Profiling of gene fusion involving targetable genes in Chinese gastric cancer.

BACKGROUND: Approximately half of all new cases of gastric cancer (GC) and related deaths occur in China. More than 80% of patients with GC are diagnosed at an advanced stage, which results in poor prognosis. Although HER2-directed therapy and immune checkpoint inhibitors have been somewhat successful, new drugs are still needed for the treatment of GC. Notably, several gene fusion-targeted drugs have been approved by the United States Food and Drug Administration for solid tumors, including GC, such as larotrectinib for NTRK fusion-positive cancers and zenocutuzumab for NRG1 fusion-positive cancers. However, gene fusions involving targetable genes have not been well characterized in Chinese patients with GC. AIM: To identify the profile of fusions involving targetable genes in Chinese patients with GC using clinical specimens and determine the distribution of patients with gene fusion variants among the molecular subtypes of GC. METHODS: We retrospectively analyzed gene fusion events in tumor tissue samples from 954 Chinese patients with GC. Clinicopathological characteristics were obtained from their medical records. Genetic alterations, such as single nucleotide variants, indels, amplifications, and gene fusions, were identified using a targeted sequencing panel containing 825 genes. Fusions were validated by fluorescence in situ hybridization (FISH) using break-apart probes. The microsatellite instability (MSI) status was evaluated using MSIsensor from the targeted sequencing panel data. Tumor mutational burden (TMB) was calculated using the total number of nonsynonymous mutations divided by the total genomic targeted region. Chi-square analysis was used to determine the enrichment of gene fusions associated with the molecular subtypes of GC. RESULTS: We found that 1.68% (16/954) of patients harbored 20 fusion events involving targetable genes. RARA fusions (n = 5) were the most common, followed by FGFR2, BRAF, MET, FGFR3, RET, ALK, EGFR, NTRK2, and NRG1 fusions. Two of the RARA fusions, EML4-ALK (E6:E20) and EGFR-SEPTIN14 (E7:E10), have been identified in other tumors but not in GC. Surprisingly, 18 gene fusion events were previously not reported in any cancer types. Twelve of the eighteen novel gene fusions included complete exons encoding functional domains of targetable genes, such as the tyrosine kinase domain of receptor tyrosine kinases and the DNA- and ligand-binding domains of RARA. Consistent with the results of detection using the targeted sequencing fusion panel, the results of FISH (fluorescence in situ hybridization) confirmed the rearrangement of FGFR2 and BRAF in tumors from patients 04 and 09, respectively. Genetic analysis indicated that the fusion genes were significantly enriched in patients with ERBB2 amplification (P = 0.02); however, there were no significant differences between fusion-positive and fusion-negative patients in age, sex, MSI status, and TMB. CONCLUSION: We characterized the landscape of fusions involving targetable genes in a Chinese GC cohort and found that 1.68% of patients with GC harbor potential targetable gene fusions, which were enriched in patients with ERBB2 amplification. Gene fusion detection may provide a potential treatment strategy for patients with GC with disease progression following standard therapy.

The role of NLRP3 inflammasome in digestive system malignancy.

Digestive system malignancies, the most common types of cancer and a major cause of death in the worldwide, are generally characterized by high morbidity, insidious symptoms and poor prognosis. NLRP3 inflammasome, the most studied inflammasome member, is considered to be crucial in tumorigenesis. In this paper, we reviewed its pro-tumorigenic and anti-tumorigenic properties in different types of digestive system malignancy depending on the types of cells, tissues and organs involved, which would provide promising avenue for exploring new anti-cancer therapies.

Sinomenine's protective role and mechanism in stress load-induced heart failure.

OBJECTIVES: This study is designed to investigate the effects and mechanisms of sinomenine (Sin) in stress load-induced heart failure in mice. METHODS: We used aortic constriction (AB) to cause pressure overload as our heart failure model. Sin was received in mice as the treatment group. Cardiac function and structural changes were detected using echocardiography. Heart-lung mass ratios were measured. The serum levels of IL-10 and IL-17 proteins were detected by using ELISA, cardiac hypertrophy markers atrial natriuretic peptide (ANP), myocardial I and III collagen mRNA levels were detected by RT-PCR. Myocardial type I and III collagen protein levels were detected by Western blotting. KEY FINDINGS: Sin significantly improved stress load-induced heart failure (P < 0.05), reduced the heart-lung mass ratio, ANP, collagen-I and -III mRNA and protein levels (P < 0.05); Sin can enhance the ratio of IL-10/IL-17. CONCLUSION: Sin may be a promising drug target to improve heart failure. Its role is related to reduce serum ANP levels, inhibit the mRNA and protein level of type I and III collagen and enhance the ratio of IL-10/IL-17.

