Identification of mitochondrial permeability transition-related gene signatures to predict lung adenocarcinoma survival and drug response

Lung cancer is the most common type of cancer, accounting for more than half of all cancer cases, with lung adenocarcinoma (LUAD) representing over half of lung cancer patients. Currently, the 5-year survival rate for metastatic LUAD patients remains low and there is an urgent need for new biomarkers as targets for targeted therapy. Go-Ichi-Ni-San 1 (GINS1), an important member of the GINS family, is closely related to the occurrence and development of human malignant tumors. This study aims to explore the role of GINS1 in glycolysis, proliferation, and metastasis of LUAD cells and the related molecular mechanisms. The expression of GINS1 was analysed using bioinformatics between LUAD patients and healthy controls. The expression levels of GINS1 in LUAD and adjacent tissues were detected by immunohistochemistry and Western blot. Western blot and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) were used to detect the expression of GINS1 in LUAD cell lines A549, SK-LU-1, Calu-3, H1299 and BEAS-2B. Stably knockdown GINS1 in A549 cells and its negative control cell line, as well as stably overexpress GINS1 in H1299 cells and its negative control cell line, were constructed by lentiviral transduction. Colony formation test was used to detect cell proliferation. Scratch test was used to detect cell migration. Transwell test was used to detect cell invasion, and the test kits were used to detect glucose consumption and lactate production. The expression levels of glycolysis-related proteins, Notch signaling pathway proteins and phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway proteins were detected by Western blot. The Notch receptor agonist Jagged1 was added to cells from the shGINS1-A549 group and the Notch receptor inhibitor LY3039478 was added to cells from the GINS1-OE-H1299 group for the regression assay. The expression of GINS1 was up-regulated in LUAD patients, tissues and cell lines, and correlated with overall survival (P<0.05). Knockdown of GINS1 significantly inhibited the proliferation, migration and invasion of A549 cells (P<0.05), while overexpression of GINS1 significantly enhanced the proliferation, migration and invasion of H1299 cells (P<0.05). Furthermore, knockdown of GINS1 resulted in reduced glucose consumption, reduced lactate production, and reduced expression levels of glycolytic-related proteins in A549 cells (P<0.05); overexpression of GINS1 enhanced glycolytic level in H1299 cells (P<0.05). The expression levels of Notch1, Notch3, phosphorylated-PI3K (p-PI3K), phosphorylated-AKT (p-AKT) and phosphorylated-mTORC1 (Ser2448)[p-mTORC1 (Ser2448)] in A549 cells were significantly decreased by GINS1 knockdown (P<0.05), while the expression levels of PI3K, AKT, mTOR and p-mTORC2 (Ser2481) were not significantly changed (P>0.05). Overexpression of GINS1 increased the levels of Notch1, Notch3 and PI3K/AKT/mTORC1 pathway phosphorylated proteins in H1299 cells (P<0.05). Jagged1 significantly reversed the inhibition of glycolysis, proliferation and metastasis induced by GINS1 knockdown in A549 cells (P<0.05), and LY3039478 significantly inhibited the enhancement of glycolysis, proliferation and metastasis induced by GINS1 overexpression in H1299 cells (P<0.05). The expression of GINS1 enhances the expression of Notch1 and Notch3 receptors, and then phosphorylates and activates the downstream PI3K/AKT/mTORC1 signaling pathway to enhance the glycolysis, proliferation and metastasis of LUAD cells.

