Roles of the WNT/β-catenin signaling pathway in the metastasis and recurrence of NSCLC

TAF1 promotes NSCLC cell epithelial-mesenchymal transition by transcriptionally activating TGFβ1

MicroRNAs (miRNAs) have been identified as important post-transcriptional regulators involved in various biological and pathological processes of cells, but their underlying mechanisms in chemosensitivity and metastasis have not been fully elucidated. The objective of this study was to identify miR-181b and its mechanism in the chemosensitivity and metastasis of NSCLC. We found that miR-181b expression levels were lower in A549/DDP cells compared with A549 cells. Functional assays showed that the overexpression of miR-181b inhibited proliferation, enhanced chemosensitivity to DDP, attenuated migration and metastatic ability in NSCLC cell lines in vitro and in vivo. TGFβR1 was subsequently identified as a novel functional target of miR-181b. TGFβR1 knockdown revealed similar effects as that of ectopic miR-181b expression, whereas overexpression of TGFβR1 rescued the function of miR-181b-mediated growth, chemosensitivity and metastasis in NSCLC cells. In addition, miR-181b could inactivate the TGFβR1/Smad signaling pathway. We also observed that decreased miR-181b expression and increased TGFβR1 expression were significantly associated with chemosensitivity to DDP and tumor metastasis in NSCLC patients. Consequently, miR-181b functions as a tumor suppressor and has an important role in proliferation, chemosensitivity to DDP and metastasis of NSCLC by targeting TGFβR1/Smad signaling pathway.

Accumulating evidence has revealed that an elevated expression level of Aurora-B is associated with metastasis in various types of malignant tumor. However, it is currently unclear whether this molecule is involved in non-small lung cancer (NSCLC) metastasis, and the molecular mechanisms associated with Aurora-B and metastasis remain unknown. In the present study, in order to investigate whether Aurora-B is involved in the development and metastasis of NSCLC, the Aurora-B protein expression in NSCLC tissues was detected by immunohistochemistry and its association with metastasis was analyzed. The results revealed that the expression levels of the Aurora-B protein in tissues obtained from NSCLC patients with lymph node metastasis were significantly higher than those without metastatic disease. Furthermore, the effect of Aurora-B inhibition on A549 cell migration and invasion, as well as the activity of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway was evaluated. Aurora-B was inhibited in the A549 cells using short hairpin RNA, and the cell migration and invasion rates were investigated using wound healing and Transwell invasion assays. In addition, the expression of the main proteins in the PI3K/Akt/nuclear factor-κB (NF-κB) signaling pathway, and matrix metalloproteinase (MMP)-2 and -9 were measured by western blot analysis. The results demonstrated that cell migration and invasion were decreased as a result of silencing Aurora-B. Furthermore, the activity of the PI3K/Akt/NF-κB signaling pathway and the expression of MMP-2 and -9 protein were suppressed by silencing Aurora-B. The results of the present study indicate that the knockdown of Aurora-B suppresses A549 cell invasion and migration via the inhibition of the PI3K/Akt signaling pathway in vitro and thus, targeting Aurora-B may present a potential treatment strategy for NSCLC.

