Anlotinib Attenuates Liver Fibrosis by Regulating the Transforming Growth Factor ß1/Smad3 Signaling Pathway.

BACKGROUND: Hepatic stellate cell hyperactivation is a central link in liver fibrosis development, transforming growth factor ß1 (TGF-ß1) is a key activator of HSCs. AIMS: This study investigated whether anlotinib attenuates CCl4 induced liver fibrosis in mice and explored its antifibrotic mechanism. METHODS: We used the human hepatic stellate cell line LX-2 for in vitro assays and used TGF-ß1 to induce hepatic fibrosis in LX-2 cells. We analyzed cytotoxicity using a cell-counting kit-8 and transwell chambers to detect the migratory ability of LX-2 cells. Western blotting was used to detect the protein levels of collagen type I, α-smooth muscle actin, and p-Smad3. In addition, mice with CCl4-induced hepatic fibrosis were used as in vivo models. Histopathological examination was performed using H&E staining, Masson's trichrome staining, and immunohistochemistry. RESULTS: Anlotinib significantly reversed TGF-ß1-induced protein levels of Col I, α-SMA and p-Smad3 and inhibits migratory and proliferative abilities in vitro using LX-2 cells. CCl4 cause F4 grade (Ishak) hepatic fibrosis, liver inflammatory scores ranged from 12 to 14 (Ishak), a mean ALT measurement of 130 U/L and a mean measurement AST value of 119 U/L in mice. However, the CCl4-induced changes were markedly attenuated by anlotinib treatment, which returned to F2 grade (Ishak) hepatic fibrosis, liver inflammatory scores ranged from 4 to 6 (Ishak), a mean ALT measurement of 40 U/L and a mean measurement AST value of 56 U/L in mice. CONCLUSIONS: Our results suggest that anlotinib-mediated suppression of liver fibrosis is related to the inhibition of TGF-ß1 signaling pathway. Hepatic stellate cell hyper activation is a central link in liver fibrosis development, transforming growth factor ß1 is a key activator of HSCs. Anlotinib is a multi-targeted tyrosine kinase inhibitor that has similar targets to nintedanib, a clinically used anti-pulmonary fibrosis drug. Our study demonstrates an FDA-approved drug-anlotinib-that could prevent liver fibrosis and inflammation. Experiments in cell cultures and mice show that anlotinib can inhibit the activation of hepatic stellate cells by down-regulating the TGFß1/smad3 pathway, thereby reversing liver fibrosis. In animal experiments, anlotinib showed protective effects on the CCl4-induced liver damage, including ameliorating liver inflammation, reversing liver fibrosis and reducing liver enzymes. This is a very good signal, anlotinib may be useful for halting or reversing the progression of liver fibrosis and could be employed in the development of novel therapeutic drugs for the management of chronic liver diseases.

Transthoracic, thoracoabdominal, and transabdominal surgical approaches for gastric cardia adenocarcinomas: a survival evaluation based on a cohort of 7103 patients.

BACKGROUND: This study compared the survival outcomes of different surgical approaches to determine the optimal approach for gastric cardia adenocarcinoma (GCA) and aimed to standardize the surgical treatment guidelines for GCA. METHODS: A total of 7103 patients with GCA were enrolled from our previously established gastric cardia and esophageal carcinoma databases. In our database, when the epicenter of the tumor was at or within 2 cm distally from the esophagogastric junction, the adenocarcinoma was considered to originate from the cardia and was considered a Siewert type 2 cancer. The main criteria for the enrolled patients included treatment with radical surgery, no radio- or chemotherapy before the operation, and detailed clinicopathological information. Follow-up was mainly performed by telephone or through home interviews. According to the medical records, the surgical approaches included transthoracic, thoracoabdominal, and transabdominal approaches. Kaplan-Meier and Cox proportional hazards regression models were applied to correlate the surgical approach with survival in patients with GCA. RESULTS: There were marked differences in age and tumor stage among the patients who underwent the three surgical approaches (P < 0.001). Univariate analysis showed that survival was related to sex, age, tumor stage, and N stage (P < 0.001 for all). Cox regression model analysis revealed that thoracoabdominal approach (P < 0.001) and transabdominal approach (P < 0.001) were significant risk factors for poor survival. GCA patients treated with the transthoracic approach had the best survival (5-year survival rate of 53.7%), and survival varied among the different surgical approaches for different tumor stages. CONCLUSION: Thoracoabdominal approach and transabdominal approach were shown to be poor prognostic factors. Patients with (locally advanced) GCA may benefit from the transthoracic approach. Further prospective randomized clinical trials are necessary.