Lung adenocarcinoma (LUAD) is a malignant tumor that occurs in the lungs. Numerous reports have substantiated the participation of long non-coding RNAs (lncRNAs) in the tumorigenesis of LUAD. Previously, lncRNA alpha-2-macroglobulin antisense RNA 1 (A2M-AS1) was confirmed to be an important regulator in the biological processes of LUAD and dysregulation of A2M-AS1 was associated with non-small cell lung cancer (NSCLC) progression. However, the precise mechanism of A2M-AS1 in LUAD has not been elucidated. Therefore, our study was designed to investigate the detailed molecular mechanism of A2M-AS1 in LUAD. Herein, the expression of lncRNA A2M-AS1, microRNA (miRNA) miR-587, and bone morphogenetic protein 3 (BMP3) in LUAD cell lines and tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. The viability, proliferation, migration and invasion of LUAD cells were tested by cell counting kit-8 (CCK-8), colony formation and Transwell assays. In vivo tumor growth was investigated by xenograft animal experiment. Interactions among A2M-AS1, miR-587 and BMP3 were measured by RNA pulldown and luciferase reporter assays. In this study, A2M-AS1 was downregulated in LUAD tissues and cells and related to poor prognosis in LUAD patients. A2M-AS1 overexpression suppressed LUAD cell proliferation, migration and invasion in vitro and inhibited tumor growth in vivo. Mechanistically, A2M-AS1 directly bound with miR-587 to promote BMP3 expression in LUAD cells. Low expression of BMP3 was found in LUAD tissues and cells and was closely correlated with poor prognosis in LUAD patients. BMP3 deficiency reserved the inhibitory influence of A2M-AS1 overexpression on LUAD cell behaviors. Overall, A2M-AS1 inhibits cell growth and aggressiveness via regulating the miR-587/BMP3 axis in LUAD.

背景与目的肺腺癌（lung adenocarcinoma, LUAD）是当前最常见的肺癌亚型，微小RNA（microRNAs, miRNAs）是一类非编码小RNA，在细胞活动中发挥核心作用。miR-30b-3p在许多类型的癌症中均起到了关键作用，但关于其在肺腺癌中如何发挥作用的研究仍然很少。本研究通过探索miR-30b-3p在肺腺癌增殖和侵袭中的作用和机制，以期为临床上抑制肺腺癌的增殖和侵袭拓展新的方向。方法利用NCBI生物数据库查找肺腺癌中差异表达明显的miRNA，并查询其差异表达及生存曲线；采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction, qRT-PCR）检测miR-30b-3p在各肺腺癌细胞系中的表达；5-乙炔基-2’脱氧尿嘧啶核苷（5-ethynyl-2'-deoxyuridine, EdU）细胞增殖实验和Transwell实验检测各组A549细胞增殖和侵袭能力的变化；使用在线预测数据网站确定miR-30b-3p的靶蛋白；Western blot验证COX6B1在不同组肺腺癌细胞中的表达情况；双荧光素酶实验证实miR-30b-3p与COX6B1是否存在结合位点。结果miR-30b-3p在肺腺癌组织和细胞中表达下调（P<0.05），低表达水平的miR-30b-3p与肺腺癌患者的不良预后有关（P=0.005,8）；过表达miR-30b-3p能够抑制肺腺癌细胞的增殖与侵袭能力（P<0.05）；双荧光素酶实验证明miR-30b-3p和COX6B1存在结合位点（P<0.05）；Western blot实验表明在肺腺癌细胞A549中，过表达miR-30b-3p能够下调COX6B1的表达（P<0.05）；EdU细胞增殖实验和Transwell侵袭实验表明，miR-30b-3p的过表达能够逆转上调COX6B1对肺腺癌细胞增殖和侵袭能力的促进作用（P<0.05）。结论miR-30b-3p在肺腺癌中起到了抑癌基因的作用，并能够通过调控COX6B1的表达来抑制肺腺癌的增殖和侵袭。