PHD3 affects gastric cancer progression by negatively regulating HIF1A.

Prolyl hydroxylase 3 (PHD3) is widely accepted as a tumor suppressor; however, the expression of PHD3 in various cancer types remains controversial. The present study aimed to investigate the association between PHD3 expression and the clinicopathological features of gastric cancer using reverse transcription­quantitative polymerase chain reaction and immunohistochemistry. The effects of PHD3 in gastric cancer cell lines were assessed using western blot analysis and transwell migration assays. The present results revealed that PHD3 expression was increased in adjacent non­cancerous tissue compared with in gastric cancer tissue, and PHD3 overexpression was correlated with the presence of well­differentiated cancer cells, early cancer stage classification and the absence of lymph node metastasis. In vitro experiments demonstrated that PHD3 may act as a negative regulator of hypoxia­inducible factor­1α and vascular endothelial growth factor, both of which participate in tumor angiogenesis. In conclusion, the present results suggested that PHD3 may act as a tumor suppressor in gastric cancer. Therefore, the targeted regulation of PHD3 may have potential as a novel therapeutic approach for the treatment of patients with gastric cancer.

The CDH1 -160C/A polymorphism is associated with breast cancer: evidence from a meta-analysis.

BACKGROUND: Numerous epidemiological studies have evaluated the association between the CDH1 -160C/A polymorphism and the risk of breast cancers. However, these studies have yielded conflicting results. To derive a more precise estimation of this association, this meta-analysis was conducted. METHODS: A comprehensive search using the keywords "CDH1," "E-Cadherin," "polymorphism," "SNP," and "variant" combined with "breast," "cancer," "tumor," or "carcinomas" was conducted. Pooled odds ratios (ORs) with 95 % confidence intervals (CIs) were appropriately calculated using a fixed effect or random effect model. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2009 checklist was used for this meta-analysis. RESULTS: Four publications including five studies were identified. It was found that the CDH1 -160C/A polymorphism was significantly associated with breast cancer risk in the dominant model (CA + AA vs. CC: OR = 1.207, 95 % CI = 1.031-1.412, P = 0.019). CONCLUSIONS: Our meta-analysis demonstrated that the -160C/A polymorphism in the CDH1 gene might contribute to breast cancer susceptibility. Further investigations using a much larger sample including different ethnicities are still needed to verify this association.

MicroRNA-145-5p inhibits gastric cancer invasiveness through targeting N-cadherin and ZEB2 to suppress epithelial-mesenchymal transition.

MicroRNA (miR)-145-5p has been reported to function as a suppressor of cancer and plays an important role in cancer invasiveness. Epithelial-mesenchymal transition (EMT) is an important process in cancer invasion and migration. However, the involvement of miR-145-5p in EMT in human gastric cancer (GC) remains unclear. In this study, we aimed to investigate the molecular mechanisms by which miR-145-5p regulates EMT in GC invasiveness. We used quantitative real-time polymerase chain reaction to investigate the miR-145-5p expression level in GC and matched normal tissues. The effects of miR-145-5p on GC cell invasion and migration abilities were evaluated using Transwell models. The relationships among miR-145-5p and zinc-finger E-box binding homeobox 2 (ZEB2), E-cadherin, and N-cadherin were analyzed by quantitative real-time polymerase chain reaction and Western blot analyses. miR-145-5p levels in primary GC tissues obtained from 60 patients were significantly downregulated, compared to those in paired normal tissues. Lauren classification, depth of tumor invasion, lymph node metastasis, lymphatic invasion, and tumor-node-metastasis stage were associated with miR-145-5p expression. miR-145-5p inhibits the expression of the candidate target gene ZEB2 to delay the invasion and migration of GC cells. ZEB2 acts as transcriptional repressor of E-cadherin, while miR-145-5p is known to suppress N-cadherin directly to regulate EMT. Therefore, we concluded that miR-145-5p may target N-cadherin and ZEB2 directly to influence EMT.

Marsdeniae tenacissimae extract (MTE) suppresses cell proliferation by attenuating VEGF/VEGFR2 interactions and promotes apoptosis through regulating PKC pathway in human umbilical vein endothelial cells.

