Blocking EGR1/TGF-ß1 and CD44s/STAT3 Crosstalk Inhibits Peritoneal Metastasis of Gastric Cancer.

Peritoneal metastasis (PM) continues to limit the clinical efficacy of gastric cancer (GC). Early growth response 1 (EGR1) plays an important role in tumor cell proliferation, angiogenesis and invasion. However, the role of EGR1 derived from the tumor microenvironment in reshaping the phenotypes of GC cells and its specific molecular mechanisms in increasing the potential for PM are still unclear. In this study, we reported that EGR1 was significantly up-regulated in mesothelial cells from GC peritoneal metastases, leading to enhanced epithelial-mesenchymal transformation (EMT) and stemness phenotypes of GC cells under co-culture conditions. These phenotypes were achieved through the transcription and secretion of TGF-ß1 by EGR1 in mesothelial cells, which could regulate the expression and internalization of CD44s. After being internalized into the cytoplasm, CD44s interacted with STAT3 to promote STAT3 phosphorylation and activation, and induced EMT and stemness gene transcription, thus positively regulating the metastasis of GC cells. Moreover, TGF-ß1 secretion in the PM microenvironment was significantly increased compared with the matched primary tumor. The blocking effect of SHR-1701 on TGF-ß1 was verified by inhibiting peritoneal metastases in xenografts. Collectively, the interplay of EGR1/TGF-ß1/CD44s/STAT3 signaling between mesothelial cells and GC cells induces EMT and stemness phenotypes, offering potential as a therapeutic target for PM of GC.

Tumor-mesothelium HOXA11-PDGF BB/TGF ß1-miR-181a-5p-Egr1 feedforward amplifier circuity propels mesothelial fibrosis and peritoneal metastasis of gastric cancer.

A proportion of gastric cancer (GC) patients suffer from peritoneal metastasis (PM) in the late stage of tumor and these patients have a poor prognosis. To provide more care for GC patient with PM, a deeper exploration of the molecular characteristics of GC-PM is needed. Here we performed the in vitro and in vivo study to illustrate the effect of HOXA11 over-expressed GC cells on peritoneal mesothelial cells (HMrSV5), transcriptomics analyses of HMrSV5 cells co-cultured with HOXA11 over-expressed GC cells, counterparts or alone, cytokine array analyses of serum-free culture medium of HOXA11 over-expressed GC cells, we validated our findings through genetic manipulation of HMrSV5 cells and neutralizing antibodies targeting cytokines secreted by HOXA11 over-expressed GC cells in vitro, as well as utilized human peritoneal metastatic lesions to validate expression of potential targets. We identified that HOXA11 over-expressed GC cells strongly propelled mesothelial fibrosis in vivo and in vitro, and HOXA11 regulated paracrine and autocrine of PDGF BB and TGF ß1 in GC cells to propel mesothelial fibrosis. Meanwhile, HOXA11 over-expressed GC cells drove PDGF BB and TGF ß1 secretion to activate developmental-process related genes in HMrSV5 cells, including Egr1, which processes dependent on miR-181a-5p. Then, Egr1 could mediate peritoneal mesothelial fibrosis. Correspondingly, Egr1 over-expressed HMrSV5 cells supported migration and peritoneal dissemination of GC cells. Together our results suggest that a feedforward amplifier circuity governing GC cells and mesothelial cells in peritoneum contribute to peritoneal metastasis of GC cells.

VPS35 promotes gastric cancer progression through integrin/FAK/SRC signalling-mediated IL-6/STAT3 pathway activation in a YAP-dependent manner.

VPS35 is a key subunit of the retromer complex responsible for recognising cytosolic retrieval signals in cargo and is involved in neurodegenerative disease and tumour progression. However, the function and molecular mechanism of VPS35 in gastric cancer (GC) remains largely unknown. Here, we demonstrated that VPS35 was significantly upregulated in GC, which was associated with poor survival. VPS35 promoted GC cell proliferation and metastasis both in vitro and in vivo. Mechanistically, VPS35 activated FAK-SRC kinases through integrin-mediated outside-in signalling, leading to the activation of YAP and subsequent IL-6 expression induction in tumour cells. What's more, combined mass spectrometry analysis of MGC-803 cell and bioinformatic analysis, we found that phosphorylation of VPS35 was enhanced in GC cells, and phosphorylated VPS35 has enhanced interaction with ITGB3. VPS35 interacted with ITGB3 and affected the recycling of ITGB3 in GC cells. Gain- and loss-of-function experiments revealed that VPS35 promoted tumour proliferation and metastasis via the IL-6/STAT3 pathway. Interestingly, we also found that STAT3 directly bound to the VPS35 promoter and increased VPS35 transcription, thereby establishing a positive regulatory feedback loop. In addition, we demonstrated that VPS35 knockdown sensitised GC cells to 5-FU and cisplatin. These findings provide evidence that VPS35 promotes tumour proliferation and metastasis, and highlight the potential of targeting VPS35- and IL-6/STAT3-mediated tumour interactions as promising therapeutic strategies for GC.

