RPTOR blockade suppresses brain metastases of NSCLC by interfering the ceramide metabolism via hijacking YY1 binding.

BACKGROUND: Ceramide metabolism is crucial in the progress of brain metastasis (BM). However, it remains unexplored whether targeting ceramide metabolism may arrest BM. METHODS: RNA sequencing was applied to screen different genes in primary and metastatic foci and whole-exome sequencing (WES) to seek crucial abnormal pathway in BM + and BM-patients. Cellular arrays were applied to analyze the permeability of blood-brain barrier (BBB) and the activation or inhibition of pathway. Database and Co-Immunoprecipitation (Co-IP) assay were adopted to verify the protein-protein interaction. Xenograft and zebrafish model were further employed to verify the cellular results. RESULTS: RNA sequencing and WES reported the involvement of RPTOR and ceramide metabolism in BM progress. RPTOR was significantly upregulated in BM foci and increased the permeability of BBB, while RPTOR deficiency attenuated the cell invasiveness and protected extracellular matrix. Exogenous RPTOR boosted the SPHK2/S1P/STAT3 cascades by binding YY1, in which YY1 bound to the regions of SPHK2 promoter (at -353 ~ -365 nt), further promoting the expression of SPHK2. The latter was rescued by YY1 RNAi. Xenograft and zebrafish model showed that RPTOR blockade suppressed BM of non-small cell lung cancer (NSCLC) and impaired the SPHK2/S1P/STAT3 pathway. CONCLUSION: RPTOR is a key driver gene in the brain metastasis of lung cancer, which signifies that RPTOR blockade may serve as a promising therapeutic candidate for clinical application.

NCAPG promotes the progression of lung adenocarcinoma via the TGF-ß signaling pathway.

BACKGROUND: Lung cancer has the highest case fatality rate among cancers because of uncontrolled proliferation and early metastasis of cancer cells in the lung tissue. This study aimed to clarify the role of the non-SMC condensin I complex, subunit G (NCAPG) in lung adenocarcinoma (LUAD), explore the mechanisms of its progression, and lay the foundation for the search for new biological markers. METHODS: We analyzed overlapping differentially expressed genes (DEGs) from three datasets; a protein-protein interaction (PPI) network was subsequently constructed and analyzed using Cytoscape. We then selected NCAPG for validation because of its poor prognosis and because it has not been sufficiently studied in the context of LUAD. Immunohistochemical analysis was used to detect the expression of NCAPG in LUAD tissues, and the relationships between NCAPG and clinical parameters were analyzed. In vitro and in vivo experiments were conducted to verify the role of NCAPG in LUAD. Finally, we studied the specific mechanism of action of NCAPG in LUAD. RESULTS: Through comprehensive analysis of the GSE43458, GSE75037, and The Cancer Genome Atlas databases, we identified 517 overlapping DEGs. Among them, NCAPG was identified as a hub gene. Immunohistochemical analysis revealed that NCAPG was strongly associated with the clinical stage, M-classification, and N-classification. Univariate and multivariate Cox regression analyses indicated that NCAPG was a prognostic risk factor for LUAD, while the in vitro experiments showed that NCAPG overexpression promoted proliferation, migration, invasion, and epithelial-mesenchymal transition. Furthermore, knockdown of NCAPG inhibited LUAD progression, both in vitro and in vivo. Mechanistically, NCAPG overexpression increased p-Smad2 and p-Smad3 expressions in the transforming growth factor ß (TGF-ß) signaling pathway. Additionally, rescue experiments indicated that TGF-ß signaling pathway inhibitors could restore the effect of NCAPG overexpression in LUAD cells. CONCLUSIONS: NCAPG may promote proliferation and migration via the TGF-ß signaling pathway in LUAD.