A novel tree shrew model of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective therapy. Animal models effectively reproducing IPF disease features are needed to study the underlying molecular mechanisms. Tree shrews are genetically, anatomically, and metabolically closer to humans than rodents or dogs; therefore, the tree shrew model presents a unique opportunity for translational research in lung fibrosis. Here we demonstrate that tree shrews have in vivo and in vitro fibrotic responses induced by bleomycin and pro-fibrotic mediators. Bleomycin exposure induced lung fibrosis evidenced by histological and biochemical fibrotic changes. In primary tree shrew lung fibroblasts, transforming growth factor beta-1 (TGF-ß1) induced myofibroblast differentiation, increased extracellular matrix (ECM) protein production, and focal adhesion kinase (FAK) activation. Tree shrew lung fibroblasts showed enhanced migration and increased matrix invasion in response to platelet derived growth factor BB (PDGF-BB). Inhibition of FAK significantly attenuated pro-fibrotic responses in lung fibroblasts. The data demonstrate that tree shrews have in vivo and in vitro fibrotic responses similar to that observed in IPF. The data, for the first time, support that the tree shrew model of lung fibrosis is a new and promising experimental animal model for studying the pathophysiology and therapeutics of lung fibrosis.

BRCA2 loss-of-function germline mutations are associated with esophageal squamous cell carcinoma risk in Chinese.

Esophageal squamous cell carcinoma (ESCC) occurs with highest frequency in China with over 90% mortality, highlighting the need for early detection and improved treatment strategies. We aimed to identify ESCC cancer predisposition gene(s). Our study included 4,517 individuals. The discovery phase using whole-exome sequencing (WES) included 186 familial ESCC patients from high-risk China. Targeted gene sequencing validation of 598 genes included 3,289 Henan and 1,228 moderate-risk Hong Kong Chinese. A WES approach identified BRCA2 loss-of-function (LOF) mutations in 3.23% (6/186) familial ESCC patients compared to 0.21% (9/4300) in the ExAC East Asians (odds ratio [OR] = 15.89, p = 2.48 × 10-10 ). BRCA2 LOF mutation frequency in the combined Henan cohort has significantly higher prevalence (OR = 10.55, p = 0.0035). Results were independently validated in an ESCC Hong Kong cohort (OR = 10.64, p = 0.022). One Hong Kong pedigree was identified to carry a BRCA2 LOF mutation. BRCA2 inactivation in ESCC was via germline LOF mutations and wild-type somatic allelic loss via loss of heterozygosity. Gene-based association analysis, including LOF mutations and rare deleterious missense variants defined with combined annotation dependent depletion score ≥30, confirmed the genetic predisposition role of BRCA2 (OR = 9.50, p = 3.44 × 10-5 ), and provided new evidence for potential association of ESCC risk with DNA repair genes (POLQ and MSH2), inflammation (TTC39B) and angiogenesis (KDR). Our findings are the first to provide compelling evidence of the role of BRCA2 in ESCC genetic susceptibility in Chinese, suggesting defective homologous recombination is an underlying cause in ESCC pathogenesis, which is amenable to therapeutic options based on synthetic lethality approaches such as targeting BRCA2 with PARP1 inhibitors in ESCC.

Molecular mechanism of tumour necrosis factor alpha regulates hypocretin (orexin) expression, sleep and behaviour.

Hypocretin 1 and hypocretin 2 (orexin A and B) regulate sleep, wakefulness and emotion. Tumour necrosis factor alpha (TNF-α) is an important neuroinflammation mediator. Here, we examined the effects of TNF-α treatment on hypocretin expression in vivo and behaviour in mice. TNF-α decreased hypocretin 1 and hypocretin 2 expression in a dose-dependent manner in cultured hypothalamic neurons. TNF-α decreased mRNA stability of prepro-hypocretin, the single precursor of hypocretin 1 and hypocretin 2. Mice challenged with TNF-α demonstrated decreased expression of prepro-hypocretin, hypocretin 1 and hypocretin 2 in hypothalamus. In response to TNF-α, prepro-hypocretin mRNA decay was increased in hypothalamus. TNF-α neutralizing antibody restored the expression of prepro-hypocretin, hypocretin 1 and hypocretin 2 in vivo in TNF-α challenged mice, supporting hypocretin system can be impaired by increased TNF-α through decreasing hypocretin expression. Repeated TNF-α challenge induced muscle activity during rapid eye movement sleep and sleep fragmentation, but decreased learning, cognition and memory in mice. TNF-α neutralizing antibody blocked the effects of TNF-α; in contrast, hypocretin receptor antagonist enhanced the effects of TNF-α. The data support that TNF-α is involved in the regulation of hypocretin expression, sleep and cognition. The findings shed some lights on the role of neuroinflammation in neurodegenerative diseases including Alzheimer's disease and Parkinson's disease.

