Migration Of Non-small Cell Lung Cancer Cells Research Articles

P300 reduces TUBB4B expression to facilitate the biological process of migration and invasion of non-small cell lung cancer cells

This article explored the mechanism of E1A binding protein p300 (P300) and beta-tubulin 4B isotype-encoding gene (TUBB4B) in regulating the migration and invasion of non-small cell lung cancer (NSCLC) cells. TUBB4B and P300 expression in NSCLC tissues and cells was monitored by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. TUBB4B function on NSCLC cell migration, invasion and epithelial-mesenchymal transition (EMT) was monitored by wound healing assay, Transwell experiment and Western blot. The regulation of P300 on TUBB4B was monitored by qRT-PCR and Western blot. Mechanism of P300 and TUBB4B in regulating NSCLC cell migration and invasion was explored by rescue experiment. A xenograft tumor model was established by using nude mouse. As a result, low TUBB4B expression and high P300 expression was discovered in NSCLC tissues and cells. TUBB4B and P300 expression showed a negative correlation in NSCLC tissues. Lower TUBB4B but higher P300 was observed in tumor tissues of NSCLC patients with metastasis. TUBB4B overexpression suppressed NSCLC cell migration, invasion and EMT. TUBB4B silencing had opposite results. P300 overexpression inhibited TUBB4B expression, and P300 silencing facilitated TUBB4B overexpression in NSCLC cells. TUBB4B overexpression counteracted the promotion of P300 overexpression on NSCLC cell invasion and migration. TUBB4B silencing abrogated the inhibition of P300 knockdown on NSCLC cell invasion and migration. TUBB4B overexpression suppressed NSCLC cell in vivo growth. Thus, TUBB4B could be reduced by P300 in NSCLC. It exerted suppression role on NSCLC cell migration, invasion and EMT. TUBB4B may be a novel target for NSCLC treatment.

Suppression of JAK2/STAT3 Pathway by Notoginsenoside R1 Reduces Epithelial-Mesenchymal Transition in Non-small Cell Lung Cancer.

It has bene reported that a novel saponin-notoginsenoside R1 (NGR1) possesses strong anti-tumor activities. This study aimed to investigate the role and mechanism of NGR1 in non-small cell lung cancer (NSCLC). NSCLC cell viability, proliferation, migration, and invasiveness were assessed using the ex vivo assays. NSCLC xenograft mouse models were constructed to confirm the role of NGR1 in vivo. Epithelial-mesenchymal transition (EMT)-related proteins and key markers in the JAK2/STAT3 pathway were examined using immunoblotting and immunohistochemistry analyses. NGR1 treatment suppressed NSCLC cell growth ex vivo and in vivo. It also decreased the migratory and invasive capacities of NSCLC cells. Additionally, NGR1 increased E-cadherin expression and reduced N-cadherin, vimentin, and snail expression in TGF-β1-treated NSCLC cells and xenograft tumors. JAK2/STAT3 pathway was inhibited by NGR1. Moreover, a specific inhibitor of JAK2, AG490, or STAT3 silencing significantly enhanced the effects of NGR1 against the EMT process in NSCLC cells. NGR1 restrains EMT process in NSCLC by inactivating JAK2/STAT3 signaling, suggesting the potential of NGR1 in anti-NSCLC therapy.

Oncogenic circ-SLC16A1 promotes progression of non-small cell lung cancer via regulation of the miR-1287-5p/profilin 2 axis