BackgroundFibronectin, an extracellular matrix protein, has been reported to be associated with heterogeneous cancer stemness, angiogenesis and progression in multiple cancer types. However, the roles and the underlying mechanism of fibronectin on the progression NSCLC need to be further elucidated.MethodsPublic dataset such as Kaplan-Meier Plotter was used to determine the prognostic significance of genes. The correlation of different protein expression in clinical and xenograft tissues was tested by immunohistochemistry experiment. Both in vitro and in vivo experiments were performed to determine the role of fibronectin on the tumor growth, metastasis, and angiogenesis in NSCLC. The activation of key signaling pathway under fibronectin was examined by WB assay. RNA-seq was applicated to screening the target gene of fibronectin. Rescue experiment was performed to confirm the role of target gene in fibronectin-mediated function in NSCLC. Finally, luciferase and CHIP assays were used to elucidate the mechanism by which fibronectin regulated the target gene.ResultsOur results revealed that fibronectin was up-regulated in cancer tissues compared with the normal ones in NSCLC patients. Dish- coated fibronectin enhanced the tumor growth, metastasis, and angiogenesis of NSCLC in vitro and in vivo by promoting EMT and maintaining stemness of NSCLC cells. As expected, fibronectin activated FAK and its downstream MAPK/ERK signaling pathway. WISP3 was screened as a potential target gene of fibronectin. Interestingly, WISP3 effectively activated Wnt signaling pathway, and knockdown of WISP3 effectively blocked the influence of fibronectin on the migration, invasion and vascular structure formation potential of NSCLC cells. Our data also manifested that fibronectin elevated the transcription of WISP3 gene by promoting the binding of HIF-1α to the promoter region of WISP3 in NSCLC cells.ConclusionsOur findings sketched the outline of the route for fibronectin exert its role in NSCLC, in which fibronectin activated downstream FAK and MAPK/ERK signaling pathways, and mediated the accumulation of HIF-1α. Then, HIF-1α enabled the transcription of WISP3, and subsequently promoted the activation of Wnt signaling pathway, and finally enhanced the tumor growth, metastasis, and angiogenesis in NSCLC.

BackgroundNon-small cell lung cancer (NSCLC) accounts for more than 85% of the total cases with lung cancer. NSCLC is characterized by easy metastasis, which often spreads to bones, brains and livers. RNA-binding motif protein 10 (RBM10) is an alternative splicing (AS) regulator frequently mutated in NSCLC. We found that there were multiple peak binding sites between RBM10 and long non-coding RNA nuclear enriched abundant transcript 1 (LncRNA Neat1) by crosslinking-immunprecipitation and high-throughput sequencing (Clip-Seq). LncRNA Neat1 plays an indispensable role in promoting cancer in a variety of tumors and produces two splicing variants: Neat1_1 and Neat1_2. This study aims to explore the mechanism of RBM10 and LncRNA Neat1 in invasion and metastasis of NSCLC.MethodsThrough histological and cytological experiments, we assessed the expression level of RBM10 protein expression. The interaction between RBM10 and Neat1 was evaluated via Clip-Seq and RNA immunoprecipitation assay. The effect of RBM10 on Neat1 and its splicing variants was identified by RT-qPCR. The effect of RBM10 and Neat1 on invasive and metastasis phenotypes of NSCLC was analyzed using transwell invasion assay and scratch test. Additionally, downstream signaling pathway of RBM10 were identified by immunofluorescence and western blot.ResultsRBM10 exhibited low levels of expression in NSCLC tissues and cells. RBM10 inhibited the invasion and metastasis of NSCLC and recruited Neat1 and Neat1_2. Overexpression of RBM10 simultaneously inhibited Neat1 and Neat1_2, and promoted the expression of Neat1_1. On the other hand, silencing RBM10 promoted Neat1 and Neat1_2, and inhibited the expression of Neat1_1. From this, we concluded that RBM10 regulated AS of Neat1, and the tumor-promoting effect of Neat1 was mainly attributed to Neat1_2. RBM10 had a negative correlation with Neat1_2. In addition, RBM10 upregulated the expression of PTEN and downregulated the phosphorylation of PI3K/AKT/mTOR through Neat1_2, which ultimately inhibited the invasion and metastasis of NSCLC.ConclusionThe RBM10 regulated AS of Neat1 to cause the imbalance of Neat1_1 and Neat1_2, and RBM10 suppressed the activation of the PTEN/PI3K/AKT/mTOR signal by downregulating Neat1_2, finally affected the invasion and metastasis of NSCLC.

