Furthermore, we aim to determine the mechanisms underlying the CUG2-induced increase in NEK2 expression in our future studies. During interphase, centrosomes are held together by a proteinaceous linker. found that CUG2 overexpression increased -catenin expression and activity. The suppression of -catenin decreased cancer stem cell (CSC)-like phenotypes, indicating that -catenin is involved in CUG2-mediated CSC-like phenotypes. Notably, CUG2 overexpression increased the phosphorylation of -catenin at Ser33/Ser37, which is known to recruit E3 ligase for -catenin degradation. PRKAA2 Moreover, CUG2 interacted with and enhanced the expression and kinase activity of never in mitosis gene A-related kinase 2 (NEK2). Recombinant NEK2 phosphorylated -catenin at Ser33/Ser37, while NEK2 knockdown decreased the phosphorylation of -catenin, suggesting that NEK2 is involved in the phosphorylation of -catenin at Ser33/Ser37. Treatment with CGK062, a small chemical molecule, which promotes the phosphorylation of -catenin at Ser33/Ser37 through protein kinase C (PKC) to induce its degradation, reduced -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 -catenin at Ser33/Ser37 by activating NEK2, thus stabilizing -catenin. CGK062 may thus have potential for use as a therapeutic drug against CUG2-overexpressing lung cancer cells. and (10-13). A number of types of cancer exhibit the accumulation of -catenin and the consequent activation of TCF/LEF-dependent gene transcription (14-16). In quiescent cells, -catenin is maintained in the cytoplasm at low levels. This is facilitated by its interaction with scaffolding proteins, such as adenomatous polyposis coli and axin, and with protein kinases, such as LY 254155 casein kinase 1a and GSK3, which phosphorylate -catenin at Ser45 and Ser33/Ser37/Thr41, respectively, leading to its ubiquitination and proteasomal degradation (17-19). Wnt and other growth stimuli induce GSK3 phosphorylation, resulting in the inactivation of -catenin phosphorylation at Ser33/Ser37/Thr41, its stabilization, and its subsequent translocation to the nucleus (20). Previous studies have demonstrated that protein kinase A (PKA) also stabilizes -catenin by phosphorylating it at Ser675 (21,22). The LY 254155 present study examined whether the overexpression of CUG2, a novel oncogene, affects the Wnt/-catenin signaling pathway, which is essential for tumorigenesis. We found that CUG2 overexpression increased -catenin activity and stability, which was regulated by never in mitosis gene A-related kinase 2 (NEK2). Treatment with CGK062 targeting -catenin through PKC inhibited CUG2-induced cancer stem cell (CSC)-like phenotypes, thus impairing tumor formation (Fig. 5C). Although the mechanisms LY 254155 underlying the effects of CGK062 on NEK2 are unknown, our results indicate that CGK062 affects both -catenin and NEK2. Open in a separate window Figure 5 CGK062 treatment decreases NEK2 expression and kinase activity in A549-CUG2 cells. (A) Lysates of A549-CUG2 cells treated with CGK062 (0, 10, 30, 40 and 50 knockdown facilitated the binding of GSK3 to -catenin, leading to its phosphorylation at Ser33/Ser37 and subsequent degradation through the E3 ligase -TrCP. However, we did not observe any change in the -catenin levels. Moreover, GSK3 inhibition or silencing did not increase the -catenin levels. In our next study, we aim to examine whether the long form of cFLIP, PCAF, or PAR-1 participates in -catenin stabilization in the presence of CUG2 overexpression. Furthermore, we aim to determine the mechanisms underlying the CUG2-induced increase in NEK2 expression in our future studies. During interphase, centrosomes are held together by a proteinaceous linker. At the onset of mitosis, this linker is dissembled to facilitate centrosome separation and bipolar spindle formation (34). NEK2 is implicated to be involved in this process, which is known as centrosome disjunction (34). Besides its cellular effects, NEK2 overexpression activates Ras-Src, PI3 kinase, and Wnt signaling pathways to promote metastasis (35). Consistently, aberrant NEK2 expression has been reported in various cancers, including hepatocellular carcinoma (36), non-small cell lung (37), colon (38), brain (39), and ovarian cancers (40). Based on these lines of clinical evidence, small-molecule drugs have been designed or screened for targeting the potentially oncogenic NEK2 (41-43). Notably, treatment with CGK062, which destabilizes -catenin through PKC, reduced the NEK2 levels. Although the.