Oncogenes expand during evolution to withstand somatic amplification.

Background: Cancer-related genes are under intense evolutionary pressure. We conjectured that gene size is an important determinant of amplification propensity for oncogenes and thus cancer susceptibility and therefore could be subject to natural selection. Patients and methods: Gene information, including size and genomic locations, of all protein-coding genes were downloaded from Ensembl (release 87). Quantification of gene amplification was based on Genomic Identification of Significant Targets in Cancer scores obtained from available The Cancer Genome Atlas studies. Results: Oncogenes are larger in size as compared with non-cancer genes (mean size: 92.1 kb versus 61.4 kb; P < 0.0001) in the human genome, which is contributed by both increased total exon size (mean size: 4.6 kb versus 3.4 kb; P < 0.0001) and higher intronic content (mean %: 84.8 versus 78.0; P < 0.01). Such non-random size distribution and intronic composition are conserved in mouse and Drosophila (all P < 0.0001). Stratification by gene age indicated that young oncogenes have been subject to a stronger evolutionary pressure for gene expansion than their non-cancer counterparts. Pan-cancer analysis demonstrated that larger oncogenes were amplified to a lesser extent. Tumor-suppressor genes also moved toward small oncogenes in the course of evolution. Conclusions: Oncogenes expand in size whereas tumor-suppressor genes move closer to small oncogenes in the course of evolution to withstand oncogenic somatic amplification. Our findings have shed new light on the previously unappreciated influence of gene size on oncogene amplification and elucidated how cancers have shaped our genome to its present configuration.

TEAD1/4 exerts oncogenic role and is negatively regulated by miR-4269 in gastric tumorigenesis.

TEA domain (TEAD) transcription factors are key components of the Hippo-YAP1 signaling pathway, but their functional role and regulatory mechanisms remain unclear. This study aims to comprehensively explore the expression pattern and functional role of TEAD family in gastric carcinogenesis and investigate its regulation by microRNAs (miRNAs). The mRNA and protein expression of TEAD family were examined by quantitative reverse transcription-PCR (qRT-PCR) and western blot. Their functional roles were determined by in vitro and in vivo studies. The clinicopathological association of TEAD4 in gastric cancer (GC) was studied using immunohistochemistry on tissue microarray. The prediction of miRNAs, which potentially target TEAD1/4, was performed by TargetScan and miRDB. The regulation of TEAD1/4 by miRNAs was confirmed by qRT-PCR, western blot and luciferase assays. TEAD1/4 were overexpressed in GC cell lines and primary GC tissues. Knockdown of TEAD1/4 induced a significant anticancer effect in vitro and in vivo. TEAD1 was confirmed to be a direct target of miR-377-3p and miR-4269, while TEAD4 was negatively regulated by miR-1343-3p and miR-4269. Among them, miR-4269 was the most effective inhibitor of TEAD1/4. Ectopic expression of these miRNAs substantiated their tumor-suppressive effects. In primary GC tumors, downregulation of miR-4269 was associated with poor disease-specific survival and showed a negative correlation with TEAD4. TEAD1 and TEAD4 are oncogenic factors, whose aberrant activation are, in part, mediated by the silence of miR-377-3p, miR-1343-3p and miR-4269. For the first time, the nuclear accumulated TEAD4 and downregulated miR-4269 are proposed to serve as novel prognostic biomarkers in GC.

Epigenetic silencing of GDF1 disrupts SMAD signaling to reinforce gastric cancer development.

Accumulating evidence reveals the effectiveness of epigenetic therapy in gastric cancer. However, the molecular mechanisms and targets underlying such therapeutic responses remain elusive. Herein, we report an aberrant yet therapeutically rectifiable epigenetic signaling in gastric carcinogenesis. Administration of DNA-demethylating drug 5-aza-2'-deoxycytidine (5-aza-dC) reduced gastric cancer incidence by ~74% (P < 0.05) in N-nitroso-N-methylurea-treated mice. Through genome-wide methylation scanning, novel promoter hypermethylation-silenced and drug-targeted genes were identified in the resected murine stomach tumors and tissues. We uncovered that growth/differentiation factor 1 (Gdf1), a member of the transforming growth factor-ß superfamily, was silenced by promoter hypermethylation in control tumor-bearing mice, but became reactivated in 5-aza-dC-treated mice (P < 0.05). In parallel, the downregulated SMAD2/3 phosphorylation in gastric cancer was revived by 5-aza-dC in vivo. Such hypermethylation-dependent silencing and 5-aza-dC-mediated reactivation of GDF1-SMAD2/3 activity was conserved in human gastric cancer cells (P < 0.05). Subsequent functional characterization further revealed the antiproliferative activity of GDF1, which was exerted through activation of SMAD2/3/4-mediated signaling, transcriptional controls on p15, p21 and c-Myc cell-cycle regulators and phosphorylation of retinoblastoma protein. Clinically, hypermethylation and loss of GDF1 was significantly associated with reduced phosphorylated-SMAD2/3 and poor survival in stomach cancer patients (P < 0.05). Taken together, we demonstrated a causal relationship between DNA methylation and a tumor-suppressive pathway in gastric cancer. Epigenetic silencing of GDF1 abrogates the growth-inhibitory SMAD signaling and renders proliferation advantage to gastric epithelial cells during carcinogenesis. This study lends support to epigenetic therapy for gastric cancer chemoprevention and identifies a potential biomarker for prognosis.