Omega-3 has been proposed as an antitumor substance that suppresses the growth and metastasis of multiple types of tumor cells, including lung cancer, but the specific mechanisms involved remain obscure. Our previous studies showed that the expression of chemokine ligand 18 was related to the migration and metastasis of non-small cell lung cancer. Here, we aim to explore whether omega-3 inhibits invasion and metastasis of NSCLC by regulating the expression of CCL18. The expression of CCL18, metastasis- and epithelial-mesenchymal transition (EMT)-related genes at mRNA and protein levels in NSCLC cell lines were detected by RT-qPCR and Western blot, respectively. The metastatic and invasive capability of NSCLC cells were evaluated by scratch wound healing and Transwell assays, respectively. Our results showed that the level of CCL18 is positively associated with metastatic ability of NSCLC cells. Docosahexaenoic acid, an important long-chain, polyunsaturated omega-3 (n-3) fatty acid, significantly inhibited invasion and metastasis of NSCLC cells, and concomitantly downregulated the expression of metastasis- and EMT-related genes and p-STAT3 signaling pathway. Additionally, we found that DHA inhibited CCL18 expression in lung cancer cells, while overexpression of CCL18 effectively reversed DHA-mediated downregulation in the expression of metastasis- and EMT-related genes and p-STAT3 signaling as well as DHA-mediated inhibitory effect on metastasis and invasion of NSCLC cells. DHA inhibits NSCLC cell invasion and metastasis possibly through targeted inhibition of CCL18/ STAT3 signaling pathway and EMT process.

Disintegrin-metalloproteinase 15(ADAM15), a member of disintegrin metalloproteinases (ADAMs), plays important roles in various cancer types. However, the underlying ADAM15 functioning in lung cancer is still unclear. In the present study, we find that ADAM15 regulates the epidermal growth factor receptor/focal adhesion kinase (EGFR/FAK) signalling pathway by interactions with integrins. Integrin αV is involved in ADAM15-mediated FAK signalling. Further, we find that ADAM15 and CD151 were co-expressed, and the presence of ADAM15 affected the integrin α3/α6-related EGFR signalling pathway by cooperating with CD151. In addition, we also prove the effect of ADAM15 on proliferation in nude mice. Finally, we show that ADAM15 is a direct target of miR-204-5p by luciferase reporter assays, qRT-PCR and western blot analyses. Our findings provide molecular and cellular evidence that ADAM15 promotes cell proliferation and metastasis in NSCLC, which might provide a potential target for NSCLC treatment.

The relationship between STMN1 and cancer metastasis is controversial. The purpose of this study was to explore the role and mechanism of STMN1 in NSCLC metastasis. In this study, we reported that STMN1 was highly expressed in NSCLC tissues and associated with poor prognosis. Both in vivo and in vitro functional assays confirmed that STMN1 promoted NSCLC metastasis. Further studies confirmed that STMN1 promoted cell migration by regulating microtubule stability. The results of Co-IP and LC‒MS/MS illustrated that STMN1 interacts with HMGA1. HMGA1 decreases microtubule stability by regulating the phosphorylation level of STMN1 at Ser16 and Ser38 after interacting with STMN1. This result suggested that STMN1 could be activated by HMGA1 to further promote NSCLC metastasis. Meanwhile, it has been found that STMN1 could promote cell migration by activating the p38MAPK/STAT1 signaling pathway, which is not dependent on microtubule stability. However, activating p38MAPK can decrease microtubule stability by promoting the dephosphorylation of STMN1 at ser16. A positive feedback loop was formed between STMN1 and p38MAPK to synergistically promote cell migration. In summary, our study demonstrated that STMN1 could promote NSCLC metastasis through microtubule-dependent and nonmicrotubule-dependent mechanisms. STMN1 has the potential to be a therapeutic target to inhibit metastasis.