Interaction of 22 risk SNPs with Helicobacter pylori infection and risk of gastric cardia adenocarcinoma.

Aim: To determine the prevalence of Helicobacter pylori infection and correlation between H. pylori infection and single nucleotide polymorphism (SNPs) identified in gastric cardia adenocarcinoma (GCA) patients. Methods: A case control study was performed. 22 risks of GCA-related SNPs were identified by genotyping assay and the relationship between susceptibility loci for GCA and H. pylori infection was further analyzed. Results: Helicobacter pylori infection was associated with GCA significantly (odds ratio: 1.40; 95% CI: 1.29-1.53 p < 0.01). Five GCA risk SNPs had their genotypes significantly different between H. pylori positive patients and H. pylori negative patients. Conclusion: The interaction between SNPs susceptibility loci and H. pylori infection is associated with an increased risk of GCA.

The alterations of cytokeratin and vimentin protein expressions in primary esophageal spindle cell carcinoma.

BACKGROUND: The accumulated evidence has indicated the diagnostic role of cytokeratin (CK) and vimentin protein immunoassay in primary esophageal spindle cell carcinoma (PESC), which is a rare malignant tumor with epithelial and spindle components. However, it is largely unknown for the expression of CK and vimentin in pathological changes and prognosis of PESC. METHODS: Eighty-two PESC patients were identified from the esophageal and gastric cardia cancer database established by Henan Key Laboratory for Esophageal Cancer Research of Zhengzhou University. We retrospectively evaluated CK and vimentin protein expressions in PESC. Clinicopathological features were examined by means of univariate and multivariate survival analyses. Furthermore, the co-expression value of cytokeratin and vimentin was analyzed by receiver operating characteristic (ROC) curve. RESULTS: The positive pan-cytokeratins AE1/AE3 (AE1/AE3 for short) staining was chiefly observed in cytoplasm of epithelial component tumor cells, with a positive detection rate of 85.4% (70/82). Interestingly, 19 cases showed AE1/AE3 positive staining both in epithelial and spindle components (23.2%). However, AE1/AE3 expression was not observed with any significant association with age, gender, tumor location, gross appearance, lymph node metastasis and TNM stage. Furthermore, AE1/AE3 protein expression does not show any effect on survival. Similar results were observed for vimentin immunoassay. However, in comparison with a single protein, the predictive power of AE1/AE3 and vimentin proteins signature was increased apparently than with single signature [0.75 (95% CI = 0.68-0.82) with single protein v.s. 0.89 (95% CI = 0.85-0.94) with AE1/AE3 and vimentin proteins]. The 1-, 3-, 5- and 7-year survival rates for PESC patients in this study were 79.3%, 46.3%, 28.0% and 15.9%, respectively. Multivariate analysis demonstrated age and TNM stage were independent prognostic factors for overall survival (P = 0.036 and 0.003, respectively). It is noteworthy that only 17.1% patients had a PESC accurate diagnosis by biopsy pathology before surgery (14/82). 72.4% PESC patients with biopsy pathology before surgery had been diagnosed as squamous cell carcinoma. CONCLUSION: The present study demonstrates that cytokeratin and vimentin protein immunoassay is a useful biomarker for PESC accurate diagnosis, but not prognosis. The co-expression of cytokeratin and vimentin in both epithelial and spindle components suggest the possibility of single clone origination for PESC.

Focal adhesion kinase signaling determines the fate of lung epithelial cells in response to TGF-ß.

Alveolar epithelial cell (AEC) injury and apoptosis are prominent pathological features of idiopathic pulmonary fibrosis (IPF). There is evidence of AEC plasticity in lung injury repair response and in IPF. In this report, we explore the role of focal adhesion kinase (FAK) signaling in determining the fate of lung epithelial cells in response to transforming growth factor-ß1 (TGF-ß1). Rat type II alveolar epithelial cells (RLE-6TN) were treated with or without TGF-ß1, and the expressions of mesenchymal markers, phenotype, and function were analyzed. Pharmacological protein kinase inhibitors were utilized to screen for SMAD-dependent and -independent pathways. SMAD and FAK signaling was analyzed using siRNA knockdown, inhibitors, and expression of a mutant construct of FAK. Apoptosis was measured using cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. TGF-ß1 induced the acquisition of mesenchymal markers, including α-smooth muscle actin, in RLE-6TN cells and enhanced the contraction of three-dimensional collagen gels. This phenotypical transition or plasticity, epithelial-myofibroblast plasticity (EMP), is dependent on SMAD3 and FAK signaling. FAK activation was found to be dependent on ALK5/SMAD3 signaling. We observed that TGF-ß1 induces both EMP and apoptosis in the same cell culture system but not in the same cell. While blockade of SMAD signaling inhibited EMP, it had a minimal effect on apoptosis; in contrast, inhibition of FAK signaling markedly shifted to an apoptotic fate. The data support that FAK activation determines whether AECs undergo EMP vs. apoptosis in response to TGF-ß1 stimulation. TGF-ß1-induced EMP is FAK- dependent, whereas TGF-ß1-induced apoptosis is favored when FAK signaling is inhibited.