BackgroundCircular RNAs (circRNAs) are single-stranded RNAs with covalently closed structures that have been implicated in cancer progression. However, the regulatory mechanisms remain largely unclear. So, the aim of this study was to reveal the role and regulatory mechanisms of circ-SLC16A1.MethodsIn this study, next-generation sequencing was used to identify abnormally expressed circRNAs between cancerous and para-carcinoma tissues. Fluorescence in situ hybridization and quantitative reverse transcription polymerase chain reaction were performed to assess the expression patterns of circ-solute carrier family 16 member 1 (SLC16A1) in non-small cell lung cancer (NSCLC) cells and tissue specimens. The dual-luciferase reporter assay was utilized to identify downstream targets of circ-SLC16A1. Transwell migration, wound healing, 5-ethynyl-2′-deoxyuridine incorporation, cell counting, and colony formation assays were conducted to assess the proliferation and migration of NSCLC cells. A mouse tumor xenograft model was employed to determine the roles of circ-SLC16A1 in NSCLC progression and metastasis in vivo.ResultsThe results found that circ-SLC16A1 was upregulated in NSCLC cells and tissues. Downregulation of circ-SLC16A1 inhibited tumor growth by reducing proliferation, lung metastasis, and lymphatic metastasis of NSCLC cells, and arrested the cell cycle in the G1 phase. Also, silencing of circ-SLC16A1 promoted apoptosis of NSCLC cells. The results of bioinformatics analysis and the dual-luciferase reporter assay confirmed that microRNA (miR)-1287-5p and profilin 2 (PFN2) are downstream targets of circ-SLC16A1. PFN2 overexpression or circ-SLC16A1 inhibition restored proliferation and migration of NSCLC cells after silencing of circ-SLC16A1. PFN2 overexpression restored migration and proliferation of NSCLC cells post miR-1287-5p overexpression.ConclusionsCollectively, these findings show that miR-1287-5p/PFN2 signaling was associated with downregulation of circ-SLC16A1 and reduced invasion and proliferation of NSCLC cells. So, circ-SLC16A1 is identified as a mediator of multiple pro-oncogenic signaling pathways in NSCLC and can be targeted to suppress tumor progression.

MicroRNA‑373‑3p inhibits the proliferation and invasion of non‑small‑cell lung cancer cells by targeting the GAB2/PI3K/AKT pathway.

MicroRNAs (miRNAs) were previously demonstrated to be involved in the pathogenesis of non-small-cell lung cancer (NSCLC); however, the roles of certain miRNAs in NSCLC remain to be elucidated. The present study aimed to investigate the functions of screened miRNAs in NSCLC and the potential mechanisms. First, expression profiles of miRNAs were downloaded from the Gene Expression Omnibus (dataset no. GSE29248) and the differentially expressed miRNAs were analyzed by bioinformatics methods. Reverse transcription-quantitative PCR was used to validate the differential expression of miR-373 in clinical samples. The association between miR-373 expression levels and clinicopathological characteristics was also investigated. To further examine how miR-373 mediates the emergence of NSCLC, western blot, Cell Counting Kit-8, cell invasion and wound-healing assays, as well as apoptosis detection and a luciferase assay were used. The results indicated significant downregulation of miR-373 in NSCLC tissues and its low expression was closely associated with the degree of differentiation, clinical stage and tumor size, and was indicative of an unfavorable prognosis for patients with NSCLC. A functional study indicated that overexpression of miR-373 inhibited the proliferation, promoted apoptosis, and suppressed invasion and migration of NSCLC cells. Bioinformatics prediction and functional assays suggested that Grb-associated binding protein 2 (GAB2) was a direct target of miR-373. In addition, GAB2 was found to be significantly upregulated in NSCLC tissues, and clinically, miR-373 was negatively associated with GAB2. Furthermore, overexpression of GAB2 blocked the tumor suppressive effects of miR-373 on NSCLC cells. Mechanistically, miR-373 mimics were able to reduce the expression of GAB2 and subsequently decrease the phosphorylation level of AKT and mTOR protein. The present results indicate that miR-373 exerts its anti-tumor effects in NSCLC cells by targeting the GAB2/PI3K/AKT pathway, suggesting that miR-373 may be a potential therapeutic target in NSCLC.