BackgroundNon-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality worldwide. Lipocalin-2 (LCN2), a pleiotropic protein implicated in tumorigenesis and cancer progression, has been associated with multiple malignancies. However, its precise role in NSCLC and the underlying molecular mechanisms remain incompletely understood. This study aimed to elucidate the function of LCN2 in NSCLC, with a particular focus on its involvement in the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway.MethodsLCN2 expression in NSCLC tissues was comprehensively analyzed using bioinformatics tools, including the Universal Analysis of Cancer (UALCAN), The Cancer Genome Atlas (TCGA), UCSC-XENA, and Gene Expression Omnibus (GEO) databases. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were employed to assess LCN2 expression levels in NSCLC cell lines. The functional impact of LCN2 on NSCLC cells, including proliferation, apoptosis, and metastasis, were assessed through a series of in vitro assays, such as Cell Counting Kit-8 (CCK-8), EdU, wound healing, and transwell migration and invasion assays. An in vivo xenograft model was established to investigate the effects of LCN2 on tumor growth and metastasis. Additionally, the involvement of the JAK2/STAT3 signaling pathway was examined using western blotting and pharmacological inhibition with AG490.ResultsLCN2 was significantly upregulated in NSCLC tissues and cell lines, and its elevated expression correlated with poor prognosis. Functional analyses demonstrated that LCN2 knockdown suppressed NSCLC cell proliferation, migration, and invasion while promoting apoptosis. Mechanistically, LCN2 was found to activate the JAK2/STAT3 pathway by interacting with SOCS3, and pharmacological blockade of this pathway effectively abrogated the oncogenic effects of LCN2 overexpression.ConclusionsThis study identifies LCN2 as a potential oncogene in NSCLC, driving tumor progression through activation of the JAK2/STAT3 signaling pathway. These findings suggest that targeting LCN2 or its downstream signaling components may represent a promising therapeutic strategy for NSCLC.

BackgroundNon-small cell lung cancer (NSCLC) frequently metastasizes to bone, leading to poor prognosis. Siglec15 has been identified as a newly discovered immune checkpoint and exists in a variety of tumors. However, the expression and function of Siglec15 in NSCLC and bone metastasis remains largely unclear.MethodsSiglec15 expression in NSCLC and the correlation between Siglec15 expression and the clinicopathological factors of patients with NSCLC were analyzed using The Cancer Genome Atlas (TCGA) dataset. Correlation analysis between Siglec15 and bone metastasis-related genes expression was based on the Molecular Signatures Database (MSigDB). Western blotting and immunohistochemistry were applied to detect Siglec15 expression in NSCLC and spinal metastasis. Human A549 and mouse CMT167 cells were transfected with Siglec15 siRNA to investigate its biological functions in NSCLC proliferation, migration, and invasion. The immune-related signaling pathways and correlations between Siglec15 and tumor-infiltrating immune cells and different immune checkpoints in the NSCLC tumor microenvironment (TME) were analyzed using Estimating Relative Subsets of RNA Transcripts (CIBERSORT) and gene set enrichment analysis (GSEA). To demonstrate Siglec15 in NSCLC cell-mediated T cell suppression and investigate the potential mechanism of Siglec15 silencing in antitumor immunity, we used a T cell killing assay in vitro and the high‑throughput sequencing approach.ResultsSiglec15 expression was positively associated with the tumor stage and lymph node metastasis, and was markedly up-regulated in NSCLC bone metastasis. Functionally, Siglec15 knockdown inhibited the proliferation, migration, and invasion of NSCLC cells (A549 and CMT167 cell lines). A total of eight kinds of tumor-infiltrating immune cells were found to have a strong association with the Siglec15 expression in NSCLC cases. The expression of previously discovered immune checkpoints was higher in the high Siglec15 expression NSCLC group. Furthermore, an in vitro T cell killing assay showed that the down-regulation of Siglec15 in tumor cells could enhance the antitumor immune responses of CD8+ T cells. High‑throughput sequencing revealed the potential molecular mechanisms underlying the Siglec15-mediated immunosuppression effect of tumor cells on immune cells.ConclusionsSiglec15 may be involved in the pathogenesis of spinal metastasis in NSCLC and provide a new potential therapeutic target for the treatment of NSCLC and bone metastasis.