[The Effect of p-JAK1/p-STAT3 Signaling Way in Intervening NAFLD by Blueberry Probiotic Serum].

OBJECTIVES: To explore the mechanism of interlukin-22 (IL-22)-mediated phosphor-Janus kinase-1(p-JAK1)/phosphor-signal transducer and activator of transcription 3 (p-STAT3) signaling way in the experiment of improving non-alcholic fatty liver disease (NAFLD) by blueberry probiotic serum. METHODS: The rat serums with low-, medium-, and high-dose of 10% blueberry probiotics, as well as saline were prepared. NAFLD model was built by inducing normal liver cell line L-02 with free fatty acid (FFA).NAFLD model cells were cultured with saline serum (model group), low-, medium-, and high-dose blueberry probiotics serums (low-, medium-, and high-dose serum groups) , respectively .Normal liver cell group (normal group) was cultured with saline serum . Oil Red O staining was used to detect the lipid deposition in the cells; the intracellular level of triglyceride (TG) was quantitatively determined; the gene and protein expressions of IL-22, p-JAK1, p-STAT3, sterol-regulatory element binding protein-1c (SREBP-1c ) were detected by RT-PCR, Western blot and immunofluorescence methods. RESULTS: Twenty-four hours after modeling, a large amount of lipid deposition could be observed in model group. Compared with normal group, model group showed lower gene and protein expression levels of IL-22, p-JAK1 and p-STAT3 (P <0.01), and higher SREBP-1c and TG levels (P <0.01).Compared with model group, TG level and the lipid deposition in low-, medium-, and high-dose blueberry probiotics serum groups were gradually reduced. High-dose serum group showed higher gene and protein expression levels of IL-22, p-JAK1, p-STAT3 and lower SREBP-1c compared with the model, low-, and medium-dose serum groups (P <0.01). No significant [CM(155.3mm]differences in gene and protein levels between low- andmedium-doseserum groups were found (P >0.05). CONCLUSION: The blueberry probiotics could antagonize the NAFLD via p-JAK1/p-STAT3 signaling way.

Genome-wide association study of gastric adenocarcinoma in Asia: a comparison of associations between cardia and non-cardia tumours.

OBJECTIVE: Genome-wide association studies (GWAS) of gastric cancer have reported differences in single-nucleotide polymorphism (SNP) associations for tumour subtypes, particularly when divided by location into the gastric cardia versus the non-cardia. DESIGN: Here we present results for a GWAS using 2350 East Asian gastric cancer cases divided as 1189 gastric cardia and 1027 gastric non-cardia cases and 2708 controls. We also included up to 3042 cardia cases, 4359 non-cardia cases and 7548 controls for replication from two Chinese studies and one Korean study. From the GWAS, we selected 12 top SNPs for each gastric cancer subtype, 4 top SNPs for total gastric cancer and 1 SNP in MUC1 for replication testing. RESULTS: We observed genome-wide significant associations for rs10074991 in PRKAA1 at 5p13.1 for cardia (p=7.36×10(-12)) and non-cardia cancers (p=2.42×10(-23)) with per allele OR (95% CI) for the combined endpoint of 0.80 (0.77 to 0.83). At 6p21.1, rs2294693 near UNC5CL was significantly associated with gastric non-cardia cancer risk (p=2.50×10(-8)), with OR (95% CI) of 1.18 (1.12 to 1.26), but there was only a nominal association for cardia cancer (p=1.47×10(-2)). We also confirmed a previously reported association for rs4072037 in MUC1 with p=6.59×10(-8) for total gastric cancer and similar estimates for cardia and non-cardia cancers. Three SNPs in PSCA previously reported to be associated with gastric non-cardia cancer showed no apparent association for cardia cancer. CONCLUSIONS: Our results suggest that associations for SNPs with gastric cancer show some different results by tumour location in the stomach.

