

Long non-coding RNA GAS5 inhibits migration and invasion in gastric cancer via interacting with p53 protein

BackgroundThe rapid progress of gastric cancer (GC) is mainly due to metastasis. Long non-coding RNA (lncRNA) GAS5 has been identified as a tumor suppressor in numerous cancers, and its downregulation in GC has already been reported. AimsIn this study, we planned to investigate the role of GAS5 in GC metastasis. MethodsGene expressions were detected by qRT-PCR. ISH staining was applied to assess GAS5 level in clinical tissues. Gain-of-function assays were conducted to evaluate the function of GAS5 in GC metastasis. RNA pull down, RIP and cycloheximide assays were performed to confirm the relationship between GAS5 and p53 protein. ResultsGAS5 expression was markedly decreased in GC tissues and cell lines, and its low expression was strongly related to GC metastasis and unsatisfactory prognosis. GAS5 overexpression repressed GC cell migration and invasion by targeting p53. Intriguingly, GAS5 relied on the exon 12 to interact with and stabilize p53 protein. ConclusionOur data implied that GAS5 is a suppressor in GC metastasis via modulating p53 signaling, suggesting GAS5 as a potential therapeutic target for GC, especially for patients with metastasis.

The YY1-CCDC43-ADRM1 Signaling Axis Promotes Tumorigenesis, Invasion and Metastasis of Gastric Cancer

Background: Previous studies have showed an association of coiled-coil domain-containing (CCDC) genes with cancers. Our studies also found that CCDC43 is highly expressed in colorectal cancer. However, the expression patterns and molecular mechanisms of CCDC43 in gastric cancer (GC) have not been elucidated. Methods: We analyzed the CCDC43 gene expression in GC samples and adjacent noncancerous mucosa tissues by immunohistochemistry, western blot and quantitative real-time PCR (qPCR) analysis. Additionally, we utilized isobaric Tag for Relative and Absolute Quantitation (iTRAQ) to identify differentially expressed proteins in CCDC43-overexpressing cells. Chromatin immunoprecipitation (ChIP) assay and luciferase reporter assay were used to investigate the role of transcription factor YY1 in promoting the expression of CCDC43 and ADRM1. Findings: CCDC43 was overexpressed in gastric tissues. CCDC43 expression was closely related to tumor differentiation, lymph-node-metastasis and prognosis of gastric cancer. Over-expression of CCDC43 promoted the proliferation, invasion and metastasis of GC cells. CCDC43 could upregulate and stabilize ADRM1, resulting in the degradation of ubiquitin-mediated proteasomal. While, inhibition of ADRM1 could reverse the function of CCDC43 in GC both in vitro and in vivo. In addition, our data demonstrated that transcription factor YY1 directly bound to the CCDC43 gene promoter, leading to over-expression of CCDC43 and ADRM1. We also confirmed that YY1, CCDC43 and ADRM1 proteins were positively and correlatively expressed in human GC tissue compared with adjacent noncancerous tissues. Interpretation: Our findings suggest that the YY1-CCDC43-ADRM1 axis provides a potential therapeutic target of GC. Funding: Natural Science Foundation of China. Declaration of Interest: The authors declare that they have no conflict of interest. Ethical Approval: Samples from surgical removal of gastric cancer from 2019 were selected from the Department of Surgery of Nanfang Hospital, Southern Medical University. The Ethics Committee of the Southern Medical University, China, approved our experimental protocols. Mice were maintained in a specific pathogen-free environment. All in vivo experiments were approved by the Institutional Animal Care and Use Committee of Southern Medical University.

Effect of inhibition to Yes-related proteins-mediated Wnt/β-catenin signaling pathway through miR-195-5p on apoptosis of gastric cancer cells.