Marsdeniae tenacissimae extract (MTE), commonly known as Xiao-Ai-Ping in China, is a traditional Chinese herb medicine capable of inhibiting proliferation and metastasis and boosting apoptosis in various cancer cells. However, little is known about the contribution of MTE towards tumor angiogenesis and the underlying mechanism. The present study aimed to evaluate the effects of MTE on the proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) and the molecular mechanism. 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium, inner salt (MTS) and PI-stained flow cytometry assays revealed that MTE dose-dependently reduced the proliferation of HUVECs by arresting cell cycle at S phase (P < 0.05). Annexin V-FITC/PI-stained flow cytometry confirmed that MTE (160 µL·L-1) enhanced the apoptosis of HUVECs significantly (P < 0.001). Real-time quantitative RT-PCR and Western blot analyses showed an increase in Bax expression and a sharply decline in Bcl-2 expression; caspase-3 was activated simultaneously in a dose-dependent manner (P < 0.05). Further study observed the dose-dependent down-regulation of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2), P2Y6 receptor (P2Y6R), and chemokine (C-C motif) ligand 2 (CCL-2), along with the activation of PKC Δ and up-regulation of p53 in a dose-dependent manner in MTE-treated selected cells (P < 0.05). Collectively, the results from the present study suggested that MTE suppressed the proliferation by attenuating CCL-2-mediated VEGF/VEGFR2 interactions and promoted the apoptosis through PKCΔ-induced p53-dependent mitochondrial pathway in HUVECs, supporting that MTE may be developed as a potent anti-cancer medicine.

Prevalence of deleterious ATM germline mutations in gastric cancer patients.

Besides CDH1, few hereditary gastric cancer predisposition genes have been previously reported. In this study, we discovered two germline ATM mutations (p.Y1203fs and p.N1223S) in a Chinese family with a history of gastric cancer by screening 83 cancer susceptibility genes. Using a published exome sequencing dataset, we found deleterious germline mutations of ATM in 2.7% of 335 gastric cancer patients of different ethnic origins. The frequency of deleterious ATM mutations in gastric cancer patients is significantly higher than that in general population (p=0.0000435), suggesting an association of ATM mutations with gastric cancer predisposition. We also observed biallelic inactivation of ATM in tumors of two gastric cancer patients. Further evaluation of ATM mutations in hereditary gastric cancer will facilitate genetic testing and risk assessment.

Recurrent TERT promoter mutations identified in a large-scale study of multiple tumour types are associated with increased TERT expression and telomerase activation.

BACKGROUND: Several somatic mutation hotspots were recently identified in the telomerase reverse transcriptase (TERT) promoter region in human cancers. Large scale studies of these mutations in multiple tumour types are limited, in particular in Asian populations. This study aimed to: analyse TERT promoter mutations in multiple tumour types in a large Chinese patient cohort, investigate novel tumour types and assess the functional significance of the mutations. METHODS: TERT promoter mutation status was assessed by Sanger sequencing for 13 different tumour types and 799 tumour tissues from Chinese cancer patients. Thymic epithelial tumours, gastrointestinal leiomyoma, and gastric schwannoma were included, for which the TERT promoter has not been previously sequenced. Functional studies included TERT expression by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), telomerase activity by the telomeric repeat amplification protocol (TRAP) assay and promoter activity by the luciferase reporter assay. RESULTS: TERT promoter mutations were highly frequent in glioblastoma (83.9%), urothelial carcinoma (64.5%), oligodendroglioma (70.0%), medulloblastoma (33.3%) and hepatocellular carcinoma (31.4%). C228T and C250T were the most common mutations. In urothelial carcinoma, several novel rare mutations were identified. TERT promoter mutations were absent in gastrointestinal stromal tumour (GIST), thymic epithelial tumours, gastrointestinal leiomyoma, gastric schwannoma, cholangiocarcinoma, gastric and pancreatic cancer. TERT promoter mutations highly correlated with upregulated TERT mRNA expression and telomerase activity in adult gliomas. These mutations differentially enhanced the transcriptional activity of the TERT core promoter. CONCLUSIONS: TERT promoter mutations are frequent in multiple tumour types and have similar distributions in Chinese cancer patients. The functional significance of these mutations reflect the importance to telomere maintenance and hence tumourigenesis, making them potential therapeutic targets.