Bioinformatics analysis and validation of CSRNP1 as a key prognostic gene in non-small cell lung cancer

Cysteine and serine-rich nuclear protein 1 (CSRNP1) has shown prognostic significance in various cancers, but its role in non-small cell lung cancer (NSCLC) remains elusive. We investigated CSRNP1 expression in NSCLC cases using bioinformatics tools from the GEO public repository and validated our findings through RT-qPCR in tumor and adjacent normal tissues. KEGG and GO enrichment analyses were employed to unveil the significant deregulation in signaling pathways. Additionally, clinical significance of CSRNP1 in NSCLC was determined through receiver operating curve (ROC) analysis, and its impact on survival was assessed using Kaplan-Meier analysis. To explore the functional impact of CSRNP1, we silenced its expression in NSCLC cells and assessed the effects on cell viability, migration, and invasion using MTT, Transwell, and wound-healing assays, respectively. Additionally, we investigated the influence of CSRNP1 silencing on the phosphorylation patterns of critical signaling proteins such as p53, p-Akt, and p-MDM2. Our results demonstrated significantly lower CSRNP1 expression in NSCLC tumor tissues (P < 0.01). ROC analysis indicated that NSCLC patients with high CSRNP1 expression exhibited extended overall survival and disease-free survival. Furthermore, CSRNP1 silencing promoted NSCLC cells viability, migration, and invasion (P < 0.05). Mechanistically, CSRNP1 silencing led to increased phosphorylation of AKT and MDM2, along with a concurrent reduction in p53 protein expression, suggesting its impact on NSCLC through deregulated cell cycle processes. In conclusion, our study underscores the significance of CSRNP1 in NSCLC pathogenesis, offering insights for targeted therapeutic interventions of NSCLC.

Discovering single cannabidiol or synergistic antitumor effects of cannabidiol and cytokine-induced killer cells on non-small cell lung cancer cells.

A multitude of findings from cell cultures and animal studies are available to support the anti-cancer properties of cannabidiol (CBD). Since CBD acts on multiple molecular targets, its clinical adaptation, especially in combination with cancer immunotherapy regimen remains a serious concern. Considering this, we extensively studied the effect of CBD on the cytokine-induced killer (CIK) cell immunotherapy approach using multiple non-small cell lung cancer (NSCLC) cells harboring diverse genotypes. Our analysis showed that, a) The Transient Receptor Potential Cation Channel Subfamily V Member 2 (TRPV2) channel was intracellularly expressed both in NSCLC cells and CIK cells. b) A synergistic effect of CIK combined with CBD, resulted in a significant increase in tumor lysis and Interferon gamma (IFN-g) production. c) CBD had a preference to elevate the CD25+CD69+ population and the CD62L_CD45RA+terminal effector memory (EMRA) population in NKT-CIK cells, suggesting early-stage activation and effector memory differentiation in CD3+CD56+ CIK cells. Of interest, we observed that CBD enhanced the calcium influx, which was mediated by the TRPV2 channel and elevated phosphor-Extracellular signal-Regulated Kinase (p-ERK) expression directly in CIK cells, whereas ERK selective inhibitor FR180204 inhibited the increasing cytotoxic CIK ability induced by CBD. Further examinations revealed that CBD induced DNA double-strand breaks via upregulation of histone H2AX phosphorylation in NSCLC cells and the migration and invasion ability of NSCLC cells suppressed by CBD were rescued using the TRPV2 antagonist (Tranilast) in the absence of CIK cells. We further investigated the epigenetic effects of this synergy and found that adding CBD to CIK cells decreased the Long Interspersed Nuclear Element-1 (LINE-1) mRNA expression and the global DNA methylation level in NSCLC cells carrying KRAS mutation. We further investigated the epigenetic effects of this synergy and found that adding CBD to CIK cells decreased the Long Interspersed Nuclear Element-1 (LINE-1) mRNA expression and the global DNA methylation level in NSCLC cells carrying KRAS mutation. Taken together, CBD holds a great potential for treating NSCLC with CIK cell immunotherapy. In addition, we utilized NSCLC with different driver mutations to investigate the efficacy of CBD. Our findings might provide evidence for CBD-personized treatment with NSCLC patients.

