EZH2 (enhancer of zeste homolog 2) is the catalytic subunit of PRC2 (polycomb repressive complex 2), which mediates histone methyltransferase activity and functions as transcriptional repressor involved in gene silencing. EZH2 is involved in malignant transformation and biological aggressiveness of several human malignancies. We previously demonstrated that non-small cell lung cancers (NSCLCs) also overexpress EZH2 and that high expression of EZH2 correlates with poor prognosis. Growing evidence indicates that EZH2 may be an appropriate therapeutic target in malignancies, including NSCLCs. Recently, an S-adenosyl-L homocysteine hydrolase inhibitor, 3-Deazaneplanocin A (DZNep), has been shown to deplete and inhibit EZH2. The aim of this study was to determine the effect of DZNep in NSCLC cells. Knockdown of EZH2 by small-interfering RNA (siRNA) resulted in decreased growth of four NSCLC cell lines. MTT assays demonstrated that DZNep treatment resulted in dose-dependent inhibition of proliferation in the NSCLC cell lines with a half maximal inhibitory concentration (IC50) ranging from 0.08 to 0.24 μM. Immortalized but non-cancerous bronchial epithelial and fibroblast cell lines were less sensitive to DZNep than the NSCLC cell lines. Soft agarose assays demonstrated that anchorage-independent growth was also reduced in all three NSCLC cell lines that were evaluated using this assay. Flow cytometry analysis demonstrated that DZNep induced apoptosis and G1 cell cycle arrest in NSCLC cells, which was partially associated with cyclin A decrease and p27Kip1 accumulation. DZNep depleted cellular levels of EZH2 and inhibited the associated histone H3 lysine 27 trimethylation. These results indicated that an epigenetic therapy that pharmacologically targets EZH2 via DZNep may constitute a novel approach to treatment of NSCLCs.

Background Delta‐like protein 3 (DLL3) is a Notch ligand that has an important role in the tumorigenesis of small cell lung cancer (SCLC). Recently, rovalpituzumab tesirine (Rova‐T), a DLL3‐targeted antibody‐drug conjugate, has been developed for treating SCLC. DLL3 is a transcriptional target of the achaete‐scute homolog‐1 (ASCL1) transcription factor, which is involved in pulmonary neuroendocrine cell development. However, the relationship between DLL3 and/or ASCL1 expression and the clinical features of SCLC remains unknown, especially for early‐stage resected SCLC. This study aimed to investigate the expression of DLL3 and ASCL1 in resected SCLC samples using immunohistochemical analysis. Materials and Methods We collected 95 surgically resected SCLC samples, which were formalin fixed and paraffin embedded. Immunohistochemistry staining was performed to investigate the correlation between the expression of either DLL3 or ASCL1 and clinicopathological features of study patients. Results Seventy‐seven (83%) of 93 immunohistochemically evaluable samples were positive for DLL3 (expression in ≥1% of tumor cells), and DLL3‐high expression (≥75%) was observed in 44 samples (47%). Sixty‐one (64%) of 95 samples were positive for ASCL1 (expression in ≥5% of tumor cells). A positive correlation was observed between DLL3 and ASCL1 expression. DLL3 and ASCL1 expression were not associated with survival in SCLC patients. DLL3 was more prevalent in patients with advanced clinical disease. Conclusion DLL3 and ASCL1 were highly expressed in patients with surgically resected SCLC. DLL3 and ASCL1 may be targets for the treatment of SCLC. Implications for Practice This article examines the relationship between delta‐like protein 3 (DLL3) and achaete‐scute homolog‐1 (ASCL1) protein expression with the clinical features of 95 surgically resected small cell lung cancer (SCLC). DLL3 is attracting attention because rovalpituzumab tesirine (Rova‐T), a DLL3‐targeted antibody‐drug conjugate, was developed recently. DLL3 and ASCL1 were highly expressed in patients with surgically resected SCLC. DLL3 and ASCL1 may be targets for the treatment of early‐stage SCLC, including with Rova‐T.

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Background:Notch receptor has an important role in both development and cancer. We previously reported that inhibition of the Notch3 by γ-secretase inhibitor (GSI) induces apoptosis and suppresses tumour proliferation in non-small-cell lung cancer. Although radiation is reported to induce Notch activation, little is known about the relationship between radiation and Notch pathway.Methods:We examined the effect of combining GSI and radiation at different dosing in three Notch expressing lung cancer cell lines. The cytotoxic effect of GSI and radiation was evaluated using MTT assay and clonogenic assay in vitro and xenograft models. Expressions of Notch pathway, mitogen-activated protein kinase (MAPK) pathway and Bcl-2 family proteins were investigated using western blot analysis.Results:We discovered that the antitumour effect of combining GSI and radiation was dependent on treatment schedule. γ-Secretase inhibitor administration after radiation had the greatest growth inhibition of lung cancer in vitro and in vivo. We showed that the combination induced apoptosis of lung cancer cell lines through the regulation of MAPK and Bcl-2 family proteins. Furthermore, activation of Notch after radiation was ameliorated by GSI administration, suggesting that treatment with GSI prevents Notch-induced radiation resistance.Conclusion:Notch has an important role in lung cancer. Treatment with GSI after radiation can significantly enhance radiation-mediated tumour cytotoxicity.

Delta‐like protein 3 ( DLL 3) is a ligand of Notch signaling, which mediates cell‐fate decisions and is tumor‐suppressive or oncogenic depending on the cellular context. Previous studies show that DLL 3 is highly expressed in small cell lung cancer ( SCLC ) but not in normal lung tissue, suggesting that DLL 3 might be associated with neuroendocrine tumorigenesis. However, its role in SCLC remains unclear. To investigate the role of DLL 3 in tumorigenesis in SCLC , we performed loss‐of‐function and gain‐of‐function assays using SCLC cell lines. In vitro analysis of cell migration and invasion by transwell assay showed that DLL 3 knockdown reduced migration and invasion of SCLC cells, whereas DLL 3 overexpression increased these activities. In addition, DLL 3 positively regulated SNAI 1 expression and knockdown of SNAI 1 attenuated the migration and invasion ability of SCLC cells. Moreover, upregulated DLL 3 expression induced subcutaneous tumor growth in mouse models. These results indicate that DLL 3 promoted tumor growth, migration and invasion in an SCLC model by modulating SNAI 1/Snail.