Genotypic variants at 2q33 and risk of esophageal squamous cell carcinoma in China: a meta-analysis of genome-wide association studies.

Genome-wide association studies have identified susceptibility loci for esophageal squamous cell carcinoma (ESCC). We conducted a meta-analysis of all single-nucleotide polymorphisms (SNPs) that showed nominally significant P-values in two previously published genome-wide scans that included a total of 2961 ESCC cases and 3400 controls. The meta-analysis revealed five SNPs at 2q33 with P< 5 × 10(-8), and the strongest signal was rs13016963, with a combined odds ratio (95% confidence interval) of 1.29 (1.19-1.40) and P= 7.63 × 10(-10). An imputation analysis of 4304 SNPs at 2q33 suggested a single association signal, and the strongest imputed SNP associations were similar to those from the genotyped SNPs. We conducted an ancestral recombination graph analysis with 53 SNPs to identify one or more haplotypes that harbor the variants directly responsible for the detected association signal. This showed that the five SNPs exist in a single haplotype along with 45 imputed SNPs in strong linkage disequilibrium, and the strongest candidate was rs10201587, one of the genotyped SNPs. Our meta-analysis found genome-wide significant SNPs at 2q33 that map to the CASP8/ALS2CR12/TRAK2 gene region. Variants in CASP8 have been extensively studied across a spectrum of cancers with mixed results. The locus we identified appears to be distinct from the widely studied rs3834129 and rs1045485 SNPs in CASP8. Future studies of esophageal and other cancers should focus on comprehensive sequencing of this 2q33 locus and functional analysis of rs13016963 and rs10201587 and other strongly correlated variants.

Novel metabolic biomarker for early detection and diagnosis to the patients with gastric cardia adenocarcinoma.

BACKGROUND: Gastric cardia adenocarcinoma (GCA) is classified as Siewert type II adenocarcinoma at the esophagogastric junction in Western countries. The majority of GCA patients do not exhibit early warning symptoms, leading to over 90% of diagnoses at an advanced stage, resulting in a grim prognosis, with less than a 20% 5-year survival rate. METHOD: Metabolic features of 276 GCA and 588 healthy controls were characterized through a widely-targeted metabolomics by UPLC-MS/MS analysis. This study encompasses a joint pathway analysis utilizing identified metabolites, survival analysis in both early and advanced stages, as well as high and negative and low expression of HER2 immunohistochemistry staining. Machine learning techniques and Cox regression models were employed to construct a diagnostic panel. RESULTS: A total of 25 differential metabolites were consistently identified in both discovery and validation sets based on criteria of p < 0.05, (VIP) ≥ 1, and FC ≥ 2 or FC ≤ 0.5. Early-stage GCA patients exhibited a more favorable prognosis compared to those in advanced stages. HER2 overexpression was associated with a more positive outcome compared to the negative and low expression groups. Metabolite panel demonstrated a robust diagnostic performance with AUC of 0.869 in discovery set and 0.900 in validation set. CONCLUSIONS: A total of 25 common and stable differential metabolites may hold promise as liquid non-invasive indicators for GCA diagnosis. HER2 may function as a tumor suppressor gene in GCA, as its overexpression is associated with improved survival. The downregulation of bile acid metabolism in GCA may offer valuable theoretical insights and innovative approaches for precision-targeted treatments in GCA patients.

Polymorphism of A133S and promoter hypermethylation in Ras association domain family 1A gene (RASSF1A) is associated with risk of esophageal and gastric cardia cancers in Chinese population from high incidence area in northern China.

