AHNAK2 Regulates NF-κB/MMP-9 Signaling to Promote Pancreatic Cancer Progression.

Early metastasis of pancreatic cancer (PaC) is a major cause of its high mortality rate. Previous studies have shown that AHNAK2 is involved in the progression of some tumors and is predicted to be an independent prognostic factor for PaC; however, the specific mechanisms through which AHNAK2 regulates PaC remain unclear. In this study, we examined the role of AHNAK2 in PaC and its potential molecular mechanisms. AHNAK2 mRNA and protein expression in PaC tissues and cells were measured using qRT-PCR and western blot analysis. After AHNAK2 knockdown using small interfering RNA, PaC cells were subjected to CCK-8 scratch, and Transwell assays to assess cell proliferation, migration, and invasion, respectively. Furthermore, the validation of the mechanistic pathway was achieved by western blot analysis. AHNAK2 mRNA and protein levels were up-regulated in PaC and silencing AHNAK2 significantly inhibited the proliferation, migration, and invasion of PaC cells. Mechanistically, AHNAK2 knockdown decreased the expression of phosphorylated p65, phosphorylated IκBα, and matrix metalloproteinase-9 (MMP-9), suggesting that activation of the NF-κB/MMP-9 signaling pathway was inhibited. Importantly, activation of NF-κB reversed the effects of AHNAK2 knockdown. Our findings indicate that AHNAK2 promotes PaC progression through the NF-kB/MMP-9 pathway and provides a theoretical basis for targeting AHNAK2 for the treatment of PaC.

Knockdown of tankyrase 1 inhibits the progression of gastric adenocarcinoma via regulating human telomerase reverse transcriptase and telomeric repeat binding factor 1.

Background: Gastric cancer is one of the most lethal cancers. Aberrant expression levels of genes are frequently associated with cell immortalization and the occurrence of tumors. In this study, we aimed to investigate the role of tankyrase 1 (TANK1) in gastric adenocarcinoma and clarify the underlying mechanism. Methods: The messenger RNA (mRNA) levels of TANK1, human telomerase reverse transcriptase (h-TERT), and telomeric repeat binding factor 1 (TRF1) in clinical specimens and SGC-7901 cells were measured via real-time quantitative polymerase chain reaction (RT-qPCR). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and immunohistochemistry (IHC) assays were utilized to observe the cell apoptosis as well as Ki67 and h-TERT expression in tumor-bearing models. The effects of TANK1 antisense oligonucleotides (TANK1 ASODN) on viability and apoptosis of SGC 7901 cells were evaluated by cell counting kit-8 and flow cytometry analysis. Results: We found that TANK1 and h-TERT were both increased in gastric adenocarcinoma, while TRF1 was decreased. Tumor-bearing models demonstrated that TANK1 ASODN appeared to be effective in inhibiting tumor growth and decreasing the expression of h-TERT. Additionally, TANK1 ASODN inhibited the viability and promoted apoptosis of SGC-7901 cells. Moreover, the mRNA levels of h-TERT and TRF1 were modulated by TANK1 ASODN. Conclusions: This study revealed that TANK1 ASODN inhibits the proliferation and induced the apoptosis of gastric adenocarcinoma cells via manipulating the expression levels of h-TERT and TRF1.

Wortmannin influences hypoxia-inducible factor-1 alpha expression and glycolysis in esophageal carcinoma cells.

