Background: Pygopus 2 (Pygo2) is a Pygo family member and an important component of the Wnt signaling transcriptional complex. Despite this data, no clinical studies investigating Pygo2 expression in lung cancer have yet been reported. Methods: In the present study, the expression patterns of Pygo2 were evaluated by immunochemistry in 168 patients with non-small cell lung cancer (NSCLC). We used small interfering RNA (siRNA) to specifically silence Pygo2, and investigated its effect on cell growth by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry analysis in human lung cancer cell lines. Results: Immunohistochemical analysis showed low expression of Pygo2 in normal lung tissues and increased nuclear expression in lung cancer tissues, either with or without perinuclear expression. Abnormal Pygo2 expression was associated with poor differentiation and a high Tumor (T), Node (N) and Metastases (M) stage in NSCLC patients, and correlated with poor prognosis. Using MTT assay we observed that Pygo2 downregulation inhibited cell proliferation; in addition, flow cytometry analysis showed that Pygo2 knockdown induced apoptosis and increased numbers of G1-phase cells and a reduction in S-phase cells. Conclusions: We therefore conclude that abnormal Pygo2 protein expression may be a marker for advanced NSCLC. Furthermore, Pygo2 knockdown suppresses cell growth.

Background: We previously reported that overexpression of Axin downregulates T cell factor-4 (TCF-4) transcription. However, the mechanism(s) by which Axin downregulates the transcription and expression of TCF-4 is not clear. It has been reported that β-catenin promotes and p53 inhibits TCF-4 transcription, respectively. The aim of this study was to investigate whether β-catenin and/or p53 is required for Axin-mediated downregulation of TCF-4. Results: Axin mutants that lack p53/HIPK2 and/or β-catenin binding domains were expressed in lung cancer cells, BE1 (mutant p53) and A549 (wild type p53). Expression of Axin or AxinΔp53 downregulates β-catenin and TCF-4, and knock-down of β-catenin upregulates TCF-4 in BE1 cells. However, expression of AxinΔβ-ca into BE1 cells did not downregulate TCF-4 expression. These results indicate that Axin downregulates TCF-4 transcription via β-catenin. Although overexpression of wild-type p53 also downregulates TCF-4 in BE1 cells, cotransfection of p53 and AxinΔβ-ca did not downregulate TCF-4 further. These results suggest that Axin does not promote p53-mediated downregulation of TCF-4. Axin, AxinΔp53, and AxinΔβ-ca all downregulated β-catenin and TCF-4 in A549 cells. Knock-down of p53 upregulated β-catenin and TCF-4, but cotransfection of AxinΔβ-ca and p53 siRNA resulted in downregulation of β-catenin and TCF-4. These results indicate that p53 is not required for Axin-mediated downregulation of TCF-4. Knock-down or inhibition of GSK-3β prevented Axin-mediated downregulation of TCF-4. Furthermore, expression of Axin and AxinΔp53, prevented the proliferative and invasive ability of BE1 and A549, expression of AxinΔβ-ca could only prevented the proliferative and invasive ability effectively. Conclusions: Axin downregulates TCF-4 transcription via β-catenin and independently of p53. Axin may also inhibits the proliferation and invasion of lung cancer cells via β-catenin and p53.

PLEKHH2 is an important FERM domain containing-protein. However, the role of PLEKHH2 in human solid tumors has not been reported yet. We report that PLEKHH2 showed enhanced cytoplasmic expression in non-small cell lung cancer (NSCLC). Its overexpression was positively correlated with high TNM stage, low differentiation, lymphatic node metastasis, and poor prognosis. In A549 and H1299 cells, high expression of PLEKHH2 significantly promoted cell proliferation, migration, invasion, and increased the expression of proliferation- and invasion-related proteins. It also enhanced the phosphorylation of FAK and promoted the activity of the PI3K/AKT pathway. Immunofluorescence and co-immunoprecipitation analyses were performed to elucidate the molecular mechanism underlying PLEKHH2-mediated regulation of proliferation and invasion in lung cancer cells. Upon transfection of full length PLEKHH2 or its FERM domain, we observed enhanced binding of PLEKHH2 to β-arrestin1, whereas FAK- β-arrestin1 binding was diminished and this led to an increase in FAK phosphorylation. PLEKHH2-mutant plasmids without the FERM domain could not effectively promote its binding to β-arrestin1, activation of FAK phosphorylation, PI3K/AKT activation, or the malignant phenotype. Our findings suggested that PLEKHH2 is an important oncogene in NSCLC. PLEKHH2 binding to β-arrestin1 through the FERM domain competitively inhibits β-arrestin1 binding to FAK, which causes the dissociation of FAK from the FAK-β-arrestin1 complex. Furthermore, the dissociation of FAK promotes its autophosphorylation, activates the PI3K/AKT signaling pathway, and subsequently promotes lung cancer cell proliferation, migration, and invasion. These results provide evidence for the potential use of PLEKHH2 inhibition as an anticancer therapy.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a poor prognosis, which is lethal in approximately 90% of cases despite advanced standard therapies. A typical feature of PDAC is the immunosuppressive tumor microenvironment with multiple immunosuppressive factors including neurotransmitters. Recently, neuromedin U (NMU), a highly conserved neuropeptide with many physiological functions, has attracted attention for its roles in tumorigenesis and metastasis in several types of cancers. However, whether NMU affects PDAC progression remains unclear. In this study, using an orthotopic mouse model of PDAC in combination with bioinformatics analysis, we found that NMU was upregulated in tumor tissues from the patients with PDAC and positively correlated with a poor prognosis of the disease. Interestingly, knockout of the Nmu gene in mice enhanced the anti‐tumor functions of tumor‐infiltrating CD8⁺ T cells in an NMU receptor 1‐dependent manner. Additionally, NMU promoted the glycolytic metabolism of mouse PDAC tumors. The activities of pyruvate kinase (PK) and lactate dehydrogenase (LDH), pivotal enzymes involved in the regulation of lactate production, were markedly reduced in tumor tissues from NMU‐knockout mice. In vitro the presence of LDHA inhibitor can reduce the production of lactic acid stimulated by NMU, which can increase the anti‐tumor activity of CD8⁺ T cells. Moreover, treatment of the pancreatic cancer cells with a phosphoinositide 3‐kinase (PI3K) inhibitor diminished NMU‐induced lactate production and the activities of PK and LDH, suggesting that NMU might regulate glycolysis via the PI3K/AKT pathway.