This clinical study aimed to explore the expression of miR-195-5p and Yes-associated protein (YAP) in gastric cancer (GC) and their relevant apoptosis mechanism. Thirty GC patients treated in our hospital were enrolled as a patient group and 30 normal people who underwent physical examination during the same period were enrolled as a normal group. The purchased GC cells and human gastric mucous membrane cells were used to transfect miR-195-5p-mimics, miR-NC, si-YAP, and si-RNA into MKN45 and SGC7901 cells. qRT-PCR was used to detect the expression of miR-195-5p and YAP in samples, and WB was used to detect the expression of YAP, Caspase-3, Caspase-9, β-catenin, c-myc, and cyclin Dl. CCK-8, the transwell, and the flow cytometry were used to detect cell proliferation, invasion, and apoptosis. The dual fluorescent enzyme reporter was used to determine the relationship between miR-195-5p and YAP. MiR-195-5p was lowly expressed in the tissues and serum of patients, but YAP was highly expressed, and the area under the miR-195-5p and YAP curves was more than 0.9. MiR-195-5p and YAP were associated with tumor diameter, TNM stage, lymph node metastasis, and differentiation in GC patients. The overexpression of miR-195-5p and the inhibition of YAP expression can inhibit cell proliferation and invasion and promote apoptosis. WB assay showed that the overexpression of miR-195-5p and the inhibition of YAP could inhibit the expression of β-catenin, c-myc, and cyclin D1 protein and promote the expression of Caspase-3, Caspase-9 protein. The dual fluorescent enzyme reporter revealed that there was a targeting relationship between miR-195-5p and YAP. The overexpression of miR-195-5p can inhibit YAP-mediated Wnt/β-catenin signaling pathway and promote cell apoptosis, so it may be a potential therapeutic target for GC.

ALKBH5 promotes invasion and metastasis of gastric cancer by decreasing methylation of the lncRNA NEAT1

N6-Methyladenosine (m6A) is the most common posttranscriptional modification of RNA and plays critical roles in cancer pathogenesis. However, the biological function of long noncoding RNA (lncRNA) methylation remains unclear. As a demethylase, ALKBH5 (alkylation repair homolog protein 5) is involved in mediating methylation reversal. The purpose of this study was to investigate lncRNA m6A modification and its role in gastric cancer (GC). Bioinformatics predicted interactions of ALKBH5 with lncRNAs. Five methods were employed to assess the function of nuclear paraspeckle assembly transcript 1 (NEAT1), including gene silencing, RT-PCR, separation of nuclear and cytoplasmic fractions, scrape motility assays, and transwell migration assays. Then, m6A RNA immunoprecipitation and immunofluorescence were used to detect methylated NEAT1 in GC cells. Rescue assays were performed to define the relationship between NEAT1 and ALKBH5. NEAT1 is a potential binding lncRNA of ALKBH5. NEAT1 was overexpressed in GC cells and tissue. Additional experiments confirmed that knockdown of NEAT1 significantly repressed invasion and metastasis of GC cells. ALKBH5 affected the m6A level of NEAT1. The binding of ALKBH5 and NEAT1 influences the expression of EZH2 (a subunit of the polycomb repressive complex) and thus affects GC invasion and metastasis. Our findings indicate a novel mechanism by which ALKBH5 promotes GC invasion and metastasis by demethylating the lncRNA NEAT1. They may be potential therapeutic targets for GC.

Long noncoding RNA MNX1-AS1 overexpression promotes the invasion and metastasis of gastric cancer through repressing CDKN1A.

Recently, long non-coding RNAs (lncRNAs) have attracted much attention for their roles in tumor progression. The aim of this study was to investigate the specific role of lncRNA MNX1-AS1 in the development of gastric cancer (GC), and to explore the underlying mechanism. MNX1-AS1 expression in both GC cells and tissue samples was detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). Moreover, the relationship between MNX1-AS1 expression and the overall survival rate of GC patients was explored. Furthermore, wound healing assay and transwell assay were conducted. In addition, the underlying mechanism of MNX1-AS1 in GC was explored by performing RT-qPCR and Western blot assay. MNX1-AS1 expression in GC samples was significantly higher than that of the corresponding normal tissues. Meanwhile, MNX1-AS1 expression was associated with the overall survival time of GC patient. Moreover, the migration and invasion of GC cells were markedly promoted after MNX1-AS1 overexpression in vitro. The mRNA and protein expressions of CDKN1A were remarkably down-regulated after MNX1-AS1 overexpression. Furthermore, the expression level of CDKN1A was negatively correlated with the expression of MNX1-AS1 in GC tissues. Our results suggested that MNX1-AS1 could enhance the metastasis and invasion of GC cells via suppressing CDKN1A. Furthermore, MNX1-AS1 might be a potential therapeutic target for GC.