BackgroundLung adenocarcinoma (LAD) is a highly aggressive malignant tumor which threatens the health and life of the population. Long non‐coding RNA X‐inactive specific transcript (XIST) and mouse double minute clone 2 (MDM2) are connected with the tumorigenesis of LAD. Nevertheless, whether MDM2 is regulated by XIST has not previously been reported in LAD.MethodsQuantitative real‐time polymerase chain reaction (qRT‐PCR) was employed to detect the expression of XIST, microRNA‐363‐3p (miR‐363‐3p) and MDM2 in LAD tissues and cells. The proliferation, migration, invasion and apoptosis of LAD cells were determined by 3‐(4, 5‐dimethylthiazol‐2‐YI)‐2, 5‐diphenyltetrazolium bromide (MTT), transwell or flow cytometry assay, respectively. MDM2 protein level was detected using western blot analysis. Dual‐luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pulldown assay were performed to determine the interaction among XIST, miR‐363‐3p and MDM2. A xenograft tumor model was constructed to validate the effect of XIST on LAD cells in vivo.ResultsWe found that XIST and MDM2 were remarkably elevated while miR‐363‐3p was reduced in LAD tissues and cells. Both XIST and MDM2 downregulation restrained proliferation, migration and invasion, and facilitated apoptosis of LAD cells in vitro. Importantly, XIST bound to miR‐363‐3p to modulate MDM2 expression in LAD cells. Moreover, miR‐363‐3p knockdown or MDM2 elevation reversed the effects of XIST downregulation on the proliferation, migration, invasion and apoptosis of LAD cells. Furthermore, XIST knockdown constrained tumor growth on LAD cells in vivo.ConclusionsXIST knockdown repressed proliferation, migration and invasion, and accelerated apoptosis of LAD cells by downregulating MDM2 expression via binding to miR‐363‐3p.Key pointsSignificant findings of the studyXIST and MDM2 were abnormally enhanced in LAD tissues and cells.Both downregulation of XIST and MDM2 repressed proliferation, migration and invasion, and boosted apoptosis of LAD cells in vitro.XIST bound to miR‐363‐3p to regulate MDM2 expression in LAD cells.Downregulation of XIST impeded tumor growth on LAD cells in vivo.What this study addsThis study confirmed that XIST was a potential target for inhibiting the development of LAD, and affords a possible strategy for the treatment of LAD in the future.

Introduction and Purpose: Recently, immune checkpoint therapy has been incorporated into treatment of solid cancers including lung cancer, resulted in prolonged survival benefit. Checkpoint inhibitors target on PD-1/PD-L1 or CTLA-4 immuno-inhibitory molecules in immune or cancer cells. However, checkpoint therapy in lung adenocarcinoma (LAD) showed less than 20% response rate, suggesting existence of other immuno-inhibitory pathways. Therefore, we focused on T cell immunoglobulin and mucin domain 3 (TIM-3) on the CD4 and CD8 T-cell surface and its ligand Galectin-9 in tumor cells, and analyzed the expression and function in LAD cells and tissues. Materials and Methods: TILs and PBMCs were obtained from 11 patients with LAD, and 194 LAD tissues resected surgically were analyzed. Expression of immuno-inhibitory molecules (PD-1, TIM-3, BTLA, LAG-3) was examined by flow cytometry using FACS on TILs and PBMCs, and PD-L1 and Galectin-9 expressions in LAD tissues was analyzed by immunohistochemistry using tissue microarray. To investigate soluble Galectin-9 released from LAD cells, EGFR-mutated PC-9 and EGFR-wild-type OU-LC-SK cells were treated with EGFR-TKI afatinib at a concentration of 10 nM for 3 days. Then, the amount of Galectin-9 in culture supernatant was measured by ELISA. To investigate T-cell apoptosis induction by Galectin-9, established XAGE1-specifiic CD8 cloned T-cells were incubated with Galectin-9 protein for 8 hrs. Results: Increased expression in TILs compared to PBMCs was observed on PD-1 and TIM-3, but not on BTLA or LAG-3 in CD4 and CD8 T-cells. In LAD tissues, the frequencies of high PD-L1 and Galectin-9 expression in the tumor cell membrane or cytoplasm were 49% and 31%, respectively (in squamous cell carcinoma, the frequencies were 32% and 16%, respectively). Furthermore, correlated expression of PD-L1, Galectin-9 and CD3 (T-cell infiltration) was observed at the periphery of the tumor nest. Those findings suggest the relevance of the PD-1/PD-L1 and TIM-3/Galectin-9 pathways in the tumor microenvironment of LAD. Next, soluble Galectin-9 released from the LAD cells was measured. Galectin-9 was detected only in the medium of EGFR-mutated LAD cells following treatment with afatinib. Moreover, apoptosis was induced in TIM-3-positive CD8 T-cell clones following interaction with Galectin-9 protein and this was inhibited by the addition of anti-Galectin-9 or an anti-TIM-3 antibody. The findings suggested that a significant amount of Galectin-9 could be released from LAD cells and induced T-cell apoptosis in tumor microenvironment. Conclusions: Our results suggested the relevance of the PD-1/PD-L1 and TIM-3/Galectin-9 immuno-inhibitory pathways in the tumor microenvironment of LAD, and that release of soluble Galectin-9 from LAD cells could negatively regulate T-cell function. For successful immune checkpoint therapy in LAD, simultaneous inhibition of TIM-3/Galectin-9 pathway may be needed. Citation Format: Mikio Oka, Yoshihiro Ohue, Koji Kurose, Yumi Nishio, Midori Isobe, Eiichi Nakayama. Immuno-inhibitory pathway, TIM-3/Galectin-9, in lung adenocarcinoma: clinical and in vitro analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2932. doi:10.1158/1538-7445.AM2017-2932