Linker Histone H1.4 Inhibits the Growth, Migration and EMT Process of Non-Small Cell Lung Cancer by Regulating ERK1/2 Expression.

H1.4 is one of the 11 variants of linker histone H1, and is associated with tumorigenesis and development of various cancers. However, it is unclear for the role of histone H1.4 in non-small cell lung cancer (NSCLC). In this study, we found that overexpression of H1.4 significantly inhibited the cell viability, migration, invasion and epithelial-mesenchymal transition (EMT) processes, whereas silencing H1.4 by shRNA knockdown promoted these processes in NSCLC cell lines A549 and H1299. We further showed that H1.4 overexpression reduced ERK1/2 expression or its phosphorylation levels, while H1.4 knockdown increased ERK1/2 expression or phosphorylation levels in NSCLC. Furthermore, we demonstrated that H1.4 bound to the promoter of ERK1/2, and acted as a transcriptional suppressor to inhibit ERK1/2 expression in A549 or H1299 cells. Importantly, we found that ERK ecto-expression can largely recovered the inhibitory effects of H1.4 on cell viability, migration, invasion and EMT processes. In summary, our study reveals that the H1.4-ERK pathway is crucial for cell viability, migration, invasion and EMT of NSCLC and could be a therapeutic target for NSCLC.

Metformin and buparlisib synergistically induce apoptosis of non-small lung cancer (NSCLC) cells via Akt/FoxO3a/Puma axis

Non-small cell lung cancer (NSCLC) is a global health issue lacking effective treatments. Buparlisib is a pan-PI3K inhibitor that shows promising clinical results in treating NSCLC. However, chemoresistance is inevitable and hampers the application of buparlisib. Studies show that a combination of phytochemicals and chemotherapeutics enhances its effectiveness. Here, we evaluated the role of metformin, an agent with multiple pharmacological properties, in enhancing the anti-tumour activities of buparlisib against NSCLC cells. Our results showed that metformin and buparlisib synergistically inhibited cell viability, migration, and invasion of NSCLC cells. In addition, co-treatment of metformin and buparlisib also induced cell cycle arrest and cell death in NSCLC cells. Mechanistically, metformin and buparlisib repressed Mcl-1 and upregulated Puma in NSCLC cells in a p53-independent manner. Moreover, they inhibited the PI3K/Akt signalling pathway, leading to activation of the FoxO3a/Puma signalling in NSCLC cells. Our findings suggest that combined treatment of metformin and buparlisib might provide a promising strategy for treating NSCLC.

METTL3-mediated the m6A modification of SF3B4 facilitates the development of non-small cell lung cancer by enhancing LSM4 expression.

Splicing factor B subunit 4 (SF3B4) has been confirmed to participate in the progression of many cancers and is considered to be a potential target for non-small cell lung cancer (NSCLC). Thus, the role and molecular mechanism of SF3B4 in NSCLC progression deserves further study. Quantitative real-time PCR and western blot were employed to detect the mRNA and protein levels of SF3B4, Sm-like protein 4 (LSM4) and methyltransferase-like 3 (METTL3). Cell proliferation, apoptosis, invasion, migration and stemness were tested by cell counting kit-8, colony formation, flow cytometry, transwell, wound healing, and sphere formation assays. The interaction between SF3B4 and METTL3 or LSM4 was confirmed by MeRIP, RIP and Co-IP assays. Mice xenograft models were constructed to assess the effects of METTL3 and SF3B4 on NSCLC tumorigenesis. SF3B4 had high expression in NSCLC tissues and was associated with the shorter overall survival of NSCLC patients. Knockdown of SF3B4 suppressed NSCLC cell proliferation, invasion, migration and stemness, while inducing apoptosis. METTL3 promoted SF3B4 mRNA stability by m6A modification, and its knockdown inhibited NSCLC cell growth, metastasis and stemness by downregulating SF3B4. SF3B4 could interact with LSM4, and sh-SF3B4-mediated the inhibition on NSCLC cell functions could be reversed by LSM4 overexpression. In addition, reduced METTL3 expression restrained NSCLC tumor growth, and this effect was reversed by SF3B4 overexpression. METTL3-stablized SF3B4 promoted NSCLC cell growth, metastasis and stemness via positively regulating LSM4.