Sirtuin 1 Activator SRT1720 Protects Against Lung Injury via Reduction of Type II Alveolar Epithelial Cells Apoptosis in Emphysema.

In chronic obstructive pulmonary disease (COPD), two major pathological changes that occur are the loss of alveolar structure and airspace enlargement. Type II alveolar epithelial cells (AECII) play a vital role in maintaining alveolar homeostasis and lung tissue repair. Sirtuin 1 (SIRT1), a NAD(+)-dependent histone deacetylase, regulates many pathophysiological processes including inflammation, apoptosis, cellular senescence and stress resistance. The main aim of this study was to investigate whether SRT1720, a pharmacological SIRT1 activator, could protect against AECII apoptosis in rats with emphysema caused by cigarette smoke exposure and intratracheal lipopolysaccharide instillation in vivo. During the induction of emphysema in rats, administration of SRT1720 improved lung function including airway resistance and pulmonary dynamic compliance. SRT1720 treatment up-regulated the levels of surfactant protein (SP)A, SPC, SIRT1 and forkhead box O 3, increased SIRT1 activity, down-regulated the level of p53 and inhibited AECII apoptosis. Lung injury caused by emphysema was alleviated after SRT1720 treatment. SRT1720 could protect against AECII apoptosis in rats with emphysema and thus could be used in COPD treatment.

Assessing the diagnostic value of serum Dickkopf-related protein 1 levels in cancer detection: a case-control study and meta-analysis.

BACKGROUND: This study aimed to summarize the potential diagnostic value of serum DKK1 levels in cancer detection. MATERIALS AND METHODS: Serum DKK1 was measured using enzyme-linked immunosorbent assay in a case-control study. Then we performed a meta-analysis and the pooled sensitivity, specificity, diagnostic odds ratio, and summary receiver operating characteristic (sROC) curves were used to evaluate the overall test performance. RESULTS: Serum DKK1 levels were found to be significantly upregulated in gastric cancer as compared to controls. ROC curve analysis revealed an AUC of 0.636, indicating the test has the potential to diagnose cancer with poor accuracy. The summary estimates of the pooled sensitivity, specificity and diagnostic odds ratio in meta-analysis were 0.55 with a 95% confidence interval (CI) (0.53-0.57), 0.86 (95%CI, 0.84-0.88) and 12.25 (95%CI, 5.31-28.28), respectively. The area under the sROC was 0.85. Subgroup analysis revealed that the diagnostic accuracy of serum DKK1 in lung cancer (sensitivity: 0.69 with 95%CI, 0.66-0.74; specificity: 0.95 with 95%CI, 0.92-0.97; diagnostic odds ratio: 44.93 with 95%CI, 26.19-77.08) was significantly higher than for any other cancer. CONCLUSIONS: Serum DKK1 might be useful as a noninvasive method for confirmation of cancer diagnosis, particularly in the case of lung cancer.

Expression of galectin-1 in carcinoma-associated fibroblasts promotes gastric cancer cell invasion through upregulation of integrin ß1.

Increased expression of galectin-1 (Gal-1) in carcinoma-associated fibroblasts (CAFs) has been reported to correlate with progression and prognosis in many cancers. However, rarely have reports sought to determine whether high Gal-1 expression in CAFs in gastric cancer is involved in the tumor process, and the specific mechanism by which it promotes the evolution of gastric cancer is still unknown. In this study, we cultured gastric cancer CAFs, which showed strong expression of Gal-1, and established a co-culture system of CAFs with gastric cancer cells. Specific siRNA and in vitro migration and invasion assays were used to explore the effects of the interaction between Gal-1 expression of CAFs and gastric cancer cells on cell migration and invasion. We found that the overexpression of Gal-1 in CAFs enhanced gastric cancer cell migration and invasion, and these stimulatory effects could be blocked by specific siRNA which reduced the Gal-1 expression level. A set of cancer invasion-associated genes were then chosen to identify the possible mechanism of Gal-1-induced cell invasion. Among these genes, integrin ß1 expression in cancer cells was considered to be associated with Gal-1 expression. Pre-blocking of the integrin ß1 expression in gastric cancer cells with siRNA could interrupt the invasion-promoting effect of CAFs with high Gal-1 expression. Furthermore, immunohistochemical assay confirmed a positive correlation between Gal-1 and integrin ß1 expression. Our results showed that high expression of Gal-1 in CAFs might facilitate gastric cancer cell migration and invasion by upregulating integrin ß1 expression in gastric cancer.