BACKGROUND: The role of tumor suppressor gene RASSF1A in the esophageal and gastric cardia carcinogenesis is still inconclusive. In this study, the polymorphism, promoter methylation and gene expression of RASSF1A were characterized in esophageal squamous cell carcinoma (ESCC) and gastric cardia adenocarcinoma (GCA). METHODS: We firstly analyzed the prevalence of RASSF1A A133S in a total of 228 cancer patients with ESCC (n=112) and GCA (n=116) and 235 normal controls by polymerase chain reaction (PCR) and restriction enzyme-digestion assay. Then, the promoter methylation status of the RASSF1A in ESCC (n=143), GCA (n=92) and corresponding adjacent normal tissues were further investigated using methylation-specific PCR (MSP) approach. Finally, the RASSF1A protein expression were determined in ESCC (n=27), GCA (n=24) and the matched adjacent normal tissues by immunohistochemical method. RESULTS: The frequency of 133Ala/Se and Ser/Ser genotype was significantly higher in GCA patients than in normal controls (19.0% vs. 10.2%, P=0.02). Compared with Ala/Ala genotype, Ala/Se and Ser/Ser genotype significantly increased susceptibility to GCA (OR=2.06, 95% CI=1.09-3.97). However, this polymorphism had no association with ESCC (P=0.69). The promoter methylation of RASSF1A gene was significantly increased the risk to both ESCC (OR=5.90, 95% CI=2.78-12.52) and GCA (OR=7.50, 95% CI= 2.78-20.23). Promoter methylation of RASSF1A gene in ESCC was also associated with age and cancer cell differentiation (for age: OR=3.11, 95% CI=1.10-8.73; for differentiation: OR=0.29, 95% CI=0.12-0.69). RASSF1A positive expression was significantly decreased the risk of GCA (OR=0.16, 95% CI=0.03-0.83). In contrast, there was no statistical significance between RASSF1A positive expression and ESCC. The expression of RASSF1A protein trend to be positively related with older GCA patients (OR=16.20, 95% CI=1.57-167.74). CONCLUSIONS: The present findings suggest that alterations of RASSF1A may play an important role in gastric cardia carcinogenesis in terms of polymorphism, promoter hypermethylation and protein expression. Whereas, RASSF1A hypermethylation may probably also be involved in esophageal squamous cell carcinogenesis.

Clinicopathological characteristics and postoperative prognosis of patients with nuclear pedigree of esophageal squamous cell carcinoma.

The aim of this work is to analyze the clinicopathological characteristics and prognostic factors of patients with nuclear pedigree of esophageal cancer. The clinicopathological data and follow-up information of 3,260 patients from different nuclear pedigree of esophageal cancer who underwent radical resection of esophageal cancer were collected, and the clinicopathological characteristics and prognostic factors of the patients were analyzed. The male to female ratio of 3,260 patients with esophageal cancer was 1.7:1. The diagnosis age was ranged from 32 to 85 (60.2 ± 8.1) years old. About 53.8% of the patients were ≥ 60 years old; About 88.8% of the patients came from the high incidence area of esophageal cancer; About 82.5% of the tumors were located in the middle and lower segments of esophagus; Poor, moderate and well differentiation accounted for 26.6%, 61.9% and 11.5% respectively; The surgical margin accounted for 94.3%; 47.6% of the tumors were shorter than 4 cm in length; Clinicopathological TNM stage (0+I) accounted for 15.2%, and stage II, III and IV accounted for 54.5%, 29.9% and 0.4%, respectively. Cox analysis showed that male, diagnosed age ≥ 60 years, tumor located in neck and upper esophageal segments, poor differentiation, tumor length ≥ 4 cm, and advanced TNM were independent risk factors for the prognosis of patients in nuclear pedigree with esophageal cancer. Gender, diagnosis age, tumor location, degree of differentiation, tumor length and TNM stage are the influencing factors for the prognosis of patients with nuclear pedigree of esophageal cancer, which will provide important data for the future study of esophageal cancer family aggregation.

Increases prognostic value of clinical-pathological nomogram in patients with esophageal squamous cell carcinoma.

Background: This study was intended to construct a brand new prognostic nomogram after combine clinical and pathological characteristics to increases prognostic value in patients with esophageal squamous cell carcinoma. Methods: A total of 1,634 patients were included. Subsequently, the tumor tissues of all patients were prepared into tissue microarrays. AIPATHWELL software was employed to explore tissue microarrays and calculate the tumor-stroma ratio. X-tile was adopted to find the optimal cut-off value. Univariate and multivariate Cox analyses were used to screen out remarkable characteristics for constructing the nomogram in the total populations. A novel prognostic nomogram with clinical and pathological characteristics was constructed on the basis of the training cohort (n=1,144). What's more performance was validated in the validation cohort (n=490). Clinical-pathological nomogram were assessed by concordance index, time-dependent receiver operating characteristic, calibration curve and decision curve analysis. Results: The patients can divide into two groups with cut-off value of 69.78 for the tumor-stroma ratio. It is noteworthy that the survival difference was noticeable (P<0.001). A clinical-pathological nomogram was constructed by combining clinical and pathological characteristics to predict the overall survival. In comparison with TNM stage, the concordance index and time-dependent receiver operating characteristic of the clinical-pathological nomogram showed better predictive value (P<0.001). High quality of calibration plots in overall survival was noticed. As demonstrated by the decision curve analysis, the nomogram has better value than the TNM stage. Conclusions: As evidently revealed by the research findings, tumor-stroma ratio is an independent prognostic factor in patients with esophageal squamous cell carcinoma. The clinical-pathological nomogram has an incremental value compared TNM stage in predicting overall survival.