AIM: To investigate the influence of phosphatidylinositol-3-kinase protein kinase B (PI3K/AKT)-HIF-1α signaling pathway on glycolysis in esophageal carcinoma cells under hypoxia. METHODS: Esophageal carcinoma cell lines Eca109 and TE13 were cultured under hypoxia environment, and the protein, mRNA and activity levels of hypoxia inducible factor-1 alpha (HIF-1α), glucose transporter 1, hexokinase-II, phosphofructokinase 2 and lactate dehydrogenase-A were determined. Supernatant lactic acid concentrations were also detected. The PI3K/AKT signaling pathway was then inhibited with wortmannin, and the effects of hypoxia on the expression or activities of HIF-1α, associated glycolytic enzymes and lactic acid concentrations were observed. Esophageal carcinoma cells were then transfected with interference plasmid with HIF-1α-targeting siRNA to assess impact of the high expression of HIF-1α on glycolysis. RESULTS: HIF-1α is highly expressed in the esophageal carcinoma cell lines tested, and with decreasing levels of oxygen, the expression of HIF-1α and the associated glycolytic enzymes and the extracellular lactic acid concentration were enhanced in the esophageal carcinoma cell lines Eca109 and TE13. In both normoxia and hypoxic conditions, the level of glycolytic enzymes and the secretion of lactic acid were both reduced by wortmannin. The expression and activities of glycolytic enzymes and the lactic acid concentration in cells were reduced by inhibiting HIF-1α, especially the decreasing level of glycolysis was significant under hypoxic conditions. CONCLUSION: The PI3K/AKT pathway and HIF-1α are both involved in the process of glycolysis in esophageal cancer cells.

HIF-1α induces VE-cadherin expression and modulates vasculogenic mimicry in esophageal carcinoma cells.

AIM: To investigate whether hypoxia inducible factor (HIF)-1α modulates vasculogenic mimicry (VM) by upregulating VE-cadherin expression in esophageal squamous cell carcinoma (ESCC). METHODS: Esophageal squamous cancer cell lines Eca109 and TE13 were transfected with plasmids harboring small interfering RNAs targeting HIF-1α or VE-cadherin. The proliferation and invasion of esophageal carcinoma cells were detected by MTT and Transwell migration assays. The formation of tubular networks of cells was analyzed by 3D culture in vitro. BALB/c nude mice were used to observe xenograft tumor formation. The relationship between the expression of HIF-1α and VE-cadherin, ephrinA2 (EphA2) and laminin5γ2 (LN5γ2) was measured by Western blot and real-time polymerase chain reaction. RESULTS: Knockdown of HIF-1α inhibited cell proliferation (32.3% ± 6.1% for Eca109 cells and 38.6% ± 6.8% for TE13 cells, P < 0.05). Both Eca109 and TE13 cells formed typical tubular networks. The number of tubular networks markedly decreased when HIF-1α or VE-cadherin was knocked down. Expression of VE-cadherin, EphA2 and LN5γ2 was dramatically inhibited, but the expression of matrix metalloproteinase 2 had no obvious change in HIF-1α-silenced cells. Knockdown of VE-cadherin significantly decreased expression of both EphA2 and LN5γ2 (P < 0.05), while HIF-1α expression was unchanged. The time for xenograft tumor formation was 6 ± 1.2 d for Eca109 cells and Eca109 cells transfected with HIF-1α Neo control short hairpin RNA (shRNA) vector, and 8.4 ± 2.1 d for Eca109 cells transfected with an shRNA against HIF-1α. Knockdown of HIF-1α inhibited vasculogenic mimicry (VM) and tumorigenicity in vivo. CONCLUSION: HIF-1α may modulate VM in ESCC by regulating VE-cadherin expression, which affects VM formation through EphA2 and LN5γ2.

Differential expression of miR-195 in esophageal squamous cell carcinoma and miR-195 expression inhibits tumor cell proliferation and invasion by targeting of Cdc42.

MicroRNAs (miRNA) have played an important role in carcinogenesis. In this study, Agilent miRNA microarray was used to identify differentially expressed miRNAs in esophageal squamous cell carcinoma (ESCC) tissues and miR-195 was downregulated in ESCC compared with normal esophageal tissues. Moreover, Cdc42 was confirmed as target gene of miR-195. Ectopic expression of miR-195 in ESCC cells significantly downregulated Cdc42 by directly binding its 3' untranslated regions, and induced G1 cell cycle arrest, leading to a significant decrease in cell growth, migration, and invasion in vitro. Therefore, our findings demonstrated that miR-195 may act as a tumor suppressor in ESCC by targeting Cdc42.