Transcriptomic atlas of GNAT family members in pulmonary epithelia under pathological conditions using single‐cell and bulk cell sequencing

CircRNAs can regulate ferroptosis and affect cancer development and are promising biomarkers and therapeutic targets in lung cancer. circSCUBE3 is expressed in lung adenocarcinoma (LUAD) tissues. In this study, our purpose was to study the role and regulatory mechanism of circSCUBE3 in LUAD ferroptosis. circSCUBE3 was identified to be significantly downregulated in LUAD samples and cell lines. The expression of biomarkers related to lipid oxidation (4-HNE) and ferroptosis (Ptgs2) was both downregulated in LUAD tissues, suggesting the ferroptosis resistance in LUAD. Erastin, a ferroptosis inducer, was used to stimulate the LUAD cells for 48 h. The cell viability, 4-HNE and Ptgs2 level of LUAD cells were decreased by exposure to erastin while the expression of circSCUBE3 was not significantly altered. We then overexpressed circSCUBE3 in LUAD cells and found it decreased the GSH level and GSH/GSSG ratio in LUAD cells. CircSCUBE3 might serve as an independent factor of ferroptosis and may induce ferroptosis in LUAD by inhibiting GSH synthesis. The loss-of-function experiments were conducted, and circSCUBE3 deficiency reversed the erastin-induced reduction in cell viability, GSH level, GSH/GSSG ratio, mitochondrial membrane potential and elevation in MDA content, Ptgs2, 4-HNE expression as well as lipid ROS production. CircSCUBE3 negatively regulated GPX4 expression in LUAD cells, and the silencing of GPX4 counteracted the impact of circSCUBE3 deficiency on LUAD cell viability as well as ferroptosis, suggesting that circSCUBE3 regulated the GPX4-mediated GSH synthesis in LUAD. CircSCUBE3 was to bind to CREB, which activated the transcription of GPX4. CircSCUBE3 negatively regulated GPX4 expression by competitively interacting with CREB. In the tumor-bearing mouse models, circSCUBE3 silencing promoted tumor growth and reversed the erastin treatment-induced inhibition on tumorigenesis in vivo. In conclusion, circSCUBE3 inhibited LUAD development by promoting ferroptosis via the CREB/GPX4/GSH axis, which might provide a novel option for the LUAD targeted therapy.