The Janus face of cathelicidin in tumorigenesis.

Cathelicidin is a host defense peptide with multiple innate immunity-related functions. Recent findings indicate that cathelicidin is frequently dysregulated in human cancers where it plays a paradoxical yet dominant role in the regulation of tumor malignancy. In this review, the regulation of malignant phenotypes by cathelicidin in relation to the activation of its receptors and intracellular signaling is discussed.

Cathelicidin protects against Helicobacter pylori colonization and the associated gastritis in mice.

Cathelicidin, an antimicrobial peptide of the innate immune system, has been shown to modulate microbial growth, wound healing and inflammation. However, whether cathelicidin controls Helicobacter pylori infection in vivo remains unexplored. This study sought to elucidate the role of endogenous and exogenous mouse cathelicidin (CRAMP) in the protection against H. pylori infection and the associated gastritis in mice. Results showed that genetic ablation of CRAMP in mice significantly increased the susceptibility of H. pylori colonization and the associated gastritis as compared with the wild-type control. Furthermore, replenishment with exogenous CRAMP, delivered via a bioengineered CRAMP-secreting strain of Lactococcus lactis, reduced H. pylori density in the stomach as well as the associated inflammatory cell infiltration and cytokine production. Collectively, these findings indicate that cathelicidin protects against H. pylori infection and its associated gastritis in vivo. Our study also demonstrates the feasibility of using the transformed food-grade bacteria to deliver cathelicidin, which may have potential clinical applications in the treatment of H. pylori infection in humans.

Epigenetic inactivation of paired box gene 5, a novel tumor suppressor gene, through direct upregulation of p53 is associated with prognosis in gastric cancer patients.

Using genome-wide methylation screening, we identified that paired box gene 5 (PAX5) is involved in human cancer development. However, the function of PAX5 in gastric cancer (GC) development is largely unclear. We analyzed its epigenetic inactivation, biological functions and clinical application in GC. PAX5 was silenced in seven out of eight GC cell lines. A significant downregulation was also detected in paired gastric tumors compared with adjacent non-cancerous tissues. The downregulation of PAX5 was closely linked to the promoter hypermethylation status and could be restored with demethylation treatment. Ectopic expression of PAX5 in silenced GC cell lines (AGS and BGC823) inhibited colony formation and cell viability, arrested cell cycle, induced apoptosis, suppressed cell migration and invasion and repressed tumorigenicity in nude mice. Consistent with the induction of apoptosis by PAX5 in vitro, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining showed significantly enhanced apoptotic cells in PAX5-expressed tumors compared with the vector control tumors. On the other hand, knockdown of PAX5 by PAX5-short hairpin RNA increased the cell viability and proliferation. The anti-tumorigenic function of PAX5 was revealed to be mediated by upregulating downstream targets of tumor protein 53 (p53), p21, BCL2-associated X protein, metastasis suppressor 1 and tissue inhibitors of metalloproteinase 1, and downregulating BCL2, cyclin D1, mesenchymal-epithelial transition factor (MET) and matrix metalloproteinase 1. Immunoprecipitation assay demonstrated that PAX5 directly bound to the promoters of p53 and MET. Moreover, PAX5 hypermethylation was detected in 77% (144 of 187) of primary GCs compared with 10.5% (2/19) of normal gastric tissues (P<0.0001). GC patients with PAX5 methylation had a significant poor survival compared with the unmethylated cases as demonstrated by Cox regression model and log-rank test. In conclusion, PAX5 is a novel functional tumor suppressor in gastric carcinogenesis. Detection of methylated PAX5 can be utilized as an independent prognostic factor in GC.