MTMR2 promotes invasion and metastasis of gastric cancer via inactivating IFN\u03b3/STAT1 signaling

BackgroundThe aberrant expression of myotubularin-related protein 2 (MTMR2) has been found in some cancers, but little is known about the roles and clinical relevance. The present study aimed to investigate the roles and clinical relevance of MTMR2 as well as the underlying mechanisms in gastric cancer (GC).MethodsMTMR2 expression was examined in 295 GC samples by using immunohistochemistry (IHC). The correlation between MTMR2 expression and clinicopathological features and outcomes of the patients was analyzed. The roles of MTMR2 in regulating the invasive and metastatic capabilities of GC cells were observed using gain-and loss-of-function assays both in vitro and in vivo. The pathways involved in MTMR2-regulating invasion and metastasis were selected and identified by using mRNA expression profiling. Functions and underlying mechanisms of MTMR2-mediated invasion and metastasis were further investigated in a series of in vitro studies.ResultsMTMR2 was highly expressed in human GC tissues compared to adjacent normal tissues and its expression levels were significantly correlated with depth of invasion, lymph node metastasis, and TNM stage. Patients with MTMR2high had significantly shorter lifespan than those with MTMR2low. Cox regression analysis showed that MTMR2 was an independent prognostic indicator for GC patients. Knockdown of MTMR2 significantly reduced migratory and invasive capabilities in vitro and metastases in vivo in GC cells, while overexpressing MTMR2 achieved the opposite results. MTMR2 knockdown and overexpression markedly inhibited and promoted the epithelial-mesenchymal transition (EMT), respectively. MTMR2 mediated EMT through the IFNγ/STAT1/IRF1 pathway to promote GC invasion and metastasis. Phosphorylation of STAT1 and IRF1 was increased by MTMR2 knockdown and decreased by MTMR2 overexpression accompanying with ZEB1 down-regulation and up-regulation, respectively. Silencing IRF1 upregulated ZEB1, which induced EMT and consequently enhanced invasion and metastasis in GC cells.ConclusionsOur findings suggest that MTMR2 is an important promoter in GC invasion and metastasis by inactivating IFNγ/STAT1 signaling and may act as a new prognostic indicator and a potential therapeutic target for GC.

BMX-ARHGAP fusion protein maintains the tumorigenicity of gastric cancer stem cells by activating the JAK/STAT3 signaling pathway

BackgroundCancer stem cells (CSCs), drug-resistant cancer cell subsets, are known to be responsible for tumor metastasis and relapse. The JAK/STAT pathway, activated by SH2 domain, is known to regulate the tumor growth in gastric cancer (GC). Now, this study was designed to examine whether BMX-ARHGAP affects the GC stem cell properties and the underlying regulatory network via JAK/STAT axis.MethodsBMX-ARHGAP expression was characterized in GC tissues and cells by RT-qPCR and western blot assay. When BMX-ARHGAP was overexpressed or silenced via plasmids or siRNA transfection, the stem cell properties were assessed by determining stem cell markers CD133, CD44, SOX2 and Nanog, followed by cell sphere and colony formation assays. Subsequently, cell proliferation and invasion were examined by conducting EdU and Transwell assays. The JAK/STAT3 signaling pathway activation was inhibited using AG490. ARHGAP12, BMX exon 10–11, BXM-SH2, JAK2 and STAT3 expression patterns were all determined to examine the regulatory network. The stem cell property in nude mice was also tested.ResultsBMX-ARHGAP was determined to be enriched in the GC. Overexpression of BMX-ARHGAP resulted in increased expression of CD133, CD44, SOX2 and Nanog protein, and accelerated proliferation and invasion of CD133+CD44+ cells as well as facilitated self-renewal potential of GC cells. However, the inhibition of the JAK/STAT3 signaling pathway reversed the stimulating effect of BMX-ARHGAP on proliferative and invasion abilities of CD133+CD44+ cells. The overexpression of BMX-ARHGAP was suggested to increase the BMX-SH2 protein expression via ARHGAP 5′UTR, and activate the JAK/STAT3 signaling pathway. Also, BMX-ARHGAP promoted tumor growth in nude mice.ConclusionsThe aforementioned results demonstrated that the BMX-ARHGAP-dependent SH2 domain-JAK/STAT3 axis mediates the maintenance of GC stem cells, benefiting the development of new potential therapeutic targets for GC.