Increasing evidence suggests that lactate is an essential compound in the tumor microenvironment, and especially for macrophage cells. However, the mechanism by which lactate affects macrophages remains unclear. This study investigated whether and how lactate affects macrophage polarization in lung adenocarcinoma (LUAD). Clinical samples of LUAD and paracancerous tissue were obtained for evaluation of lactate dehydrogenase A (LDHA) expression. LUAD cell lines and THP-1 induced macrophages were used in this study. Quantitative real-time PCR (QPCR), western blotting, and immunohistochemical (IHC) staining were performed to detect gene expression. Flow cytometry and ELISA assays were used to detect the levels of M1 macrophage and M2 macrophage biomarkers. LDHA was highly expressed in the LUAD tissues. Culture medium supernatants derived from LUAD cells (CM) promoted macrophage M2 polarization, and lactate levels were elevated in the CM. Inhibition of LDHA in LUAD cells decreased lactate levels and suppressed M2 macrophage polarization. Moreover, overexpression of GPR132 in macrophages promoted, while GPR132 knockdown in macrophages suppressed M2 macrophage polarization and cAMP (Cyclic Adenosine 3',5'-Monophosphate)/PKA (Protein Kinase) pathway activation induced by lactate. The effect of GPR132 overexpression was reversed by a PKA inhibitor (H-89). Collectively, our results confirmed that lactate released by LUAD cells promoted M2 macrophage polarization via the GPR132/cAMP/PKA pathway.

The late diagnosis and easy metastasis of lung adenocarcinoma (LADC) remains a challenge. SGPP2 is reported to modulate cell processes in many cancers. However, the roles and molecular mechanisms of SGPP2 in LADC are unclear. Online bioinformatics tools GEPIA, CPTAC, and K-M plotter were used to analyze the expression of SGPP2 and the prognosis in LADC. JASPAR and PROMO were used to predict the transcription factors of SGPP2. Real-time quantitative reverse transcription PCR and western blot were used to detect the levels of SGPP2 in LADC cell lines and tissues. Cell counting kit-8, colony formation, flow cytometry, and transwell assay were used to detect cell proliferation, apoptosis, and invasion. The anti-cancer effect of SGPP2 silence was evaluated in the LADC xenograft model. It was found that SGPP2 was highly expressed and related to the poor prognosis of LADC patients. Elevated SGPP2 expression was detected in LADC cell lines and tissues. The chi-square test indicated that the expression of SGPP2 was positively related to tumor, node, metastasis grades and lymph node metastasis. Knocking down SGPP2 significantly inhibited LADC cell viability, and invasion, but induced apoptosis. The anti-tumor effects of SGPP2 were verified in vivo. The upstream transcription factor of SGPP2 was predicted to be SP1, which was highly expressed in LADC tissues and cell lines. Overexpression of SP1 partly rescued the inhibition of SGPP2-shRNA in cell growth, colony formation, and invasion capabilities, and decreased apoptotic cell number in LADC cells. This study demonstrated that SGPP2, activated by SP1, promotes LADC cell proliferation and invasion, and suppresses apoptosis in LADC.

Lung adenocarcinoma (LUAD) is a high aggressive human cancer which usually diagnosed at advanced stages. Accumulating evidences indicate that long noncoding RNAs (lncRNAs) are crucial participants in LUAD progression. In the present study, we found that lncRNA LINC00968 was significantly down-regulated in LUAD tissues and cell lines. LINC00968 level was positively correlated to survival rate, and negatively correlated to tumor node metastasis (TNM) stage, tumor size and lymph node metastasis of LUAD patients. We over-expressed LINC00968 in LUAD cells using lentivirus, inhibited proliferation and cell cycle arrest at G1 phase were detected. LINC00968 over-expression also suppressed migration, invasion and epithelial mesenchymal transition. We further validated that LINC00968 localized in cytoplasm and acted as an upstream regulator of microRNA miR-22-5p, which was up-regulated in LUAD tissues and cell lines. Besides, elevated miR-22-5p expression abolished the effect of LINC00968 over-expression on LUAD progression including in vivo tumor growth. In addition, we first validated that cell division cycle 14A (CDC14A), which was down-regulated in LUAD tissues, was a downstream target of miR-22-5p. We over-expressed CDC14A in LUAD cells and miR-22-5p induced LUAD progression was partially reversed. In conclusion, our study demonstrated that LINC00968 inhibited proliferation, migration and invasion of LUAD by sponging miR-22-5p and further restoring CDC14A. This novel regulatory axis might provide us with promising diagnostic and therapeutic target in LUAD treatment.

