Yes-associated protein (YAP), the nuclear effector of the Hippo pathway, is a key regulator of organ size and a candidate human oncogene. This study aimed to assess the clinical significance and biological functions of YAP in non-small-cell lung cancer (NSCLC). We investigated the expression of YAP in 92 cases of NSCLC tissue by immunohistochemistry and found that YAP was expressed in 66.3% (61 ⁄ 92) cases and predominantly presented in the nucleus. The expression of YAP in NSCLC was significantly correlated with p-TNM stage (P = 0.0037) and lymph node metastasis (P = 0.0093). Importantly, YAP expression was associated with short overall survival. Further study in NSCLC cell lines in which YAP was either overexpressed or depleted confirmed that YAP markedly promoted cell proliferation and invasion. These results indicate that YAP plays an important role in NSCLC and might be a useful therapeutic target of NSCLC. (Cancer Sci 2010; 101: 1279-1285 L ung cancer is one of the leading causes of malignancyrelated deaths. (1,2) Non-small-cell lung cancer (NSCLC) accounts for up to 80% of all lung cancer cases. Despite progress made in the past decades, the 5-year survival rate for lung cancer remains under 15%. (3) Recent studies suggest that conventional therapies may have reached a therapeutic plateau, (3) so the demanding tasks are to elucidate the mechanisms of tumor biology and to identify new therapeutic targets.The Hippo signaling pathway, initially identified in Drosophila, is a highly conserved potent regulator of cell growth and apoptosis. (4,5) Acting downstream of the Hippo pathway, yes-associated protein (YAP) functions as a transcription co-activator (5,6) which interacts with the PPXY-motif-containing transcription factors, including ErbB4, P73, TEAD, P53BP-2, and Runx2. (7)(8)(9)(10)(11)(12)(13) In mammals, the Hippo pathway kinase cascade phosphorylates YAP and induces its cytoplasmic translocation; thus attenuating its interaction with the nuclear binding partners and inhibiting its function. (4)(5)(6) YAP is a key regulator of organ size by orchestrating cell proliferation and death. YAP induced the transcription of genes that promote proliferation in murine livers including Ki-67, c-myc, SOX4, and AFP. At the same time, it induced expression of negative regulators of apoptosis, such as the IAP family members BIRC5 ⁄ survivin and BIRC2 ⁄ cIAP1, and the BCL2 family gene MCL1 . (4) In addition, YAP is vitally required for development as mice deficient in YAP have an embryonic lethal phenotype. (14) Recently, YAP has been shown to be a candidate oncogene in the human chromosome 11q22 amplicon. (15)(16)(17) YAP expression and nuclear localization was found to be elevated in hepatocellular carcinoma, prostate cancer, colon cancer, ovary cancer, and breast cancer. (18)(19)(20) Moreover, some studies have further confirmed the oncogenic function of YAP in vivo and in vitro. YAP overexpression was able to overcome cell contact inhibition, (20) therefore providing a growth advantage for YAP-overexpressing canc...

BackgroundTwo mature microRNAs (miRNAs), hsa-miR-125a-3p and hsa-miR-125a-5p (collectively referred to as hsa-miR-125a-3p/5p), are derived from 3' and 5' ends of pre-miR-125a, respectively. Although impaired regulation of hsa-miR-125a-5p has been observed in some tumors, the role of this miRNA in invasion and metastasis remains unclear, and few studies have examined the function of hsa-miR-125a-3p. In order to characterize the functions of hsa-miR-125a-3p/5p in invasion and metastasis of non-small cell lung cancer (NSCLC), we investigated the relationships between hsa-miR-125a-3p/5p expression and lymph node metastasis in NSCLC tissues. We also explored the impact of expression of these miRNAs on invasive and migratory capabilities of lung cancer cells.MethodsExpression of hsa-miR-125a-3p/5p in NSCLC tissues was explored using real-time PCR. The relationships between hsa-miR-125a-3p/5p expression and pathological stage or lymph node metastasis were assessed using the Spearman correlation test. For in vitro studies, lung cancer cells were transfected with sense and antisense 2'-O-methyl oligonucleotides for gain-of-function and loss-of-function experiments. Transwell experiments were performed to evaluate cellular migration and invasion.ResultsExpression of hsa-miR-125a-3p/5p was lower in NSCLC tissues than in adjacent normal lung tissues (LAC). Furthermore, the results from the Spearman correlation test showed a negative relationship between hsa-miR-125a-3p expression and pathological stage or lymph node metastasis and an inverse relationship between hsa-miR-125a-5p expression and pathological stage or lymph node metastasis. In vitro gain-of-function experiments indicated that hsa-miR-125a-3p and hsa-miR-125a-5p function in an opposing manner, suppressing or enhancing cell migration and invasion in A549 and SPC-A-1 cell lines, respectively. These opposing functions were further validated by suppression of hsa-miR-125a-3p and hsa-miR-125a-5p expression in loss-of-function experiments.ConclusionHsa-miR-125a-3p and hsa-miR-125a-5p play distinct roles in regulation of invasive and metastatic capabilities of lung cancer cells, consistent with the opposing correlations between the expression of these miRNAs and lymph node metastasis in NSCLC. These results provide new insights into the roles of miR-125a family members in the development of NSCLC.