Survival influence of gender on 42,345 patients with gastric cardia adenocarcinoma.

PURPOSE: Some studies indicated that gender is associated with prognostic of cancer, However, currently the prognostic value of gender for gastric cardia adenocarcinoma (GCA) survival is unclear. The aim of our study is to reveal the influence of gender on the prognosis of patients with GCA. PATIENTS AND METHODS: A total of 42,345 cases Chinese GCA patients were enrolled from our previously established GCA and esophageal cancer databases. The clinicopathological characteristics were retrieved from medical records in hospital. The follow-up was performed through letter, telephone or home interview. Among GCA patients, there were 32,544 (76.9%) male patients with the median age 62 years (range 17-97) and 9,801 (23.1%) female patients with the median age 61 years (range 17-95 years). The Chi-square test and Kaplan-Meier method were used to compare the continuous variables and survival. Cox proportional hazards model was used for competing risk analyses, hazard ratios (HRs) and 95% confidence intervals (CIs) were evaluated. RESULTS: Men had shorter GCA-specific survival than women by multivariate analysis (HR 1.114; 95% CI 1.061 to 1.169; P < 0.001). Whether premenopausal, perimenopausal or postmenopausal, the survival of women was better than that of men (premenopausal vs. male, P < 0.001; perimenopausal vs. male, P < 0.001; postmenopausal vs. male, P = 0.035). It was worth noting that in patients with stages I, II, III, and IV, female patients survive longer than male patients (P = 0.049; P = 0.011; P < 0.001; P = 0.044, respectively). CONCLUSION: Gender is an independent prognostic factor for patients with GCA. In comparison with men, women have a significantly better outcome. Smoking and drinking may be protective factors for male GCA patients.

Krüppel-like Factor 13 Promotes HCC Progression by Transcriptional Regulation of HMGCS1-mediated Cholesterol Synthesis.

Background and Aims: Krüppel-like factor (KLF) has a role in the occurrence, development and metabolism of cancer. We aimed to explore the role and potential molecular mechanism of KLF13 in the growth and migration of liver cancer cells. Methods: The expression of KLF13 in hepatocellular carcinoma (HCC) tissues was higher than that in normal tissues according to analysis of The Cancer Genome Atlas (TCGA) database. Lentiviral plasmids were used for overexpression and plasmid knockdown of KLF13. Real-time quantitative polymerase chain reaction (qPCR) and western blotting were used to detect mRNA and protein expression in HCC tissues and cells. Cell counting kit-8 (CCK-8), colony formation, cell migration and invasion, and flow cytometry assays were used to assess the in vitro function of KLF13 in HCC cells. The effect of KLF13 on xenograft tumor growth in vivo was evaluated. The cholesterol content of HCC cells was determined by an indicator kit. A dual-luciferase reporter assay and chromatin immunoprecipitation sequencing (ChIP-seq) revealed the binding relationship between KLF13 and HMGCS1. Results: The expression of KLF13 was upregulated in HCC tissues and TCGA database. KLF13 knockdown inhibited the proliferation, migration and invasion of HepG2 and Huh7 cells and increased the apoptosis of Huh7 cells. The opposite effects were observed with the overexpression of KLF13 in SK-Hep1 and MHCC-97H cells. The overexpression of KLF13 promoted the growth of HCC in nude mice and KLF13 transcription promoted the expression of HMGCS1 and the biosynthesis of cholesterol. KLF13 knockdown inhibited cholesterol biosynthesis mediated by HMGCS1 and inhibited the growth and metastasis of HCC cells. Conclusions: KLF13 acted as a tumor promoter in HCC by positively regulating HMGCS1-mediated cholesterol biosynthesis.

Increased prognostic value of clinical-reproductive model in Chinese female patients with esophageal squamous cell carcinoma.