Paracrine activin B-NF-κB signaling shapes an inflammatory tumor microenvironment in gastric cancer via fibroblast reprogramming.

BACKGROUND: Important roles of INHBB in various malignancies are increasingly identified. The underlying mechanisms in gastric cancer (GC) microenvironment are still greatly unexplored. METHODS: The clinical significance of INHBB and the correlation between INHBB and p-p65 in GC were assessed through analyzing publicly available databases and human paraffin embedded GC tissues. The biological crosstalk of INHBB between GC cells and fibroblasts was explored both in vitro and in vivo. RNA-seq analyses were performed to determine the mechanisms which regulating fibroblasts reprogramming. Luciferase reporter assay and chromatin immunoprecipitation (CHIP) assay were used to verify the binding relationship of p65 and INHBB in GC cells. RESULTS: Our study showed that INHBB level was significantly higher in GC, and that increased INHBB was associated with poor survival. INHBB positively regulates the proliferation, migration, and invasion of GC cells in vitro. Also, activin B promotes the occurrence of GC by reprogramming fibroblasts into cancer-associated fibroblasts (CAFs). The high expression of INHBB in GC cells activates the NF-κB pathway of normal gastric fibroblasts by secreting activin B, and promotes fibroblasts proliferation, migration, and invasion. In addition, activin B activates NF-κB pathway by controlling TRAF6 autoubiquitination to induce TAK1 phosphorylation in fibroblasts. Fibroblasts activated by activin B can induce the activation of p65 phosphorylation of GC cells by releasing pro-inflammatory factors IL-1ß. p65 can directly bind to the INHBB promoter and increase the INHBB transcription of GC cells, thus establishing a positive regulatory feedback loop to promote the progression of GC. CONCLUSIONS: GC cells p65/INHBB/activin B and fibroblasts p65/IL-1ß signal loop led to the formation of a whole tumor-promoting inflammatory microenvironment, which might be a promising therapeutic target for GC.

Neratinib for HER2-positive breast cancer with an overlooked option.

Positive human epidermal growth factor receptor 2 (HER2) expression is associated with an increased risk of metastases especially those to the brain in patients with advanced breast cancer (BC). Neratinib as a tyrosine kinase inhibitor can prevent the transduction of HER1, HER2 and HER4 signaling pathways thus playing an anticancer effect. Moreover, neratinib has a certain efficacy to reverse drug resistance in patients with BC with previous HER2 monoclonal antibody or targeted drug resistance. Neratinib, as monotherapy and in combination with other therapies, has been tested in the neoadjuvant, adjuvant, and metastatic settings. Neratinib with high anticancer activity is indicated for the prolonged adjuvant treatment of HER2-positive early BC, or in combination with other drugs including trastuzumab, capecitabine, and paclitaxel for the treatment of advanced HER2-positive BC especially cancers with central nervous system (CNS) metastasis to reduce the risk of BC recurrence. This article reviewed the pharmacological profiles, efficacy, safety, tolerability, and current clinical trials pertaining to neratinib, with a particular focus on the use of neratinib in patients with metastatic breast cancer (MBC) involving the CNS. We further discussed the use of neratinib for HER2-negative and HER2-mutant breast cancers, and mechanisms of resistance to neratinib. The current evidence suggests that neratinib has promising efficacy in patients with BC which is at least non-inferior compared to previous therapeutic regimens. The most common AE was diarrhea, and the incidence, severity and duration of neratinib-related grade 3 diarrhea can be reduced with loperamide. Of note, neratinib has the potential to effectively control and prevent brain metastasis in patients with advanced BC, providing a therapeutic strategy for HER2-positive BC.