High expression of TCF12 contributes to gastric cancer development via being target regulated by miR-183 and activating PI3K/AKT pathway.

This study aimed to explore the role of transcription factor 12 (TCF12) in the process of gastric cancer (GC) and to elucidate its possible regulatory mechanism. The expression data of GC tissues and matched normal tissues were downloaded from The Cancer Genome Atlas (TCGA) database. Survival analysis for GC patients with different levels of TCF12 was performed by the Kaplan-Meier analysis. In addition, TCF12 was suppressed in human GC cell lines AGS and MKN-45, followed by detecting cell biological processes (proliferation, apoptosis, migration, and invasion). Moreover, the association between TCF12 and the phosphatidylinositol 3-kinase (PI3K)/AKT signal was elucidated. Besides, the potential micro RNAs that could target TCF12 expression were explored. The results showed that TCF12 was highly expressed in GC tissues and TCF12 upregulation was associated with poor prognosis of GC patients. In addition, suppression of TCF12 significantly inhibited the proliferation, migration, and invasion of both AGS and MKN45 cells, as well as induced apoptosis of the two cell lines. Moreover, suppression of TCF12 significantly decreased the expression of p-AKT, cyclin D1, p-P70, and β-catenin in both AGS and MKN45 cells. Besides, TCF12 was target regulated by miR-183 in GC cells. Our findings reveal that TCF12 is upregulated in GC and its upregulation is associated with poor prognosis of GC patients. To sum up, downregulation of TCF12 may inhibit GC development via being target regulated by miR-183 and inhibiting the PI3K/AKT signal. TCF12 may function as a potential therapeutic target for GC.

MIR-1265 regulates cellular proliferation and apoptosis by targeting calcium binding protein 39 in gastric cancer and, thereby, impairing oncogenic autophagy

Increasing evidence indicates that microRNAs (miRNAs) play an important role in various tumors by regulating downstream target genes and diverse signaling pathways. Herein, we confirmed miR-1265 expression in gastric cancer (GC) using the Cancer Genome Atlas (TCGA) database and assessed the level of miR-1265 expression in clinical specimens and cell lines. We found that miR-1265 expression was negatively correlated with tumor size. Further functional analysis revealed that miR-1265 suppresses cellular proliferation and autophagy while inducing apoptosis in GC cells. A luciferase reporter assay was used to identify an miR-1265 targeted gene, calcium binding protein 39 (CAB39), which is an essential upstream regulator in the AMPK-mTOR signaling pathway. Upregulation or downregulation of CAB39 expression reversed the effects of miR-1265 overexpression or inhibition, respectively. Notably, the knockdown of autophagy-related gene 12 (ATG12) impaired the effects of miR-1265 inhibition or CAB39 overexpression in GC. MiR-1265 also suppressed the growth of GC cells in vivo and that of human gastric organoids. Altogether, our results show that miR-1265 suppresses GC progression and oncogenic autophagy by reducing CAB39 expression and regulating the AMPK-mTOR signaling pathway. Therefore, miR-1265 may represent a potential therapeutic target for GC.