FTO-mediated autophagy inhibition promotes non-small cell lung cancer progression by reducing the stability of SESN2 mRNA

The role of fat mass and obesity-associated protein (FTO), an N6-methyladenosine (m6A) demethylase, in non-small cell lung cancer (NSCLC) has recently received widespread attention. However the underlying mechanisms of FTO-mediated autophagy regulation in NSCLC progression remain elusive. In this study, we found that FTO was significantly upregulated in NSCLC, and downregulation of FTO suppressed the growth, invasion and migration of NSCLC cells by inducing autophagy. FTO knockdown resulted in elevated m6A levels in NSCLC cells. Methylated RNA immunoprecipitation sequencing showed that sestrin 2 (SESN2) was involved in m6A regulation during autophagy in NSCLC cells. Interestingly, m6A modifications in exon 9 of SESN2 regulated its stability. FTO deficiency promoted the binding of insulin-like growth factor 2 mRNA-binding protein 1 to SESN2 mRNA, enhancing its stability and elevating its protein expression. FTO inhibited autophagic flux by downregulating SESN2, thereby promoting the growth, invasion and migration of NSCLC cells. Besides, the mechanism by which FTO blocked SESN2-mediated autophagy activation was associated with the AMPK-mTOR signaling pathway. Taken together, these findings uncover an essential role of the FTO–autophagy–SESN2 axis in NSCLC progression.

Circ_BLNK is a Unique Molecular Marker in Non-small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) patients are characterized by distant metastasis and poor prognosis. Growing evidence has implied that circular RNAs (circRNAs) are involved in multiple tumor progression, including NSCLC. The objective of the present study was to functionally dissect the role and mechanism of circ_BLNK in NSCLC development and progression. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of circ_BLNK, miR-942-5p, and forkhead box protein O1 (FOXO1) in NSCLC tissues and cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay and colony formation assay detected cell proliferation; the protein expression levels were tested by western blot assay; cell apoptosis was measured by flow cytometry, and transwell assay detected cell migration and invasion. The molecular targeting relationship was determined by dual-luciferase reporter assay. The effect of circ_BLNK overexpression on tumor growth was detected by in vivo experiments and immunohistochemistry. Circ_BLNK was dramatically decreased in NSCLC, and overexpression of circ_BLNK inhibited proliferation, migration, and invasion of NSCLC cells and promoted cell apoptosis. Circ_BLNK level was negatively correlated with miR-942-5p expression and positively correlated with FOXO1 expression. Moreover, circ_BLNK acted as a sponge for miR-942-5p, which targeted FOXO1. Rescue assays presented that miR-942-5p reversed the anticancer action of circ_BLNK in NSCLC. Besides that, miR-942-5p inhibition suppressed the oncogenic behaviors, which were attenuated by FOXO1 knockdown. Animal experiments exhibited that circ_BLNK upregulation repressed tumor growth in vivo. Our study demonstrated a novel regulatory mechanism that circ_BLNK/miR-942-5p/FOXO1 axis adjusted non-small cell lung cancer development.

CircCPA4 induces ASCT2 expression to promote tumor property of non-small cell lung cancer cells in a miR-145-5p-dependent manner.