Plasma Exosome Proteins ILK1 and CD14 Correlated with Organ-Specific Metastasis in Advanced Gastric Cancer Patients.

The exosome plays important roles in driving tumor metastasis, while the role of exosome proteins during organ-specific metastasis in gastric cancer has not been fully understood. To address this question, peripheral blood samples from 12 AGC patients with organ-specific metastasis, including distant lymphatic, hepatic and peritoneal metastasis, were collected to purify exosomes and to detect exosome proteins by Nano-HPLC-MS/MS. Gastric cancer cell lines were used for in vitro experiments. Peripheral blood sample and ascites sample from one patient were further analyzed by single-cell RNA sequencing. GO and KEGG enrichment analysis showed different expression proteins of hepatic metastasis were correlated with lipid metabolism. For peritoneal metastasis, actin cytoskeleton regulation and glycolysis/gluconeogenesis could be enriched. ILK1 and CD14 were correlated with hepatic and peritoneal metastasis, respectively. Overexpression of CD14 and ILK1 impacted the colony formation ability of gastric cancer and increased expression of Vimentin. CD14 derived from immune cells in malignant ascites correlated with high activation of chemokine- and cytokine-mediated signaling pathways. In summary, biological functions of plasma exosome proteins among AGC patients with different metastatic modes were distinct, in which ILK1 and CD14 were correlated with organ-specific metastasis.

Circulating neutrophils activated by cancer cells and M2 macrophages promote gastric cancer progression during PD-1 antibody-based immunotherapy.

Aims: To analyze the correlation between the neutrophil-to-lymphocyte ratio (NLR) and prognosis of advanced gastric cancer (AGC) patients treated by PD-1 antibody-based therapy and to delineate molecular characteristics of circulating neutrophils by single-cell RNA sequencing (scRNA-seq). Methods: The clinicopathological information of 45 AGC patients receiving PD-1 antibody-based regimens at the Department of Oncology, Ruijin Hospital, was reviewed. Treatment outcomes including objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) were recorded. The correlation between NLR and efficacy of PD-1 antibody-based treatment was analyzed. Single-cell RNA sequencing (scRNA-seq) analysis was performed based on multisite biopsy samples from two AGC patients to explore the molecular characteristics of circulating neutrophils and their pro-tumor mechanisms. Tissue samples from 88 gastric cancer patients who underwent radial gastrectomy were collected for immunochemistry staining. Results: A high posttreatment NLR was associated with poor outcomes of AGC patients receiving PD-1 antibody-based regimens. scRNA-seq analysis showed that an increased number of circulating neutrophils were found in peripheral blood samples after treatment in which neutrophil cluster 1 (NE-1) was the major subcluster. NE-1 was featured with a neutrophil activation phenotype with the high expression of MMP9, S100A8, S100A9, PORK2, and TGF-ß1. NE-1 displayed an intermediate state in pseudotime trajectory analysis with gene function enrichment found in neutrophil activation, leukocyte chemotaxis, and negative regulation of MAP kinase activity. Cellular interaction analysis showed that the chemokine signaling pathway is the major interactional pathway of NE-1 between subclusters of malignant epithelial cells (EP-4) and M2 macrophages (M2-1 and M2-2). In turn, the MAPK signaling pathway and Jak-STAT signaling pathway of EP-4, including IL1B/IL1RAP, OSM/OSMR, and TGFB1/TGFBR2 axes, were identified as interacting pathways between EP-4 and NE-1. The high expression of OSMR in tumor cells was closely correlated with lymph node metastasis of gastric cancer. Conclusion: The posttreatment NLR could be a poor prognostic marker of AGC patients treated with immune checkpoint inhibitors (ICIs). Subclusters of circulating neutrophils activated by tumor cells and M2 macrophages could participate in gastric cancer progression through signaling interactions with tumor cells.

HNF4A-BAP31-VDAC1 axis synchronously regulates cell proliferation and ferroptosis in gastric cancer.

B cell receptor associated protein 31 (BAP31) is closely associated with tumor progression, while the role and mechanism of BAP31 in gastric cancer (GC) remains unknown. This study explored that BAP31 was upregulated in GC tissues and high expression indicated poor survival of GC patients. BAP31 knockdown inhibited cell growth and induced G1/S arrest. Moreover, BAP31 attenuation increased the lipid peroxidation level of the membrane and facilitated cellular ferroptosis. Mechanistically, BAP31 regulated cell proliferation and ferroptosis by directly binding to VDAC1 and affected VDAC1 oligomerization and polyubiquitination. HNF4A was bound to BAP31 at the promoter and increased its transcription. Furthermore, knockdown of BAP31 inclined to make GC cells vulnerable to 5-FU and ferroptosis inducer, erastin, in vivo and in vitro. Our work suggests that BAP31 may serve as prognostic factor for gastric cancer and act as potential therapeutic strategy for gastric cancer.