Lung adenocarcinoma (LUAD) is a highly malignant tumor. In this study, we examined the role of miR-3646 and its underlying mechanism in the progression of LUAD. The expression of miR-3646 and sorbin and SH3 domain-containing protein 1 (SORBS1) in LUAD tissues and cells was evaluated by quantitative reverse transcription-polymerase chain reaction. LUAD cell adhesion, proliferation, apoptosis was determined. The targeting relationship between SORBS1 and miR-3646 was verified by dual luciferase and RNA pull-down assays. In vivo assays were performed to verify the in vitro results. The expression of miR-3646 was found to be upregulated in LUAD tissues and cells. MiR-3646 overexpression stimulated the proliferation and adhesion of LUAD cells but inhibited apoptosis, whereas a miR-3646 inhibitor produced the opposite results. Furthermore, the inhibitory effect of miR-3646 inhibitor was verified in vivo. SORBS1, a target gene identified downstream of miR-3646, was downregulated in LUAD tissues and cells. Additionally, increased SORBS1 inhibited the malignant phenotypes of LUAD cells, which was restored by miR-3646 upregulation. Additionally, western blot analysis revealed that SORBS1 ectopic expression disrupted the JNK signaling pathway, and this effect was restored by miR-3646 overexpression. Thus, this study revealed that miR-3646 promotes LUAD cell proliferation and adhesion, and reduces apoptosis by directly downregulating SORBS1 via the JNK signaling pathway. Investigation of the molecular mechanism of LUAD carcinogenesis revealed that miR-3646 may serve as a biomarker for LUAD treatment.in vivo

We examined the expression of oncoprotein 18 (Op18) in 93 lung adenocarcinomas and 10 uninvolved lung samples using quantitative two-dimensional PAGE analysis with confirmation by mass spectrometry and two-dimensional Western blot analysis. mRNA expression was examined using oligonucleotide microarrays, and the cellular localization of the Op18 protein was examined using immunohistochemical analysis of tissue microarrays. Three phosphorylated forms and one unphosphorylated form of the Op18 protein were identified and found to be overexpressed in lung adenocarcinomas as compared with normal lung. The percentage of phosphorylated to total Op18 protein isoforms increased from 3.2% in normal lung to 7.9% in lung tumors. Both the phosphorylated and unphosphorylated Op18 proteins were significantly increased in poorly differentiated tumors as compared with moderately or well differentiated lung adenocarcinomas (p<0.03), suggesting that up-regulated expression of Op18 reflects a poor differentiation status and higher cell proliferation rates. This was further verified in A549 and SKLU1 lung adenocarcinoma cell lines by examining Op18 levels and phosphorylation status following treatment that altered either cell proliferation or differentiation. The increased expression of Op18 protein was significantly correlated with its mRNA level indicating that increased transcription likely underlies elevated expression of Op18. The overexpression of Op18 proteins in poorly differentiated lung adenocarcinomas and the elevated expression of the phosphorylated forms of Op18 may offer a new target for drug- or gene-directed therapy and may have potential utility as a tumor marker.