NETO2 promotes invasion and metastasis of gastric cancer cells via activation of PI3K/Akt/NF-\u03baB/Snail axis and predicts outcome of the patients

Aberrant expression of neuropilin and tolloid-like 2 (NETO2) has been observed during the progression of some human carcinomas. However, the expression pattern and clinical relevance of NETO2 in gastric cancer (GC) remain to be elucidated. In this study, we found that NETO2 expression was higher in GC tissues compared with paired non-cancerous tissues. Moreover, the expression of NETO2 was positively correlated with clinical stage, invasion depth, lymph node metastasis, and tumor size, but inversely correlated with overall and disease-free survival rates. Cox regression analysis identified NETO2 as an independent prognostic indicator for GC patients. Overexpression of NETO2 facilitated migration and invasion of GC cells in vitro and metastasis in vivo in association with induction of epithelial-mesenchymal transition. Conversely, knockdown of NETO2 had the opposite effects. Mechanistically, silencing NETO2 reduced the phosphorylation of PI3K, AKT, and NF-κB p65 as well as the expression of Snail, whereas NETO2 overexpression achieved the opposite results. Furthermore, we identified TNFRSF12A as a mediator for NETO2 to activate PI3K/AKT/NF-κB/Snail axis. Collectively, our results demonstrate that NETO2 promotes invasion and metastasis of GC cells and represents a novel prognostic indicator as well as a potential therapeutic target in GC.

Long non-coding antisense RNA GAS6-AS1 supports gastric cancer progression via increasing GAS6 expression

ObjectiveAs one broader class of non-coding RNAs (lncRNAs), non-coding antisense (AS) transcripts are functionally characterized to play pivotal roles in various pathophysiological processes, including tumor biology. MethodsIn this study, the exact biological functions and regulation mechanisms of GAS6-AS1 in gastric cancer (GC) was examined. ResultsThe expression of GAS6-AS1 was markedly upregulated in GC tissues and is associated with advanced stage (III + IV) of GC patients. Gain-of-function and loss-of-function experiments showed that GAS6-AS1 promoted cell proliferation, migration, invasion ability in vitro and xenograft tumor growth in vivo by promoting entry into S-phase. The mechanistic investigations showed that GAS6-AS1 can control the expression of its cognate sense gene GAS6 at the transcriptional or translational levels by forming a RNA-RNA duplex, consequently inducing an increase of AXL level and driveling AXL signaling pathway activation. ConclusionsTaken together, our studies indicate that GAS6-AS1 significantly driving the aggressive phenotype in GC through activating its cognate sense gene GAS6, and provides a more complete understanding of GAS6-AS1 as a potential therapeutic target for GC.

MiR-384 targets metadherin gene to suppress growth, migration, and invasion of gastric cancer cells.

ObjectiveMicroRNA-384 (miR-384) has been reported to function as a tumor suppressor in multiple cancers; however, its role in gastric cancer (GC) remains unclear.MethodsWe measured expression levels of miR-384 in GC cell lines and in a normal gastric cell line (GES-1). The association between miR-384 and the metadherin gene (MTDH) was assessed by luciferase reporter assay and western blot. The effects of the miR-384/MTDH axis on GC cell behaviors were measured by CCK-8, wound-healing, and transwell invasion assays.ResultsmiR-384 was significantly downregulated in GC cell lines compared with normal gastric cells. MTDH was identified as a direct target of miR-384 by bioinformatics analysis, luciferase assay, and western blot. Functional assays demonstrated that miR-384 inhibited GC cell proliferation, migration, and invasion through targeting MTDH.ConclusionThese results reveal that miR-384 acts as a tumor suppressor in GC and suggest that the miR-384/MTDH axis may be a potential therapeutic target for GC.

Asporin promotes cell proliferation via interacting with PSMD2 in gastric cancer.