Camrelizumab and metronomic capecitabine for patients with treatment-refractory solid tumors (McCREST trial).

This is an open-label, single-center, multi-cohort phase Ib trial, which consists of three cohorts, including cohort 1 (HER2 negative gastric or gastric esophageal junction adenocarcinoma), cohort 2 (esophageal squamous cell carcinoma and head and neck squamous cell carcinoma) and cohort 3 (hepato-biliary-pancreatic and non-stomach non-esophagi gastrointestinal carcinoma). All eligible patients will be treated by camrelizumab (200 mg, every 2 weeks) and capecitabine (500 mg, twice a day, per os). The primary end point is the safety profiles of camrelizumab plus metronomic capecitabine according to CTCAE v5.0. The secondary end points are progression free survival, overall survival, objective response rate, disease control rate and duration of response. Planned enrollment is 20 subjects for each cohort. Total duration of this trial is expected to be 2 years.

Immune checkpoint inhibitors (ICIs) such as PD-1 inhibitors have been used to treat gastrointestinal cancer patients in clinical practices. Combination with other drugs can improve the efficacy of ICIs. Metronomic chemotherapy using low dose and high frequency of cytotoxic drugs has multi-targeted anti-tumor effects and can be a potential partner of ICIs. In this study, the authors assess the safety and efficacy of a combination of a PD-1 inhibitor (camrelizumab) and an oral chemotherapy drug (capecitabine with metronomic dose) in patients with metastatic treatment-refractory solid tumors.

CRISPR/Cas9-mediated knockout of SGLT1 inhibits proliferation and alters metabolism of gastric cancer cells.

BACKGROUND: The roles played by sodium/glucose cotransporters 1 (SGLT1) that transport glucose in cells independent of extracellular glucose concentration in gastric cancer are unknown. METHODS: The expression of SGLT1 in 75 primary gastric cancer and paired adjacent normal tissue specimens was determined. Also, the underlying mechanism of the altered SGLT1 expression and its impact on the proliferation of the gastric cancer cells and their metabolism were investigated. RESULTS: SGLT1 expression was found to be positively associated with pT, pN, TNM staging, histological differentiation, and a worse overall survival. CRISPR/Cas9 mediated knockout of SGLT1 could inhibit proliferation of gastric cancer cells, promote their apoptosis, and could also alter the metabolism of gastric cancer cells. Mechanistically, the transcription activity of SGLT1 could be negatively regulated by p53. CONCLUSIONS: Besides identifying the important role of SGLT1 in gastric cancer, the underlying regulation mechanism in play was also elucidated. These make SGLT1 a promising new molecular target for the design of novel therapeutic modalities to control gastric cancer.

Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis.

Background: The AT-rich interaction domain 1A (ARID1A) is thought to be a tumor suppressive gene, and most of its mutations result in loss of expression of ARID1A protein. Combined with SIRPα on the surface of macrophages, CD47 on the surface of cancer cells can send an antiphagocytic "Don't eat me" signal to the immune system that helps to avoid immune surveillance. However, the relationship between ARID1A and CD47 expression and their prognostic value in gastric cancer (GC) are still unknown. Methods: In this study, we evaluated ARID1A and CD47 expression in 154 GC patients' tissues using tissue microarray. Expressions of ARID1A and CD47 in GC cell lines were determined by western blot and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) techniques, and cell membranous CD47 expression was quantified by flow cytometry. In addition, chromatin immunoprecipitation (ChIP)-qPCR was used to determine the aspects of regulation of CD47 by ARID1A. The proportions of tumor-infiltrating immune cells were estimated on The Cancer Genome Atlas (TCGA) data set by using quanTIseq and EPIC algorithms. The infiltration of M1-polarized macrophages, M2-polarized macrophages, and regulatory T cells (Tregs) in GC tissues was determined by multispectral immunofluorescence. Results: A significant correlation was found between loss of ARID1A and high expression of CD47 at protein level in GC. By integrating 375 bulk RNA sequencing samples from TCGA data set, we found that mutated ARID1A correlated with high CD47 expression. In GC cell lines, knockdown of ARID1A significantly increased CD47 expression both at protein and mRNA levels as measured by western blot, qRT-PCR, and flow cytometry. Moreover, ChIP-qPCR revealed that CD47 was a direct downstream target gene of ARID1A in GC. Utilizing univariate and multivariate survival analyses, we found that patients with ARID1AlossCD47high expression had a worse prognosis. Estimation of infiltrating immune cells on TCGA data set showed that a higher infiltration proportion of M2 macrophages and Tregs was found in ARID1A mutated CD47 high expression subgroup. Furthermore, application of multispectral immunofluorescence revealed a higher infiltration proportion of M2 macrophages and Tregs in ARID1AlossCD47high GC tissues. Conclusion: Loss of ARID1A is strongly correlated with high CD47 expression in GC, and combination of ARID1A and CD47 is a promising prognosis factor in GC.