BackgroundThe incidence rate and mortality rate of lung adenocarcinoma (LUAD) are in the forefront of malignant tumors in recent years. Due to its atypical early manifestations, many patients are diagnosed at advanced stages and miss the opportunity for timely intervention, resulting in a poor 5-year overall survival rate. Therefore, identifying specific targets in LUAD is essential for molecular precision therapy. This study integrates clinical and basic research to elucidate the diagnostic value and clinical significance of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) in LUAD.MethodsImmunohistochemistry (IHC) and immunofluorescence (IF) were first employed to detect the protein expression and localization of NFATc1 in LUAD and adjacent tissues. Receiver operating characteristic (ROC) curve analysis was then performed to evaluate the diagnostic potential of NFATc1 for LUAD, and to analyze the correlation between NFATc1 expression and clinical data of LUAD patients. Subsequently, NFATc1 messenger RNA (mRNA) and protein expression in A549 LUAD cells were measured using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assays. The effects of NFATc1 silencing on cell proliferation, migration, and invasion were assessed using Cell Counting Kit-8 (CCK-8), colony formation, wound healing, and Transwell assays. Finally, the signaling pathway, underlying mechanisms and gene alterations types through which NFATc1 influences LUAD progression were predicted and validated.ResultsNFATc1 expression was higher in LUAD tissues than in adjacent normal tissues and was localized in both the cytoplasm and nucleus. Area under the curve (AUC) of NFATc1 in diagnosing LUAD was 0.8020. NFATc1 expression was related to clinical stage, T stage, N stage, pathological grading, and maximum diameter of LUAD. NFATc1 expression in human LUAD (A549) cell line was significantly higher than in human normal lung epithelial BEAS-2B cell line. Silencing NFATc1 reduced the proliferation, migration, and invasion of A549 cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis suggested that NFATc1 may be associated with the Ras/Raf/p38 mitogen-activated protein kinase (MAPK) pathway. Pretreatment with the inhibitor SB202190 decreased the expression of proteins related to Ras/Raf/p38 MAPK and significantly suppressed the proliferation, migration, and invasion of A549 cells. Public database analyses revealed that the NFATc1 alterations in LUAD primarily involve gene deletions and gene mutations.ConclusionsNFATc1 is highly expressed in LUAD tissues, and its expression level is closely related to clinical characteristics. NFATc1 may promote the proliferation, migration, and LUAD cell invasion via the Ras/Raf/p38 MAPK pathway, providing a new therapeutic target for LUAD.

Neural precursor cell expressed developmentally down-regulated 4-like protein (NEDD4L), an E3 ubiquitin ligase, exerts an important role in diverse biological processes including development, tumorigenesis, and tumor progression. Although the role of NEDD4L in the pathogenesis of lung adenocarcinoma (LUAD) has been described, the mechanism by which NEDD4L promotes LUAD progression remains poorly understood. In the study, the correlation between NEDD4L level and clinical outcome in LUAD patients was analysed using the data from The Cancer Genome Atlas (TCGA) database. NEDD4L expression in LUAD cell lines and tissue samples was assessed through quantitative real-time PCR (qRT-PCR). The biological function of NEDD4L on regulating LUAD cell proliferation was tested with Cell Counting Kit-8 (CCK-8) assay in vitro, and mouse xenograft tumor model in vivo. We found that NEDD4L expression was significantly decreased in LUAD tissues and cell lines. Lower expression of NEDD4L exhibited a significantly poorer overall survival. Functionally, NEDD4L knockdown in H1299 cells accelerated cell growth, whereas NEDD4L overexpression in A549 cells repressed cell proliferation. NEDD4L overexpression also inhibited tumor xenograft growth in vivo. Mechanistically, NEDD4L decreased the protein stability of notch receptor 2 (Notch2) through facilitating its ubiquitination and degradation by ubiquitin-proteasome system. Consequently, NEDD4L negatively regulated Notch signaling activation in LUAD cells, and RO4929097 (a Notch inhibitor) treatment effectively repressed the effect of NEDD4L knockdown on LUAD cell proliferation. Taken together, these results demonstrate that down-regulated NEDD4L facilitates LUAD progression by activating Notch signaling, and NEDD4L may be a promising target to treat LUAD.

CHPF promotes lung adenocarcinoma proliferation and anti-apoptosis via the MAPK pathway