Inhibition of Cell-surface Molecular GPR87 With GPR87-suppressing Adenoviral Vector Disturb Tumor Proliferation in Lung Cancer Cells.
- 作者列表："Kita Y","Go T","Nakashima N","Liu D","Tokunaga Y","Zhang X","Nakano T","Nii K","Chang SS","Yokomise H
BACKGROUND/AIM:GPR87 is a member of the cell surface molecular G protein-coupled receptors (GPCR) family and suggested to contribute to the viability of human tumor cells. Its tumor-specific expression and cell surface location make it a potential molecule for targeted therapy. In the present study, we aimed to examine the effect of silencing GPR87 expression and explore the possibility of establishing gene therapy against GPR87-overexpressing lung cancer. MATERIALS AND METHODS:Twenty malignant cell lines were investigated and GPR87-overexpressing H358 and PC9 lung cancer cells were subjected to inhibiting experiments. A short hairpin siRNA targeting the GPR87 gene was transformed into an adenoviral vector (Ad-shGPR87). Real-time RT-PCR and western blot analyses were performed to evaluate gene and protein expression. Tumors derived from human H358 cells were subcutaneously implanted in nude mice for in vivo experiments. RESULTS AND CONCLUSION:About 50% (10/20) malignant cells showed GPR87-overexpression, especially for lung cancer cells (70%, 7/10). Ad-shGPR87 effectively down-regulated the GPR87 expression, and significantly inhibited the cell proliferation in GPR87-overexpressing H358 and PC9 cells. Treatment with Ad-shGPR87 exerted a significant antitumor effect against the GPR87-expressing H358 xenografts. In addition, the gene expression of H3.3, a recently proved activator for GPR87 transcription, was positively correlated with GPR87 gene expression. Furthermore, a significant decrease of KRAS and c-Myc expression was observed in both cell lines after Ad-shGPR87 infection. In conclusion, GPR87 may play a critical role in cancer cell proliferation, and indicate its potential as a novel target for lung cancer treatment.
背景/目的: GPR87 是细胞表面分子g蛋白偶联受体 (GPCR) 家族的成员，被认为有助于人类肿瘤细胞的生存。其肿瘤特异性表达和细胞表面位置使其成为靶向治疗的潜在分子。在本研究中，我们旨在检测沉默GPR87 表达的作用，并探索建立针对GPR87-overexpressing肺癌的基因治疗的可能性。 材料和方法: 研究了 20 种恶性细胞系，并对GPR87-overexpressing H358 和PC9 肺癌细胞进行了抑制实验。将靶向GPR87 基因的短发夹siRNA转化到腺病毒载体中 (Ad-shGPR87)。进行实时rt-pcr和蛋白质印迹分析以评估基因和蛋白质表达。将源自人H358 细胞的肿瘤皮下植入裸鼠中用于体内实验。 结果与结论: GPR87-overexpression大约有 50% (10/20) 的恶性细胞，尤其是肺癌细胞 (70%，7/10)。Ad-shGPR87 有效下调GPR87 的表达，并显著抑制GPR87-overexpressing H358 和PC9 细胞的增殖。用Ad-shGPR87 处理对GPR87-expressing H358 异种移植物表现出显著的抗肿瘤作用。此外，最近证实的GPR87 转录激活因子H3.3 的基因表达与GPR87 基因表达呈正相关。此外，在Ad-shGPR87 感染后，在两种细胞系中都观察到KRAS和c-Myc表达的显著降低。总之，GPR87 可能在癌细胞增殖中发挥关键作用，并表明其作为肺癌治疗的新靶点的潜力。
METHODS::Pulmonary artery sling is a rare congenital anomaly of the origin and course of the left pulmonary artery. Patients with this condition typically present with respiratory failure in young infancy, and asymptomatic cases are uncommon. We describe the case of an adult patient with a lung adenocarcinoma of the right upper lobe, extending into the hilum and superior mediastinum, and with a previously unknown pulmonary artery sling anomaly. The local invasiveness of the tumor and the peculiar vascular anatomy contributed to a unique surgical scenario, wherein multiple reconstructive procedures were required.
METHODS::Patients with idiopathic pulmonary fibrosis (IPF) have higher risk of developing lung cancer, for example, squamous cell carcinoma (SCC), and show poor prognosis, while the molecular basis has not been fully investigated. Here we conducted DNA methylome analysis of lung SCC using 20 SCC samples with/without IPF, and noncancerous lung tissue samples from smokers/nonsmokers, using Infinium HumanMethylation 450K array. SCC was clustered into low- and high-methylation epigenotypes by hierarchical clustering analysis. Genes hypermethylated in SCC significantly included genes targeted by polycomb repressive complex in embryonic stem cells, and genes associated with Gene Ontology terms, for example, "transcription" and "cell adhesion," while genes hypermethylated specifically in high-methylation subgroup significantly included genes associated with "negative regulation of growth." Low-methylation subgroup significantly correlated with IPF (78%, vs. 17% in high-methylation subgroup, p = 0.04), and the correlation was validated by additional Infinium analysis of SCC samples (n = 44 in total), and data from The Cancer Genome Atlas (n = 390). The correlation between low-methylation subgroup and IPF was further validated by quantitative methylation analysis of marker genes commonly hypermethylated in SCC (HOXA2, HOXA9 and PCDHGB6), and markers specifically hypermethylated in high-methylation subgroup (DLEC1, CFTR, MT1M, CRIP3 and ALDH7A1) in 77 SCC cases using pyrosequencing (p = 0.003). Furthermore, low-methylation epigenotype significantly correlated with poorer prognosis among all SCC patients, or among patients without IPF. Multivariate analysis showed that low-methylation epigenotype is an independent predictor of poor prognosis. These may suggest that lung SCC could be stratified into molecular subtypes with distinct prognosis, and low-methylation lung SCC that significantly correlates with IPF shows unfavorable outcome.
METHODS::The role of Fyn-related kinase (FRK) in malignant tumors remains controversial. Our study investigated the function of FRK in lung cancer. Immunohistochemistry staining and generating a knockout of FRK by CRISPR/Cas9 in H1299 (FRK-KO-H1299) cells were strategies used to explore the role of FRK. Immunohistochemistry staining indicated that FRK expression was elevated in 223 lung cancer tissues compared to 26 distant normal lung tissues. FRK contributed to poor survival status in lung cancer patients and acted as a predictor for poor prognosis of lung cancer. Knockout of FRK by CRISPR/Cas9 markedly inhibited proliferation, invasion, colony formation and epithelial-mesenchymal transition (EMT) process in the lung cancer cell line H1299. Further exploration indicated that FRK-KO damaged the stemness phenotype of H1299 by inhibiting CD44 and CD133 expression. Seahorse detection and a U-13 C flux assay revealed that FRK-KO induced metabolism reprogramming by inhibiting the Warburg effect and changing the energy type in H1299 cells. Epidermal growth factor stimulation recovered the expression of FRK and biological functions, metabolic reprogramming and stemness phenotype of H1299 cells. FRK plays an oncogenic role in lung cancer cells via a novel regulation mechanism of enhancing the stemness of H1299 cells by inducing metabolism reprogramming, which finally promotes EMT and metastasis. Our study also indicates that FRK could be used as a potential therapeutic target for drug development.