CXCR4/SDF-1 axis regulates the effect of human lung adenocarcinoma PC-9 cells on function of in vitro blood-brain barrier model formed by Bends cells / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the influences of human lung adenocarcinoma PC-9 cells on tight junction proteins of blood brain barrier (BBB) under CXCR4/SDF-1 axis by establishing a model of BBB in vitro. Methods: The immortalized mouse brain microvascular endothelial Bends cells were used to establish a model of BBB in vitro by monolayer culture; Subsequently, transendothelial electric resistance (TEER) and fluorescein sodium permeability experiment were used to detect the function of in vitro BBB model and observe the effect of PC-9 cells on the function of BBB model, respectively. Western blotting was used to detect the effect of PC-9 cells on function of BBB model and expressions of endothelial tight junction proteins under the treatment of single or combined AMD3100 and SDF-1 (1 μg/ml AMD3100,100 ng/ml SDF-1, AMD3100+SDF-1). Transwell assay was used to detect the influence of CXCR4/SDF-1 axis on the ability of PC-9 cells transmigrating the cell layer of BBB model. Results: Monolayer culture of Bends cells can form tightly connected BBB withhighTEER,which reached (182.13±5.19) Ω.cm2 at the 96 h; in the meanwhile, fluorescein sodium permeability experiment showed that BBB had significantly lower permeability than that of control group ([40.31±2.43]% vs [150.10±3.17]%, P<0.05). The TEER of BBB decreased to (46.7±4.35) Ω·cm2 after coculture with PC-9 cells for 24 h, and at the same time the sodium fluorescein permeability of BBB significantly increased than that of pre-treatment ([136.32±4.93]% vs [50.24±6.21]%, P<0.05). PC-9 cells up-regulated the expressions of tight junction proteins of Bends cells under the treatment of AMD3100 (P<0.05). The number of PC-9 cells transmigrating the BBB inAMD3100 treatment group was significantly lower than that of CON group (43±2 vs 81±2, P<0.05). Conclusion: AMD3100 can reduce the ability of PC-9 cells destroying the tight junction of the BBB model established in vitro by Bends cells.

A case-control study on prognosis between EGFR gene mutant-and wild-type non-small cell lung cancer with brain metastasis / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨表皮生长因子受体（epidermal growth factor receptor，EGFR）基因突变与非小细胞肺癌（non-small cell lung cancer，NSCLC）伴脑转移患者预后的相关性，为改善NSCLC合并脑转移患者预后、指导个体化治疗提供临床依据。方法：回顾性分析福建省立医院2013年1月1日至2018年9月30日期间收治的88例NSCLC合并脑转移患者的临床资料，随访取得患者的死亡时间，随访截止日期为2019年10月31日。收集和分析的临床资料包括性别、年龄、吸烟史、病理类型、基因检测、治疗情况、无进展生存期（progression free survival，PFS）、总生存期（overall survival，OS）等。运用生存分析（Kaplan-Meier生存时间曲线）评价EGFR突变型患者的预后，以单因素分析（log-rank检验）预测影响EGFR-TKI治疗效果的因素。结果：88例NSCLC脑转移患者有57例为EGFR突变型，其中位PFS（MPFS）为13.0个月（95%CI：11.951~14.049），明显高于EGFR野生型患者（P=0.003），患者中位生存期（median survival time，MST）为29.0个月（95%CI：20.531~37.468），明显高于EGFR野生型（P=0.001）。EGFR突变型中，Exon19-del突变组患者较Exon21 L858R突变组患者OS有延长趋势（P=0.05），Exon19-del+Exon20T790M突变组患者OS较Exon21 L858R突变组有延长趋势（P=0.077）。结论：EGFR突变组较野生型组NSCLC脑转移患者预后相对好些，且携带19外显子单一缺失突变的患者预后最好。