The prognostic value of HGF-c-MET signaling pathway in Gastric Cancer: a study based on TCGA and GEO databases.

Gastric cancer is a heterogeneous tumor that underlying molecular mechanisms are largely unclear. This study aimed to elucidate the expression level of HGF-c-MET in gastric cancer patients and to investigate the prognostic and diagnostic value of HGF-c-MET. In silico analysis of the TCGA and GEO database found that HGF and c-MET mRNA expression are significantly higher in gastric cancer tissues than those in peritumor tissues. Both higher mRNA expression of HGF and c-MET were associated with a poorer prognosis. c-MET expression was modulated by methylation in the promoter regions. HGF was positively correlated with CD8+ T cell, CD4+ T cell, macrophage, neutrophil and dendritic cell. Furthermore, functional enrichment analysis and protein-protein interaction networks further shown that HGF-c-MET and related proteins mainly participated in growth factor receptor binding, protein tyrosine kinase activity and signaling receptor binding. Finally, outcome of GSEA analysis showed 13 shared KEGG pathways enriched in high expressed group of HGF and c-MET.

Stearoyl-CoA desaturase 1 (SCD1) facilitates the growth and anti-ferroptosis of gastric cancer cells and predicts poor prognosis of gastric cancer.

Cancer cells are characterized by metabolic alterations. Thereinto, Stearoyl-CoA Desaturase 1 (SCD1), an enzymatic node located in the conversion of saturated fatty acids into monounsaturated fatty acids (MUFAs), has been reported to accelerate the tumorigenesis of multiple cancers. However, its role in the metabolic process of gastric cancer remains largely unexplored. In this study, by in vitro, in vivo and in silico assessments, our results revealed that SCD1 exhibited the ability to promote tumor growth, migration and anti-ferroptosis of gastric cancer. The underlying mechanism might involve the alteration of cancer stemness and modulation of cell cycle-related proteins. Moreover, based on our findings, high expression of SCD1 might predict poor prognosis in gastric cancer patients. Our study provided new insights into the potential of SCD1 as a biomarker as well as a therapeutic target in the treatment of gastric cancer.

Low expression of transferrin receptor 2 predict poor prognosis in gastric cancer patients.

This study was to detect the expression level of transferrin receptor 2 (TfR2) in gastric cancer and to analyze its value in predicting the prognosis of gastric cancer patients. Real-time PCR (RT-qPCR) was applied to detect the mRNA expression of TfR2 in gastric cancer tissues and paired adjacent nontumorous tissues. Immunohistochemistry (IHC) and western blotting were used to determine the protein expression of TfR2 in gastric cancer, and the relationship with the prognosis of gastric cancer patients was analyzed. The results were: (1) The mRNA and protein levels of TfR2 in gastric cancer tissues were remarkably lower than those in adjacent nontumorous gastric tissue (P < .05). (2) Clinicopathological analysis showed that the expression level of TfR2 was significantly correlated with TNM stage of patients (P = .047). (3) The results of univariate survival analysis showed that histological grade, T stage, N stage, M stage, Lauren's classification, and TfR2 expression level influenced the overall survival of gastric cancer patients. Multivariate survival analysis showed that low TfR2 expression (HR = 1.671, 95%CI: 1.006-2.774, P = .047), poor differentiation (HR = 2.123, 95%CI: 1.188-3.795, P = .011), and M1 stage (HR = 8.541, 95%CI: 3.416-21.353, P < .001) were independent risk factors affecting the overall survival of gastric cancer patients. In conclusion, TfR2 exhibited low expression in gastric cancer tissue, and is an independent prognostic factor of gastric cancer patients.

