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The mechanisms through which cancer-upregulated gene 2 (CUG2), a novel oncogene, affects Wnt/β-catenin signaling, essential for tumorigenesis, are unclear. In this study, we aimed to elucidate some of these mechanisms in A549 lung cancer cells. Under the overexpression of CUG2, the protein levels and activity of β-catenin were evaluated by western blot analysis and luciferase assay. To examine a biological consequence of β-catenin under CUG2 overexpression, cell migration, invasion and sphere formation assay were performed. The upregulation of β-catenin induced by CUG2 overexpression was also accessed by xenotransplantation in mice. We first found that CUG2 overexpression increased β-catenin expression and activity. The suppression of β-catenin decreased cancer stem cell (CSC)-like phenotypes, indicating that β-catenin is involved in CUG2-mediated CSC-like phenotypes. Notably, CUG2 overexpression increased the phosphorylation of β-catenin at Ser33/Ser37, which is known to recruit E3 ligase for β-catenin degradation. Moreover, CUG2 interacted with and enhanced the expression and kinase activity of never in mitosis gene A-related kinase 2 (NEK2). Recombinant NEK2 phosphorylated β-catenin at Ser33/Ser37, while NEK2 knockdown decreased the phosphorylation of β-catenin, suggesting that NEK2 is involved in the phosphorylation of β-catenin at Ser33/Ser37. Treatment with CGK062, a small chemical molecule, which promotes the phosphorylation of β-catenin at Ser33/Ser37 through protein kinase C (PKC)α to induce its degradation, reduced β-catenin levels and inhibited the CUG2-induced features of malignant tumors, including increased cell migration, invasion and sphere formation. Furthermore, CGK062 treatment suppressed CUG2-mediated tumor formation in nude mice. Taken together, the findings of this study suggest that CUG2 enhances the phosphorylation of β-catenin at Ser33/Ser37 by activating NEK2, thus stabilizing β-catenin. CGK062 may thus have potential for use as a therapeutic drug against CUG2-overexpressing lung cancer cells.

Overexpression of HIF-1α, a transcription factor responsive to hypoxia, is frequently observed in malignant tumors, which sometimes show resistance to chemotherapy and radiation therapy. Consequently, decrease of HIF-1α through virotherapy offers a logical strategy for the treatment of aggressive tumors. In this study, we found that infection with the oncolytic H-1 parvovirus decreased HIF-1α protein levels in pancreatic cancer cells under CoCl2 or hypoxia. The H-1 virus-induced decrease of HIF-1α was regulated by a proteasome-mediated pathway. Suppression of VHL, an E3 ligase and a critical regulator of HIF-1α, or enforced expression of UCP, an E2 ubiquitin-conjugating enzyme, failed to inhibit the H-1 virus-induced decrease of HIF-1α. Furthermore, siRNA-mediated suppression of RACK1, another regulator of HIF-1α, did not prevent H-1 viral infection from lowering HIF-1α protein levels. Although decrease of HIF-1α was observed after H-1 viral infection, constitutive expression of HIF-1α limited H-1 viral replication. After combined treatment with H-1 parvovirus and YC-1, an inhibitor of HIF-1α, the apoptosis of pancreatic cancer cells was greater than after treatment with H-1 virus alone or YC-1 alone. Accordingly, we propose that H-1 parvovirus could be used with YC-1 as a potential therapeutic agent against aggressive tumors exhibiting hypoxia and increased levels of HIF-1α.

We showed in our previous study that cancer upregulated gene (CUG) 2, a novel oncogene, confers resistance to infection of oncolytic vesicular stomatitis virus (VSV) by activating Stat1-mediated signal transduction. Since many studies have reported that autophagy is involved in virus replication, we investigated whether autophagy also plays a role in the antiviral activity in A549 cells overexpressing CUG2 (A549-CUG2). We suppressed Atg5 or Beclin 1 expression using siRNA and examined its effect on the susceptibility of cells to infection by oncolytic VSV. We found that A549-CUG2 cells treated with Atg5 or Beclin 1 siRNA became susceptible to VSV infection, whereas A549-CUG2 cells treated with control siRNA were resistant. This result suggests that autophagy is involved in the antiviral response of A549-CUG2 cells. Further investigation revealed that autophagy impairment enhanced the generation of reactive oxygen species (ROS), which resulted in inactivation of S6 kinase. Under these conditions, the levels of ISG15 transcript and protein decreased, which conferred on A549-CUG2 cell susceptibility to VSV infection. Finally, we found that overloading of H₂O₂ sensitized control A549-CUG2 cells to VSV-induced apoptosis. Taken together, these results indicate that autophagy impairment induces excessive ROS formation, which decreases S6 kinase activity and ISG15 expression, ultimately rendering the A549-CUG2 cells susceptible to VSV infection. We propose that autophagy impairment is a potential strategy for successful VSV virotherapy of CUG2-overexpressing tumors.