Asporin (ASPN), a member of small leucine-rich repeat proteoglycan (SLRP) family of proteins, serves important roles in diverse biological responses and disease conditions. We tested the hypothesis that ASPN regulated proliferation of gastric cancer (GC) cells and identified its down-stream regulators. ASPN promoted the proliferation of GC cells. We identified the effector of this effect as proteasome 26S subunit non-ATPase 2 (PSMD2) which is known to regulate proliferation through suppression of DUSP7, WIP1 and PTEN and then inducing the phosphorylation of ERK, P38 and AKT. PSMD2 co-immunoprecipitated with ASPN from GC cell lysates and co-localized with PSMD2 inside GC cells. Moreover, knockdown of ASPN significantly increased the expression of DUSP7, WIP1 and PTEN and led to a repression in the phosphorylation of ERK, P38 and AKT. These changes were counteracted by knockdown of PSMD2. In conclusion, ASPN promotes cell proliferation by interacting with PSMD2, and down-regulation of its effectors and serves as a potential therapeutic target in GC.

MiR-1254 inhibits cell proliferation, migration, and invasion by down-regulating Smurf1 in gastric cancer

Gastric cancer (GC) is one of the most frequent malignancies, and increasing evidence supports the contribution of microRNA (miRNAs) to cancer progression. miR-1254 has been confirmed to participate in the regulation of various cancers, while the function of miR-1254 in GC remains unknown. In this study, we investigated the role of miR-1254 in GC. The expression of miR-1254 was detected in human GC specimens and cell lines by miRNA RT-PCR. The effects of miR-1254 on GC proliferation were determined by CCK-8 proliferation assays, colony formation assays, 5-ethynyl-2′-deoxyuridine (EdU) incorporation, and cell-cycle assays. The ability of migration and invasion was examined by transwell and wound-healing assay. Dual Luciferase reporter assay was used to validate the interaction of miR-1254 with its target gene. The xenograft mouse models were conducted to investigate the effects of miR-1254 in vivo. The signaling pathways and epithelial–mesenchymal transition (EMT)-related proteins were detected with western blot. The results showed that miR-1254 inhibited the proliferation, migration and invasion in vitro and suppressed tumorigenesis in vivo. Smurf1 was shown to be the direct target of miR-1254. Overexpressing Smurf1 could partially counteract the effects caused by miR-1254. Similarly, the effects of the miR-1254-inhibitor were also rescued by Smurf1-shRNA. Furthermore, we found that miR-1254 inhibited EMT and decreased the PI3K/AKT signaling pathway through downregulating Smurf1. In summary, overexpression of miR-1254 could suppress proliferation, migration, invasion, and EMT via PI3K/AKT signaling pathways by downregulation of Smurf1 in GC, which suggests a potential therapeutic target for GC.

In silico analyses for potential key genes associated with gastric cancer.

BackgroundUnderstanding hub genes involved in gastric cancer (GC) metastasis could lead to effective approaches to diagnose and treat cancer. In this study, we aim to identify the hub genes and investigate the underlying molecular mechanisms of GC.MethodsTo explore potential therapeutic targets for GC,three expression profiles (GSE54129, GSE33651, GSE81948) of the genes were extracted from the Gene Expression Omnibus (GEO) database. The GEO2R online tool was applied to screen out differentially expressed genes (DEGs) between GC and normal gastric samples. Database for Annotation, Visualization and Integrated Discovery was applied to perform Gene Ontology (GO) and KEGG pathway enrichment analysis. The protein-protein interaction (PPI) network of these DEGs was constructed using a STRING online software. The hub genes were identified by the CytoHubba plugin of Cytoscape software. Then, the prognostic value of these identified genes was verified by gastric cancer database derived from Kaplan-Meier plotter platform.ResultsA total of 85 overlapped upregulated genes and 44 downregulated genes were identified. The majority of the DEGs were enriched in extracellular matrix organization, endodermal cell differentiation, and endoderm formation. Moreover, five KEGG pathways were significantly enriched, including ECM-receptor interaction, amoebiasis, AGE-RAGE signaling pathway in diabetic complications, focal adhesion, protein digestion and absorption. By combining the results of PPI network and CytoHubba, a total of nine hub genes including COL1A1, THBS1, MMP2, CXCL8, FN1, TIMP1, SPARC, COL4A1, and ITGA5 were selected. The Kaplan-Meier plotter database confirmed that overexpression levels of these genes were associated with reduced overall survival, except for THBS1 and CXCL8.ConclusionsOur study suggests that COL1A1, MMP2, FN1, TIMP1, SPARC, COL4A1, and ITGA5 may be potential biomarkers and therapeutic targets for GC. Further study is needed to assess the effect of THBS1 and CXCL8 on GC.