A self-enforcing HOXA11/Stat3 feedback loop promotes stemness properties and peritoneal metastasis in gastric cancer cells.

Rationale: Peritoneal metastasis is one of the most common and life-threatening metastases in gastric cancer patients. The disseminated gastric cancer cells forming peritoneal metastasis exhibit a variety of characteristics that contrast with those of adjacent epithelial cell of gastric mucosa and even primary gastric cancer cells. We hypothesized that the gene expression profiles of peritoneal foci could reveal the identities of genes that might function as metastatic activator. Methods: In this study, we show, using in vitro, in vivo, in silico and gastric cancer tissues studies in humans and mice, that Homoebox A11 (HOXA11) potently promote peritoneal metastasis of gastric cancer cells. Results: Its mechanism of action involves alternation of cancer stemness and subsequently enhancement of the adhesion, migration and invasion and anti-apoptosis. This is achieved, mainly, through formation of a positive feedback loop between HOXA11 and Stat3, which is involved in the stimulation of Stat3 signaling pathway. Conclusions: These observations uncover a novel peritoneal metastatic activator and demonstrate the association between HOXA11, Stat3 and cancer stemness of gastric cancer cells, thereby revealing a previously undescribed mechanism of peritoneal metastasis.

Expression pattern of CDK12 protein in gastric cancer and its positive correlation with CD8+ cell density and CCL12 expression.

Background: The aims of this study were to investigate the expression pattern of CDK12 protein in gastric cancer, and to analyze the correlations of CDK12 expression between CD8+ cell density and CCL12 expression. Methods: Eighty-six paired tumor and non-tumor samples were collected from patients who underwent radical surgery and had pathological confirmed gastric adenocarcinoma. Immunohistochemistry was used to assess CDK12 expression and CD8+ cell density. Expression of CDK12 and CCL21 mRNA was detected by quantitative reverse transcription-polymerase chain reaction. Results: CDK12 expression in gastric tumor tissues was significantly higher than it in paired non-tumor tissues (P<0.001). High expression of CDK12 was identified in 43 cases (50%), and it was significantly correlated with Lauren's classification (diffuse type) and number of metastatic lymph nodes (≥15). High CDK12 protein level indicated a relative poorer overall survival than patients with CKD12 low expression, while it was not identified as an independent prognostic factor. Median number of CD8+ cells in tumor tissues was 51 (range: 0-292). Number of CD8+ cells was positively correlated with CDK12 expression score in tumor tissues (r=0.243, P=0.024). Positive correlation was also found between CDK12 and CCL21 mRNA expression (r=0.419, P=0.017). Conclusion: High CDK12 expression was detected in gastric cancer which was correlated with malignant phenotypes and worse outcome. Positive correlations of CD8+ cell number and CCL21 mRNA expression with CDK12 level were identified.

High promoter activity of cytokeratin-19 gene in cholangiocarcinoma.

Conditionally replicating adenoviral vectors constructed with tumor-specific promoters (TSPs) offers a viable tool for the treatment of cholangiocarcinoma. The aim of the present study was to investigate cholangiocarcinoma-specific TSPs that remain active in adenoviral constructs in gene therapy. The mRNA expressions of cyclooxygenase-2, cytokeratin-19 (CK19), mucin-1, midkine and telomerase reverse transcriptase were determined in human cholangiocarcinoma cell lines, primary human hepatocytes and cholangiocytes using reverse transcription-quantitative polymerase chain reaction. The candidate promoters constructed in adenoviral vectors were analyzed for their activities in cholangiocarcinoma cell lines, primary human hepatocytes and cholangiocytes using dual-luciferase reporter assays. The mRNA expression of CK19 was markedly higher in the QBC939 cell line, indicating specificity to cholangiocarcinoma. Moreover, the promoter activity of CK19 in the adenoviral vector in infected cholangiocarcinoma cells was found to be significantly stronger compared with that in infected primary human hepatocytes and cholangiocytes. CK19 may be implicated in the pathogenesis of cholangiocarcinoma, as demonstrated by the stronger activity of its promoter, as well as the higher expression of mRNA in tumor cells. Therefore, the use of the promoter sequence of the CK19 gene may represent a potential tool in cholangiocarcinoma-specific adenoviral gene therapy.