BACKGROUND: In China, it has been well recognized that some female patients with esophageal squamous cell carcinoma (ESCC) have different overall survival (OS) time, even with the same tumor-node-metastasis (TNM) stage, challenging the prognostic value of the TNM system alone. An effective predictive model is needed to accurately evaluate the prognosis of female ESCC patients. AIM: To construct a novel prognostic model with clinical and reproductive data for Chinese female patients with ESCC, and to assess the incremental prognostic value of the full model compared with the clinical model and TNM stage. METHODS: A new prognostic nomogram incorporating clinical and reproductive features was constructed based on univariatie and Cox proportional hazards survival analysis from a training cohort (n = 175). The results were recognized using the internal (n = 111) and independent external (n = 85) validation cohorts. The capability of the clinical-reproductive model was evaluated by Harrell's concordance index (C-index), Kaplan-Meier curve, time-dependent receiver operating characteristic (ROC), calibration curve and decision curve analysis. The correlations between estrogen response and immune-related pathways and some gene markers of immune cells were analyzed using the TIMER 2.0 database. RESULTS: A clinical-reproductive model including incidence area, age, tumor differentiation, lymph node metastasis (N) stage, estrogen receptor alpha (ESR1) and beta (ESR2) expression, menopausal age, and pregnancy number was constructed to predict OS in female ESCC patients. Compared to the clinical model and TNM stage, the time-dependent ROC and C-index of the clinical-reproductive model showed a good discriminative ability for predicting 1-, 3-, and 5-years OS in the primary training, internal and external validation sets. Based on the optimal cut-off value of total prognostic scores, patients were classified into high- and low-risk groups with significantly different OS. The estrogen response was significantly associated with p53 and apoptosis pathways in esophageal cancer. CONCLUSION: The clinical-reproductive prognostic nomogram has an incremental prognostic value compared with the clinical model and TNM stage in predicting OS in Chinese female ESCC patients.

Identification of a seven-cell cycle signature predicting overall survival for gastric cancer.

While genetic alterations in several regulators of the cell cycle have a significant impact on the gastric carcinogenesis process, the prognostic role of them remains to be further elucidated. The TCGA-STAD training set were downloaded and the mRNA expression matrix of cell cycle genes was extracted and corrected for further analysis after taking the intersection with GSE84437 dataset. Differentially expressed mRNAs were identified between tumor and normal tissue samples in TCGA-STAD. Univariate Cox regression analysis and lasso Cox regression model established a novel seven-gene cell cycle signature (including GADD45B, TFDP1, CDC6, CDC25A, CDC7, SMC1A and MCM3) for GC prognosis prediction. Patients in the high-risk group shown significantly poorer survival than patients in the low-risk group. The signature was found to be an independent prognostic factor for GC survival. Nomogram including the signature shown some clinical net benefit for overall survival prediction. The signature was further validated in the GSE84437 dataset. In tissue microarray, CDC6 and MCM3 protein expression were significant differences by the immunohistochemistry-based H-score between tumor tissues and adjacent tissues, and CDC6 is an independent prognostic factor for GC. Interestingly, our GSEA revealed that low-risk patients were more related to cell cycle pathways and might benefit more from therapies targeting cell cycle. Our study identified a novel robust seven-gene cell cycle signature for GC prognosis prediction that may serve as a beneficial complement to clinicopathological staging. The signature might provide potential biomarkers for the application of cell cycle regulators to therapies and treatment response prediction.

Focal adhesion kinase-related non-kinase ameliorates liver fibrosis by inhibiting aerobic glycolysis via the FAK/Ras/c-myc/ENO1 pathway.

BACKGROUND: Hepatic stellate cell (HSC) hyperactivation is a central link in liver fibrosis development. HSCs perform aerobic glycolysis to provide energy for their activation. Focal adhesion kinase (FAK) promotes aerobic glycolysis in cancer cells or fibroblasts, while FAK-related non-kinase (FRNK) inhibits FAK phosphorylation and biological functions. AIM: To elucidate the effect of FRNK on liver fibrosis at the level of aerobic glycolytic metabolism in HSCs. METHODS: Mouse liver fibrosis models were established by administering CCl4, and the effect of FRNK on the degree of liver fibrosis in the model was evaluated. Transforming growth factor-ß1 was used to activate LX-2 cells. Tyrosine phosphorylation at position 397 (pY397-FAK) was detected to identify activated FAK, and the expression of the glycolysis-related proteins monocarboxylate transporter 1 (MCT-1) and enolase1 (ENO1) was assessed. Bioinformatics analysis was performed to predict putative binding sites for c-myc in the ENO1 promoter region, which were validated with chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. RESULTS: The pY397-FAK level was increased in human fibrotic liver tissue. FRNK knockout promoted liver fibrosis in mouse models. It also increased the activation, migration, proliferation and aerobic glycolysis of primary hepatic stellate cells (pHSCs) but inhibited pHSC apoptosis. Nevertheless, opposite trends for these phenomena were observed after exogenous FRNK treatment in LX-2 cells. Mechanistically, the FAK/Ras/c-myc/ENO1 pathway promoted aerobic glycolysis, which was inhibited by exogenous FRNK. CONCLUSION: FRNK inhibits aerobic glycolysis in HSCs by inhibiting the FAK/Ras/c-myc/ENO1 pathway, thereby improving liver fibrosis. FRNK might be a potential target for liver fibrosis treatment.