PPARgamma is essential for protection against nonalcoholic steatohepatitis.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a transcription factor that regulates lipid metabolism and inflammatory responses. Certain PPARgamma ligands improve nonalcoholic steatohepatitis (NASH). The role of PPARgamma itself in NASH remains poorly understood. The functional consequences of PPARgamma in the development of steatohepatitis through gene deficiency or gene overexpression of PPARgamma delivered by adenovirus (Ad-PPARgamma) were examined. Our results show that PPARgamma-deficient (PPARgamma(+/-)) mice fed the methionine- and choline-deficient (MCD) diet developed more severe steatohepatitis than wild-type mice, and were unaffected by PPARgamma ligand rosiglitazone. Overexpression of PPARgamma delivered by Ad-PPARgamma attenuated steatohepatitis. This effect was associated with redistribution of fatty acid from liver to adipose tissue by enhancing expression of fatty acid uptake genes (fatty acid binding protein-4 (aP2), fatty acid translocase (CD36), lipoprotein lipase (LPL) and fatty acid transport protein-1 (FATP-1)) and lipogenic genes (sterol regulatory element binding protein isoform-1 (SREBP-1) and stearoyl-CoA desaturase isoform-1 (SCD-1)) in adipose tissue and to a lesser extent in liver. The anti-steatohepatitis action of PPARgamma was also mediated via regulating adipokines through suppressing tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) and inducing adiponectin. Moreover, PPARgamma activation suppressed hepatic lipoperoxide and reduced hepatic pro-inflammatory cytokines (TNF-alpha and IL-6) production. In conclusion, PPARgamma is an important endogenous regulator and potential therapeutic target for nutritional steatohepatitis.

Warburg effect revisited: an epigenetic link between glycolysis and gastric carcinogenesis.

In cancer cells, glucose is often converted into lactic acid, which is known as the 'Warburg effect'. The reason that cancer cells have a higher rate of aerobic glycolysis, but not oxidative phosphorylation, remains largely unclear. Herein, we proposed an epigenetic mechanism of the Warburg effect. Fructose-1,6-bisphosphatase-1 (FBP1), which functions to antagonize glycolysis was downregulated through NF-kappaB pathway in Ras-transformed NIH3T3 cells. Restoration of FBP1 expression suppressed anchorage-independent growth, indicating the relevance of FBP1 downregulation in carcinogenesis. Indeed, FBP1 was downregulated in gastric carcinomas (P<0.01, n=22) and gastric cancer cell lines (57%, 4/7). Restoration of FBP1 expression reduced growth and glycolysis in gastric cancer cells. Moreover, FBP1 downregulation was reversed by pharmacological demethylation. Its promoter was hypermethylated in gastric cancer cell lines (57%, 4/7) and gastric carcinomas (33%, 33/101). Inhibition of NF-kappaB restored FBP1 expression, partially through demethylation of FBP1 promoter. Notably, Cox regression analysis revealed FBP1 promoter methylation as an independent prognosis predicator for gastric cancer (hazard ratio: 3.60, P=0.010). In summary, we found that NF-kappaB functions downstream of Ras to promote epigenetic downregulation of FBP1. Promoter methylation of FBP1 can be used as a new biomarker for prognosis prediction of gastric cancer. Such an important epigenetic link between glycolysis and carcinogenesis partly explains the Warburg effect.

Expression of cyclooxygenase-2 in chronic hepatitis B and the effects of anti-viral therapy.

BACKGROUND: Cyclooxygenase-2 may play a role in the development of hepatocellular carcinoma, but the relationship between cyclooxygenase-2 and chronic hepatitis B is unknown. AIM: To investigate the expression and cellular localization of cyclooxygenase-2 in chronic hepatitis B patients and the effects of anti-viral therapy. METHODS: Using immunohistochemistry, in situ hybridization, Western blot and reverse transcription polymerase chain reaction, protein and messenger RNA expression and cellular localization of cyclooxygenase-2 in 35 chronic hepatitis B patients were assessed. Fourteen histologically normal and non-viral-infected livers were used as controls. The cyclooxygenase-2 immunoreactivities of paired liver biopsies from 12 patients receiving anti-viral therapy were compared. RESULTS: Immunohistochemistry and in situ hybridization revealed that cyclooxygenase-2 expression was confined to hepatocytes. Patients with chronic hepatitis B had significantly higher cyclooxygenase-2 expression compared with controls. The cyclooxygenase-2 expression of hepatitis B e antigen-positive and -negative chronic hepatitis B patients was not significantly different, although the necro-inflammatory activity of the latter group was significantly lower. Over-expression of cyclooxygenase-2 in patients with chronic hepatitis B was further confirmed by Western blot and reverse transcription polymerase chain reaction. Twelve hepatitis B e antigen-positive chronic hepatitis B patients received anti-viral therapy: lamivudine in seven and interferon in five. Despite hepatitis B e antigen seroconversion, disappearance of hepatitis B virus DNA in serum, normalization of liver enzymes and a significant reduction in necro-inflammatory activity in all 12 patients, no significant change in cyclooxygenase-2 expression was found. CONCLUSIONS: Chronic hepatitis B is associated with elevated cyclooxygenase-2 levels in hepatocytes, and the over-expression of this enzyme does not reflect inflammatory activity. Up-regulation of cyclooxygenase-2 persists after successful anti-viral therapy.