HGF-mediated crosstalk between cancer-associated fibroblasts and MET-unamplified gastric cancer cells activates coordinated tumorigenesis and metastasis.

Cancer-associated fibroblasts (CAFs) are important components of tumor stroma and play a key role in tumor progression. CAFs involve in crosstalk with tumor cells through various kinds of cytokines. In the present study, we screened hepatocyte growth factor (HGF) as a cytokine predominantly originating from CAFs. CAFs-derived HGF was found to promote MET-unamplified gastric cancer (GC) proliferation, migration, and invasion through the activation of HGF/c-Met/STAT3/twist1 pathway. It also activated interleukin (IL)-6/IL-6R/JAK2/STAT3/twist1 pathway by up-regulating IL-6R expression. As IL-6 was also found to upregulate c-Met expression, we identified the cooperation of HGF and IL-6 in enhancing the characteristics of CAFs. In vivo experiments revealed that CAFs-derived HGF promoted tumorigenesis and metastasis of MET-unamplified GC. Gene set enrichment analysis (GSEA) was performed to confirm our findings. Our study found that the increased expression of HGF in CAFs induced by MET-unamplified GC contributed to the malignant phenotype of both MET-unamplified GC and CAFs in tumor microenvironment.

HGF derived from cancer­associated fibroblasts promotes vascularization in gastric cancer via PI3K/AKT and ERK1/2 signaling.

Cancer­associated fibroblasts (CAFs) are predominate cells in tumor stroma and play a key role in tumor progression. Hepatocyte growth factor (HGF) is a cytokine mainly derived from fibroblasts. In the present study, we reported that HGF significantly promoted angiogenesis of human umbilical vein endothelial cells (HUVECs) and vasculogenic mimicry (VM) formation of gastric cancer cells, respectively, by increasing cell proliferation and migration. In addition, mosaic vessels formed by HUVECs and gastric cancer cells were also increased with treatment of recombinant human HGF and conditioned medium from CAFs. The opposite results were achieved in HGF­neutralized groups. In accordance with these observations, we determined that phosphorylation of AKT and ERK1/2 were upregulated in HUVECs and gastric cancer cells with HGF treatment and co­culture with CAFs. Both AKT inhibitor LY294002 and ERK1/2 inhibitor U0126 reduced the ability of angiogenesis and VM formation, as well as mosaic vessel formation induced by HGF. Gene Set Enrichment Analysis and correlation analysis were performed to confirm our findings. In conclusion, CAF­derived HGF promotes angiogenesis, VM and mosaic vessel formation via PI3K/AKT and ERK1/2 signaling in gastric cancer and HGF may serve as a potential therapeutic target for cancer anti­vascular treatment.

Comparison of Different Dosages of Rocuronium on Abdominal Wound Closure / 昆明医科大学学报

Objective To evaluate the effects of different dosages of rocuronium on abdominal wound closure under general anesthesia.Methods Ninety ASA class I or II patients scheduled for elective abdominal surgery under general anesthesia were randomized into three groups with 30 cases each.Each group received rocuronium 1 time of ED95 (0.3 mg/kg),0.5 time of ED95 (0.15 mg/kg),0.2 time of ED95 (0.06 mg/kg) respectively when closing the abdominal wall.TOF recovered to 25%, 75%, 90%, time of eye-opening on command, time of head-lift for more than 5 seconds,extubation time, the rate of upper airways obstruction after extubation and the satisfaction score for the muscle relaxation to general surgeons were recorded.Results TOF values had no significantly different among the three groups before abdominal wound closure (P＞0.05).Compared with group A and group B,time of TOF in group C was significangtly increased after administration of rocuronium (P＜0.05) . Time of TOF recovery to 25%, 75%, 90%in group C were significangtly decreased (P＜0.01).Compared with group A and group B, time of eye-opening on command ,head-lift for more than 5 seconds and extubation time were significantly shorter in group C.There were 8,4,0 patients undwent upper airways obstruction among each group respectively after extubation (P＜0.05).The satisfaction score for muscle relaxation among three groups had no significant difference.Conclusion An additional administration of 0.2 time of ED95 (0.06 mg/kg) rocuronium before abdominal wound closure in abdominal surgery can achieve satisfactory muscle relaxation for surgeons.