Non-small cell lung cancer (NSCLC) is a type of lung cancer that occurs in the cells of the respiratory tract, and its development is influenced by the regulation of circular RNAs (circRNAs). However, the role of circRNA carboxypeptidase A4 (circCPA4) in the progression of NSCLC and the underlying mechanism remain relatively clear. The study utilized both real-time quantitative polymerase chain reaction (RT-qPCR) and western blot techniques to evaluate the levels of circCPA4, microRNA-145-5p (miR-145-5p), alanine, serine, or cysteine-preferring transporter 2 (ASCT2). To assess cell proliferation, cell counting kit-8 (CCK8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were performed. Apoptosis was determined using flow cytometry, while cell migration and invasive capacity were evaluated through transwell and wound-healing assays. Intracellular levels of glutamine, glutamate, and α-KG were measured using specific kits. The relationship between miR-145-5p and circCPA4 or ASCT2 was confirmed using dual-luciferase reporter assay and RNA immunoprecipitation assay. CircCPA4 and ASCT2 RNA levels were elevated, while miR-145-5p was downregulated in both NSCLC tissues and cells. Depletion of circCPA4 significantly inhibited NSCLC cell proliferation, migration, invasion, and intracellular levels of glutamine, glutamate, and α-KG, and promoted apoptosis. Moreover, circCPA4 knockdown delayed tumor growth in vivo. Furthermore, circCPA4 was found to bind to miR-145-5p, thereby regulating the progression of NSCLC in vitro. ASCT2 was also identified as a downstream target of miR-145-5p, and its upregulation rescued the effects of miR-145-5p overexpression on NSCLC cell processes. CircCPA4 knockdown inhibited tumor property of NSCLC cells by modulating the miR-145-5p/ASCT2 axis.

TRAIL predisposes non-small cell lung cancer to ferroptosis by regulating ASK-1/JNK1 pathway

ObjectiveOur current study aimed to assess the relationship between TNF-related apoptosis-inducing ligand (TRAIL) and ferroptosis in non-small cell lung cancer (NSCLC) development.MethodsThe expression of TRAIL was detected by western blot, RT-qRCR and immunohistochemistry. The viability of NSCLC cells was analyzed by CCK-8 kit. The migration and invasion of NSCLC cells were detected by wound healing assay and transwell assay, respectively. Labile iron pool (LIP) was detected based on the calcein-acetoxymethyl ester method. Ferrous iron (Fe2+) and iron levels were assessed by detection kits. The levels of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were measured using corresponding detection kits. Mice tumor xenograft models were used for the in vivo research.ResultsThe expression of TRAIL was reduced in H1299, NCL-H1395, and A549 cells compared with BEAS-2B cells. The up-regulation of TRAIL expression significantly reduced cell viability, invasion, and migration of H1299 and A549 cells. TRAIL reduced the expression of ferroptosis-related genes (FTH1, GPX4, and SLC7A11), increased the levels of LIP, iron, and Fe2+, and promoted lipid peroxidation, thereby predisposing NSCLC cells to ferroptosis. TRAIL up-regulated the expression of phosphate modification of ASK-1 and JNK. ASKI-1 inhibitor GS-4977 attenuated the effects of TRAIL on the viability, migration, invasion, and ferroptosis of H1299 cells. Furthermore, TRAIL further suppressed tumor growth and ferroptosis in mice tumor xenograft models.ConclusionWe indicated that overexpression of TRAIL induced ferroptosis in NSCLC cells and exerted anti-tumor effects. Mechanistically, TRAIL promoted ferroptosis by the activation of the ASK-1/JNK1 pathway. Our results may provide new therapeutic strategies for NSCLC.

Inhaled delivery of cetuximab-conjugated immunoliposomes loaded with afatinib: A promising strategy for enhanced non-small cell lung cancer treatment.