HVEM as a tumor-intrinsic regulator in non-small cell lung cancer: Suppression of metastasis via glycolysis inhibition and modulation of macrophage polarization.

Objective: This study was designed to predict non-small cell lung cancer metastasis and explore mechanism of lung cancer cell proliferation, metastasis and migration in vitro and vivo. Methods: Firstly, Adenylate cyclase-associated protein 1, CAP1 were determined by real-time PCR and western blot analysis and IHC in 82 lung cancer specimens. Secondly, BM group (n =50) and non-BM group (n =70) were treated and followed up by 4 years. Thirdly, we characterized the expression of CAP1 in lung cell by using Western blot and RT-PCR analyses. In addition, Si-CAP1, empty vector, over-CAP1 in cultured A549,95-C,95-D by using MTT, wound Healing, Transwell invasive, Western blot, and IHC analyses and nude mouse. Results: We provided evidence that overexpression of CAP1 in lung cancer cells, particularly at BM [figure 1]. In addition,These findings suggest that CAP1 promotes the lung cancer cell proliferation and migration in vitro as well as its growth and metastasis in vivo via LIMK/cofilin I [figure 2]. Conclusion: CAP1 is associated with metastasis of NSCLC especially in BM patients and promotes the lung cancer cell proliferation and migration in vitro and vivo by limk1-cofilin.

BackgroundThis study aims to investigate potential biomarkers associated with NSCLC-BM and elucidate their regulatory roles in critical pathways involved in cerebral metastatic dissemination.MethodsThe identified DEGs were subjected to functional enrichment analysis. PPI networks were predicted using the STRING database and visualized with Cytoscape. Hub genes were subsequently screened from the PPI network to construct a transcription TF-miRNA regulatory network. Subsequent analyses included: survival analysis, immune infiltration assessment and comprehensive mutational profiling.ResultsAmong the 56 identified DEGs, 19 were upregulated while 37 were downregulated. GOntology enrichment analysis revealed significant enrichment in immune response, signaling receptor binding, and extracellular region. KEGG pathway analysis demonstrated predominant involvement in cytokine-cytokine receptor interaction and chemokine signaling pathway. Through Cytoscape-based screening, we identified 10 hub genes: CD19, CD27, IL7R, SELL, CCL5, CCR5, PRF1, GZMK, GZMA, and TIGIT. The TF-miRNA regulatory network analysis uncovered 6 transcription factors (STAT5A/B, NFKB1, EGR1, RELA, and CTCF) and 4 miRNAs(hsa-miR-204, hsa-miR-148b, hsa-miR-618, and hsa-miR-103) as critical transcriptional and post-transcriptional regulators of DEGs.Integrated analyses including Kaplan-Meier survival curves, immune infiltration profiling, and comprehensive mutational analysis demonstrated significant associations with brain metastatic progression in the studied cohort.ConclusionThis study provides novel biomarkers from a unique perspective for the diagnosis, prognosis, and development of molecular-targeted therapies or immunotherapies for brain metastasis in NSCLC.