Involvement of c-KIT mutation in the development of gastrointestinal stromal tumors through proliferation promotion and apoptosis inhibition.

The aim of this study was to discuss the role of c-KIT mutation in the pathogenesis of gastrointestinal stromal tumors (GISTs) and analyze its correlation with proliferation and apoptosis. c-KIT and PDGFRA genotypes were examined by deoxyribonucleic acid sequencing. Immunohistochemistry was performed to determine the expression levels of Kit, Ki-67 (proliferation marker), and apoptotic protease-activating factor (APAF)-1 (apoptosis marker) and the relationship between their three genes. In the 68 cases examined, 44 cases (64.7%) showed mutations in one of the four exons of c-KIT. The mutations were most frequently found in exon 11 (30 cases [44.1%]), followed by exon 9 (ten cases [14.7%]) and exon 13 (four cases [5.9%]). c-KIT mutation showed no association with prognostic factors using the classification of risk of aggressive behavior in GIST proposed by Fletcher et al. No cases had mutated exon 17 of c-KIT, and neither did exon 12, 14, or 18 of PDGFRA in our present study. There was a positive correlation between the expression level of Kit and Ki-67 (R=0.282, P=0.020). Conversely, a negative correlation was found between the expression levels of Kit and APAF1 (R=-0.243, P=0.046). In conclusion, most GISTs with Kit expression showed c-KIT mutation. Kit expression has a positive correlation with Ki-67 and a negative correlation with APAF1, showing that c-KIT is involved in GIST occurrence and development through proliferation promotion and apoptosis inhibition.

Up-regulated miR-199a-5p in gastric cancer functions as an oncogene and targets klotho.

BACKGROUND: Recent studies have shown that miR-199a-5p plays opposite roles in cancer initiation and progression of different cancer types, acting as oncogene for some cancer types but as tumor suppressor gene for others. However, the role and molecular mechanism of miR-199a-5p in gastric cancer are largely unknown. METHODS: In this study, miR-199a-5p expression level in gastric cancer was first analyzed by qPCRand then validated in 103 gastric cancer patients by in situ hybridization (ISH). Gastric cancer cell lines were transfected with miR-199a-5p inhibitor and mimic, and underwent in vitro transwell assays. Target genes (klotho) were identified using Luciferase reporter assay. Immunohistochemical staining was also used to investigate on how miR-199a-5p regulates the tumour-suppressive effects of klotho in gastric cancer. RESULTS: In our present study, we found that miR-199a-5p level was significantly increased in gastric cancer tissues compared to paired normal tissues. We observed that miR-199a-5p could promote migration and invasion of gastric cancer cells. In situ hybridization of miR-199a-5p also confirmed that higher miR-199a-5p expression level was associated with increased likelihood of lymph node metastasis and later TNM stage. Luciferase reporter assay and immunohistochemistry revealed that klotho might be the downstream target of miR-199a-5p. CONCLUSIONS: Our present study suggests that miR-199a-5p acts as an oncogene in gastric cancer and functions by targeting klotho.

Proliferative and apoptotic effects of andrographolide on the BGC-823 human gastric cancer cell line.

BACKGROUND: Andrographolide has been shown to have anticancer activity on diverse cancer cell lines representing different types of human cancers. The aim of this research was to investigate the anticancer and apoptotic effects of andrographolide on the BGC-823 human gastric cancer cell line. METHODS: Cell proliferation and IC50 were evaluated using MTT assay, cell-cycle analysis with flow cytometry apoptotic effects with Annexin-V/propidium iodide double-staining assay, and morphologic structure with transmission electron microscopy. Immunohistochemistry and reverse-transcription PCR was used to analyze Bcl-2, Bax, and caspase-3 expressions. RESULTS: Andrographolide showed a time- and concentration-dependent inhibitory effects on BGC-823 cell growth. Compared to controls, the number of cells in the G0-G1-phase increased significantly, S and G2-M-phase cells decreased after 48 hours of treatment with andrographolide, and both early and late apoptotic rates increased significantly compared to the controls, all in a concentration-dependent manner. Bax and caspase-3 expressions were markedly increased, and Bcl-2 expression was decreased. CONCLUSIONS: Andrographolide inhibits BGC-823 cell growth and induces BGC-823 cell apoptosis by up-regulating Bax and caspase-3 expressions and down-regulating Bcl-2 expression. Andrographolide may be useful as a potent and selective agent in the treatment of human gastric cancers.