Afatinib (AT), an FDA-approved aniline-quinazoline derivative, is a first-line treatment for metastatic non-small cell lung cancer (NSCLC). Combining it with cetuximab (CX), a chimeric human-murine derivative immunoglobulin-G1 monoclonal antibody (mAb) targeting the extracellular domain of epidermal growth factor receptor (EGFR), has shown significant improvements in median progression-free survival. Previously, we developed cetuximab-conjugated immunoliposomes loaded with afatinib (AT-MLP) and demonstrated their efficacy against NSCLC cells (A549 and H1975). In this study, we aimed to explore the potential of pulmonary delivery to mitigate adverse effects associated with oral administration and intravenous injection. We formulated AT-MLP dry powders (AT-MLP-DPI) via freeze drying using tert-butanol and mannitol as cryoprotectants in the hydration medium. The physicochemical and aerodynamic properties of dry powders were well analyzed firstly. In vitro cellular uptake and cytotoxicity study revealed concentration- and time-dependent cellular uptake behavior and antitumor efficacy of AT-MLP-DPI, while Transwell assay demonstrated the superior inhibitory effects on NSCLC cell invasion and migration. Furthermore, in vivo pharmacokinetic study showed that pulmonary delivery of AT-MLP-DPI significantly increased bioavailability, prolonged blood circulation time, and exhibited higher lung concentrations compared to alternative administration routes and formulations. The in vivo antitumor efficacy study carried on tumor-bearing nude mice indicated that inhaled AT-MLP-DPI effectively suppressed lung tumor growth.

Farnesoid X receptor promotes non-small cell lung cancer metastasis by activating Jak2/STAT3 signaling via transactivation of IL-6ST and IL-6 genes

Metastasis accounts for the majority of cases of cancer recurrence and death in patients with advanced non-small cell lung cancer (NSCLC). Farnesoid X Receptor (FXR) is a bile acid nuclear receptor that was recently found to be upregulated in NSCLC tissues. However, whether and how FXR regulates NSCLC metastasis remains unclear. In the present study, it was found that FXR promoted the migration, invasion, and angiogenic ability of NSCLC cells in vitro, and increased NSCLC metastasis in a mouse model in vivo. Mechanistic investigation demonstrated that FXR specifically bound to the promoters of IL-6ST and IL-6 genes to upregulate their transcription, thereby leading to activation of the Jak2/STAT3 signaling pathway, which facilitated tumor migration, invasion, and angiogenesis in NSCLC. Notably, Z-guggulsterone, a natural FXR inhibitor, significantly reduced FXRhigh NSCLC metastasis, and decreased the expression of FXR, IL-6, IL-6ST, and p-STAT3 in the mouse model. Clinical analysis verified that FXR was positively correlated with IL-6, IL-6ST and p-STAT3 expression in NSCLC patients, and was indicative of a poor prognosis. Collectively, these results highlight a novel FXR-induced IL-6/IL-6ST/Jak2/STAT3 axis in NSCLC metastasis, and a promising therapeutic means for treating FXRhigh metastatic NSCLC.

CircEPB41L2 blocks the progression and metastasis in non-small cell lung cancer by promoting TRIP12-triggered PTBP1 ubiquitylation

The metastasis of non-small cell lung cancer (NSCLC) is the leading death cause of NSCLC patients, which requires new biomarkers for precise diagnosis and treatment. Circular RNAs (circRNAs), the novel noncoding RNA, participate in the progression of various cancers as microRNA or protein sponges. We revealed the mechanism by which circEPB41L2 (hsa_circ_0077837) blocks the aerobic glycolysis, progression and metastasis of NSCLC through modulating protein metabolism of PTBP1 by the E3 ubiquitin ligase TRIP12. With ribosomal RNA-depleted RNA seq, 57 upregulated and 327 downregulated circRNAs were identified in LUAD tissues. circEPB41L2 was selected due to its dramatically reduced levels in NSCLC tissues and NSCLC cells. Interestingly, circEPB41L2 blocked glucose uptake, lactate production, NSCLC cell proliferation, migration and invasion in vitro and in vivo. Mechanistically, acting as a scaffold, circEPB41L2 bound to the RRM1 domain of the PTBP1 and the E3 ubiquitin ligase TRIP12 to promote TRIP12-mediated PTBP1 polyubiquitylation and degradation, which could be reversed by the HECT domain mutation of TRIP12 and circEPB41L2 depletion. As a result, circEPB41L2-induced PTBP1 inhibition led to PTBP1-induced PKM2 and Vimentin activation but PKM1 and E-cadherin inactivation. These findings highlight the circEPB41L2-dependent mechanism that modulates the “Warburg Effect” and EMT to inhibit NSCLC development and metastasis, offering an inhibitory target for NSCLC treatment.