Hsa_circ_0000467 promotes cancer progression and serves as a diagnostic and prognostic biomarker for gastric cancer.

Emerging evidence indicates that dysregulation of circular RNAs (circRNAs) is implicated in the development of malignancies. However, the diagnostic value and functional role of circRNAs in gastric cancer (GC) remain largely elusive. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to detect the expression of hsa_circ_0000467 in tissues, cell lines, and plasma. A receiver operating characteristic (ROC) curve was constructed to estimate the diagnostic value of hsa_circ_0000467. The association between the expression level of hsa_circ_0000467 and the clinicopathological features was analyzed. Moreover, cell functional assays were performed. Hsa_circ_0000467 was significantly upregulated in GC tissue compared to adjacent nontumor tissue (n=51, P<0.05). Similar results were detected in the HGC-27, MGC-803, AGS, NUGC-3, GES-1 cell lines (n=15, P<0.001), and in the plasma samples from GC patients (n=20, P<0.05). The area under the ROC curve of hsa_circ_0000467 was 0.790, which is superior to commonly used biomarkers including CEA and CA-724. We found that the expression levels of hsa_circ_0000467 in the same patient were significantly lower after surgery (n=20, P<0.05). Moreover, the hsa_circ_0000467 expression level is closely associated with TNM stage. Additionally, Cox multivariate analysis showed that hsa_circ_0000467 is a novel independent prognostic factor. Furthermore, in vitro experiments demonstrated that knockdown of hsa_circ_0000467 markedly inhibited the proliferation, migration, and invasion of GC cells. Moreover, hsa_circ_0000467 silencing increased tumor apoptosis in vitro. Hsa_circ_0000467 can act as a novel noninvasive biomarker for the diagnosis and prognosis of GC and may be a potential therapeutic target for GC.

Prognostic Value of Excision Repair Cross-Complementing mRNA Expression in Gastric Cancer.

Except for excision repair cross-complementing 1 (ERCC1), mRNA expression of the remaining ERCC genes has not been investigated in the prognosis of gastric cancer (GC). The present study aimed to explore the mRNA expression and prognostic values of each member of the ERCC family in GC patients by using the Kaplan–Meier (KM) plotter tool. The details of each ERCC family member were entered into a database and GC patients were separated into high and low expression to draw survival plots using the KM plotter. In the present study, we observed that high expression of ERCC1 mRNA was significantly associated with longer overall survival (OS) for all GC patients (hazard ratio [HR]=0.77, 95% confidence intervals [CI]=0.63–0.95, P=0.016) compared with low expression. High expression of ERCC4 and ERCC6 mRNA indicated a worse OS for all GC patients (HR=1.28, 95% CI=1.02–1.6, P=0.035 and HR=1.25, 95% CI=1.02–1.54, P=0.029, respectively) and especially for patients with intestinal-type GC (HR=1.87, 95% CI=1.26–2.79, P=0.0018 and HR=1.62, 95% CI=1.04–2.54, P=0.033, respectively). High ERCC8 mRNA expression indicated a worse OS for all GC patients (HR=1.34, 95% CI=1.02–1.76, P=0.034) and especially for patients with diffuse-type GC (HR=2.25, 95% CI=1.36–3.75, P=0.0013). In conclusion, our findings indicate that ERCC4, ERCC6, and ERCC8 may be potential biomarkers for GC prognosis and may serve as potential therapeutic targets for GC. However, these findings still need further verification.