SIRT4 is well-known for its deacetylase activity in energy metabolism, but little is known about its roles in carcinogenesis. We demonstrated that SIRT4 was decreased in 70 out of 133 non-small cell lung cancer (NSCLC) cases by immunohistochemical staining and localized in the mitochondria using confocal microscopy. Low levels of SIRT4 expression was correlated with tumor node metastasis (TNM) stage, histological type of tumor (adenocarcinoma), lymph nodal status, Ki-67 (proliferation index) and poor overall survival. We also studied the biological role of SIRT4 in lung cancer cell lines transfected with SIRT4 plasmid or SIRT4-siRNA. SIRT4 inhibited lung cancer cell proliferation, blocked the cell cycle and repressed cell invasion and migration. Mitochondrial dynamics has been implicated in malignant properties of cells, particularly metastasis that is the major cause of death in patients diagnosed with cancer including lung cancer. This is the first study to identify an association between SIRT4 expression and decreased mitochondrial fission, which was driven by Drp1. SIRT4 inhibited Drp1 phosphorylation and weakened Drp1 recruitment to the mitochondrial membrane via an interaction with Fis-1. SIRT4 expression was lower in nodal metastatic tumor samples than their corresponding primary tumors, and cases with low expression of SIRT4 tended to have high p-Drp1 labeling. Also, MEK/ERK activity appeared to be hampered by SIRT4 expression, which may have implications for cells' invasive capacities. In conclusion, our findings suggest that SIRT4 functions as an important antitumor protein in NSCLC, and should be investigated further with respect to future anticancer strategies.

MicroRNA-449a is expressed at a low level in several tumors and cancer cell lines, and induces G1 arrest, apoptosis, and senescence. To identify the function of miR-449a in non-small cell lung cancer (NSCLC), we discussed the potential relevance of miR-449a to clinicopathological characteristics and prognosis in NSCLC. We also investigated the impact of miR-449a on migration and invasion in NSCLC cells. The expression of miR-449a in NSCLC tissues and cell lines was detected using RT-qPCR. In vitro, gain-of-function, loss-of-function experiments, and fluorescence assays were performed to identify the potential target of miR-449a and the function of miR-449a in NSCLC cells. MiR-449a was downregulated in both NSCLC tissues and cell lines. Moreover, a low expression level of miR-449a appeared to be correlated with lymph node metastasis and poor survival. In vitro, miR-449 regulated cell migration and invasion in NSCLC cells as a potential tumor suppressor, at least in part by targeting c-Met. Furthermore, reciprocal expression of miR-449a and c-Met was shown in NSCLC tissue samples. This study indicates that miR-449a might be associated with NSCLC progression, and suggests a crucial role for miR-449a in NSCLC.

These authors contributed equally to this workBackground: Lung cancer is the leading cause of cancer-related death worldwide. Although the macrophages can affect the development of tumor, the contribution of macrophages to the prognosis of non-small-cell lung cancer (NSCLC) is still controversial. Moreover, anti-PD-1 therapy can redirect macrophages from an M2 to an M1 phenotype, suggesting that tumor PD-L1 may affect the prognostic role of macrophages. Therefore, in this study, we aimed to display a macrophage landscape to clarify the function of macrophages, considering the localization and polarization of the macrophages, and evaluate the effect of M2 macrophages and tumor PD-L1 in combination on the prognosis of NSCLC. Methods: We performed multiplex quantitative immunofluorescence staining of pan-cytokeratin (CK), CD68, CD163, PD-L1, and DAPI on one tissue specimen simultaneously from 137 NSCLC patients. Results: M2 macrophages, involved marginM2 (M2 macrophages in tumor stroma), and centralM2 (M2 macrophages infiltrating into tumor islets) increased as the tumor stage increased. More macrophages were found in lung squamous cell carcinoma (LUSC) patients, patients with wild-type EGFR, and smokers than in patients with lung adenocarcinoma (LUAD), patients with EGFR mutations, and non-smokers. Infiltration of centralM2 was an independent prognostic factor of poor overall survival (OS) and disease-free survival (DFS) for NSCLC patients (P<0.05), which was superior to total macrophages and total M2 macrophages. Moreover, patients with centralM2 less PD-L1 − tumors showed the best OS and DFS, while the patients with centralM2 more PD-L1 + tumors showed the worst OS and DFS, and the two groups with centralM2 less PD-L1 + and centralM2 more PD-L1 − were in the middle (P=0.002, 0.034, respectively). Conclusion: Tumor islet-infiltrating M2 macrophages influence the prognosis of NSCLC patients. The analysis of M2 macrophages and tumor PD-L1 in combination may enhance the accuracy of prognostic prediction. This study provides a new understanding of macrophages in the development of NSCLC through the analysis of macrophage landscape.