Novel ADAM-17 inhibitor ZLDI-8 inhibits the metastasis of hepatocellular carcinoma by reversing epithelial-mesenchymal transition in vitro and in vivo.
新型ADAM-17 抑制剂ZLDI-8 通过逆转上皮-间质转化在体内外抑制肝细胞癌的转移。
- 作者列表："Lu HY","Chu HX","Tan YX","Qin XC","Liu MY","Li JD","Ren TS","Zhang YS","Zhao QC
AIMS:Epithelial-mesenchymal transition (EMT) is one of the important regulators of metastasis in advanced hepatocellular carcinoma (HCC). Blocking the Notch signaling pathway and then reversing the EMT process is a hot spot in clinical tumor research. Here, we aimed to investigate the effect and underlying mechanisms of ADAM-17 (a key cleavage enzyme of Notch pathway) inhibitor ZLDI-8 we found before on the metastasis of hepatocellular carcinoma in vitro and in vivo. MAIN METHODS:The cell viability of HCC cells was evaluated by MTT and colony formation assays. Migration and invasion were assessed respectively with wound healing and transwell assays. The expression and location of proteins were detected by western blot and immunofluorescence, respectively. The effects of ZLDI-8 on metastasis of liver cancer in vivo were investigated in a tail vein injection model. KEY FINDINGS:In the present work, ZLDI-8 significantly inhibited proliferation, migration, invasion and EMT phenotype of highly aggressive MHCC97-H and LM3 cells. Moreover, ZLDI-8 could inhibit the migration and invasion of HepG2 and Bel7402 cells induced by TGF-β1. ZLDI-8 suppressed the protein expression of interstitial markers and increased that of epithelial markers. Meanwhile, ZLDI-8 decreased the expression of proteins in the Notch signaling pathway. Finally, ZLDI-8 blocks metastasis in the lung metastasis model in vivo. SIGNIFICANCE:ZLDI-8 suppressed the metastasis of hepatocellular carcinoma, which was associated with reversing the EMT process and regulating Notch signaling pathway. The study laid the foundation for the discovery of drugs that reverse EMT to inhibit advanced HCC metastasis.
目的: 上皮-间质转化 (EMT) 是晚期肝细胞癌 (HCC) 转移的重要调节因子之一。阻断Notch信号通路进而逆转EMT过程是临床肿瘤研究的热点。在此，我们旨在研究ADAM-17 (Notch通路的关键裂解酶) 的作用和潜在机制我们在体外和体内发现了抑制剂ZLDI-8 对肝癌转移的影响。 主要方法: 通过MTT和集落形成试验评估HCC细胞的细胞活力。分别用伤口愈合和transwell试验评估迁移和侵袭。蛋白的表达和定位分别通过western blot和免疫荧光检测。在尾静脉注射模型中研究了ZLDI-8 对体内肝癌转移的影响。 主要发现: 在本工作中，ZLDI-8 显著抑制高度侵袭性MHCC97-H和LM3 细胞的增殖、迁移、侵袭和EMT表型。ZLDI-8 可抑制TGF-β1 诱导的HepG2 和Bel7402 细胞的迁移和侵袭。ZLDI-8 抑制了间质标志物的蛋白表达，增加了上皮标志物的蛋白表达。同时，ZLDI-8 降低了Notch信号通路中蛋白的表达。最后，ZLDI-8 在体内阻断肺转移模型中的转移。 意义: ZLDI-8 抑制肝细胞癌的转移，这与逆转EMT过程和调节Notch信号通路有关。该研究为发现逆转EMT以抑制晚期HCC转移的药物奠定了基础。
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.