Suppressive effect of Tet-on mediated livin RNA interference on growth of lung carcinoma xenegraft in a rat model / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate he effect of tetracycline- (Tet-on) mediated livin RNA interference on growth of lung carcinoma xenegrafts, and find a better regulatory way to interfere the development on lung cancer. Methods: livin shRNA lentiviral vectors were constructed; and the lung cancerA549 cells were subcutaneously injected into right upper back of nude mice to establish xenegraft model. The livin shRNAlentiviral vectors were injected into xenografts to interfere the expression of livin, then tetracycline was injected intraperitoneally for the induction. The suppressive effect of Tet-on mediated livin RNA interference efficiency was investigated and lung cancer xenograft development was observed. Results: After the induction with Tet-on, livin gene expression was significantly inhibited by livin shRNAcompared with the control group and Tet-on-NC group; the xenograft volume in Tet-on- livin shRNAgroup was significantly smaller than that in control group and Tet-on-NC group ([5.31±0.86]g vs [8.22±0.63]g and [7.17±0.54] g, P<0.05). Moreover, little body toxicity was observed and no nude mice died in this study. Conclusion: The Tet-on mediated livin shRNA could suppress the growth of lung cancer development with good targeting and controllable characteristics, which might provide a potent tool for treating lung cancer with livin protein as target.

AMD3100 inhibits brain-specific metastasis in lung cancer via suppressing the SDF-1/CXCR4 axis and protecting blood-brain barrier.

Lung cancer represents the foremost cause of cancer-related mortality in both men and women throughout the world. Metastasis to the brain constitutes a major problem in the management of patients with lung cancer. However, the mechanism of brain-specific metastasis in lung cancer has not been fully elucidated. Chemokines and their receptors have emerged as attractive targets regulating the cancer metastasis. It has been discovered that the stromal cell-derived factor 1 (SDF-1)/CXCR4 axis plays a critical role in determining the metastatic destination of tumor cells. In this study, strong expression of SDF-1 was observed in highly metastatic brain tissues, and CXCR4 overexpressed in PC-9 lung cancer cells and tumor foci. Therefore, we chose to block SDF-1/CXCR4 axis with AMD3100, which led to the increased tight junction protein level, less damage, and decreased permeability of blood-brain barrier (BBB). Consequently, the process of lung cancer metastasis to the brain was significantly slowed down. These findings were further validated by in vivo experiments, which showed that AMD3100 can effectively inhibit lung cancer brain metastasis and extend the survival of nude mice model, suggesting that it is a potential drug candidate for inhibiting the lung cancer metastasis to brain. These findings provided valuable information for designing new therapeutic strategies for the treatment of lung cancer brain metastasis.

Silencing of Livin inhibits tumorigenesis and metastasis via VEGF and MMPs pathway in lung cancer.

Livin, an inhibitor of apoptosis protein (IAP), is overexpressed in various cancers and decreases tumor sensitivity to chemotherapy and radiotherapy. However, the effect of Livin on lung adenocarcinoma metastasis and the specific mechanism involved remain unclear. RNAi technology was used to stably silence Livin in A549 cells in the present study. The effect of Livin on tumor growth and invasion was investigated in lung cancer cells in vitro and animal models were established to determine the anti-metastasis ability of Livin silencing in vivo. The results indicated that Livin knock-down suppressed cell proliferation and inhibited cell invasion, accompanied by downregulation of VEGF and MMP-2/-9. Silencing of Livin resulted in the prevention of xenograft tumor formation. Seventy-five immunodeficient male BALB/C nude mice were randomly divided into three groups, the relative ratio of the areas with pulmonary nodules in the experimental group decreased from 46.71±7.27% to 11.07±2.94% compared with the negative control group (P<0.001), indicating the interaction between Livin, VEGF and MMPs. The xenograft tumor model of intravenous injection of tumor cells were successfully established and applied for the analysis of lung cancer tumorigenesis and metastasis in a time-dependent manner for the first time. Based on the reliable and reproducible animal model, our findings indicate that knock-down of Livin inhibits cell growth and invasion through blockade of the VEGF and MMPs pathways in lung cancer cells in vitro, and inhibits tumorigenesis and metastasis of lung cancer in vivo, suggesting that Livin is a promising antitumor target.