Despite advances in the understanding of tumor biology in recent years, lung cancer remains the leading cause of cancer death worlwide. Non small cell lung cancer (NSCLC; adenocarcinoma, squamous carcinoma, and large cell carcinoma) is the most common type, representing more than 80% of lung cancer diagnoses. Clinical improvements following treatment with traditional chemotherapy have reached a plateau in patients with NSCLC and 5 year survival still remains at 15%. Moreover, the molecularly targeted therapies having already progressed to clinical trials have not proved successful [1,2]. Although NSCLC is very often extremely resistant to chemotherapeutic agents, chemotherapy remains an established treatment for advanced NSCLC. Platinum-based chemotherapy frequently combined with other agents and/or approaches remain the standard in the first-line treatment of patients with advanced NSCLC [3,4]. Methylating agents such as temozolomide, an oral SN1 methylating agent which crosses the blood brain barrier achieving effective concentrations in the Central Nervous System (CNS), have already been used combined with platinum-based regimen and whole brain radiotherapy as prophylaxis against brain metastasis in NSCLC and/or as second-line treatment in NSCLC patients with brain metastases [5–8]. Methylating agents of SN1 type, including temozolomide (TMZ), procarbazine, dacarbazine and streptozotocine, constitute a widely used class of anticancer drugs. These DNA damaging agents are highly cytotoxic, mutagenic, recombinogenic and clastogenic inducing about a dozen DNA methylation adducts [9]. Among them, O6-methylguanine (O6-meG), induced in small amounts (maximally 8% of total methylation products), is the most biologically significant lesion; it mispairs with thymine during DNA replication finally generating G to A transitions in the second round of replication. The DNA mismatches are recognized by the Methyl-directed Mismatch Repair (MMR) system that removes the mis-incorporated base opposite the O6-meG lesion. Repair resynthesis of DNA leads to reinsertion of thymine opposite O6-meG and re-initiation of futile mismatch repair. The DNA double-strand breaks (DSBs) resulting from this process are thought to initiate a series of events including prolonged G2 arrest leading to cell death [9]. A critical factor influencing the cellular response to methylating agents is O6-methylguanine- DNA methyltransferase (MGMT), the DNA repair protein that stoichiometrically and selectively removes methyl lesions from the O6 position of guanine and returns the DNA to its pre-lesioned state [10]. Pre-replicative repair by MGMT as well as post-replicative MMR determine the level of methylating agent-induced genotoxicity and cell death [11–13]. Chemotherapeutic agents inducing DNA damage, such as SN1 methylating agents and cisplatin, may activate cell death by apoptosis or necrosis. They could also induce autophagy, senescence or mitotic catastrophe, which may then be followed by apoptosis or necrosis [13–15]. The molecular basis underlying the decision- making process is currently the subject of intense investigation because a deeper understanding of how a given chemotherapy affects all of the signalling pathways involved in cell death is highly relevant in order to develop more effective therapeutics. Despite their use in combination therapies, the effect of SN1 methylating agents on human NSCLC has not been studied thoroughly. We thus investigated the mechanism of the cell death induced by a model SN1 methylating agent, N-methyl-N-nitrosourea (MNU) in two human NSCLC cell lines, A549 (p53wt) and H157 (p53null) [13] through a time course gene expression profiling study 24, 48 and 72 hours after treatment. The list of differentiated genes, biological processes and cellular pathways were identified using appropriate bioinformatics tools and the results were further validated through RT-PCR of selected genes. MNU induced cell death through distinct responses at the gene expression level in the above cell lines. Our results overall support the use of SN1 methylating agents in platinum-based combination regimen against advanced NSCLC.

The mechanism by which tumor-associated macrophages (TAMs) affect cancer progression is not fully understood. This study developed a microfluidic-based co-culture device to mimic the tumor microenvironment to assess TAM effects on invasion and metastasis in NSCLC. The results showed lung carcinoma cells could cause macrophages to show the M2 (a TAM-like) phenotype, and these M2 macrophages promoted lung cancer cell EMT and invasion. Proteomic analysis by the iTRAQ quantitation strategy and GO ontology of the cancer cells indicated that αB-Crystallin (CRYAB) might be involved in this process. Further, we confirmed the role of CRYAB in cancer invasion and metastasis through cell and animal experiments, as well as human cancer tissue assessment. Overall, we demonstrated that M2 macrophages promote malignancy in lung cancer through the EMT by upregulating CRYAB expression and activating the ERK1/2/Fra-1/slug signaling pathway.