KIAA1429-mediated RXFP1 attenuates non-small cell lung cancer tumorigenesis via N6-methyladenosine modification.

N6-methyladenosine (m6A) modification has been associated with non-small cell lung cancer (NSCLC) tumorigenesis. This study aimed to determine the functions of Vir-like m6A methyltransferase-associated (KIAA1429) and relaxin family peptide receptor 1 (RXFP1) in NSCLC. A quantitative real-time polymerase chain reaction was used to analyze the mRNA levels of KIAA1429 and RXFP1 in NSCLC. After silencing KIAA1429 or RXFP1 in NSCLC cells, changes in the malignant phenotypes of NSCLC cells were assessed using cell counting kit-8, colony formation, and transwell assays. Finally, the m6A modification of RXFP1 mediated by KIAA1429 was confirmed using luciferase, methylated RNA immunoprecipitation, and western blot assays. KIAA1429 and RXFP1 were upregulated and downregulated in NSCLC, respectively. Silencing of KIAA1429 attenuated the viability, migration, and invasion of NSCLC cells, whereas silencing of RXFP1 showed the opposite function in NSCLC cells. Moreover, RXFP1 expression was inhibited by KIAA1429 via m6A-modification. Therefore, silencing RXFP1 reversed the inhibitory effect of KIAA1429 knockdown in NSCLC cells. Our findings confirmed that the KIAA1429/RXFP1 axis promotes NSCLC tumorigenesis. This is the first study to reveal the inhibitory function of RXFP1 in NSCLC via KIAA1429-mediated m6A-modification. These findings may help identify new biomarkers for targeted NSCLC therapy.

Acidity-induced ITGB6 promote migration and invasion of lung cancer cells by epithelial-mesenchymal transition and focal adhesion

Non-small cell lung cancer (NSCLC) is a prevalent tumor and acidic tumor microenvironment provides an energy source driving tumor progression. We previously demonstrated significantly upregulated Integrin β6 (ITGB6) in NSCLC cells. This study was designed to investigate the role of ITGB6 in NSCLC metastasis and explore the potential mechanisms. The expression of ITGB6 was evaluated in patients with NSCLC. Migration and invasion assays were utilized to investigate the role of ITGB6, and ChIP-qPCR and dual-luciferase reporter experiments preliminarily analyzed the relationship between ETS proto-oncogene 1 (ETS1) and ITGB6. Bioinformatics analysis and rescue models were performed to explore the underlying mechanisms. The results demonstrated that ITGB6 was upregulated in NSCLC patients and the difference was even more pronounced in patients with poor prognosis. Functionally, acidity-induced ITGB6 promoted migration and invasion of NSCLC cells in vitro, and epithelial-mesenchymal transition (EMT) and focal adhesion were the important mechanisms responsible for ITGB6-involved metastasis. Mechanistically, we revealed ETS1 enriched in the ITGB6 promoter region and promoted transcription to triggered the activation of subsequent signaling pathways. Moreover, ChIP-qPCR and dual-luciferase reporter experiments demonstrated that ETS1 played an important role in directly mediating ITGB6 expression. Furthermore, we found ITGB6 was responsible for the acidic microenvironment-mediated migration and invasion processes in NSCLC by performing rescue experiments with ITGB6 knockdown. Our findings indicated acidic microenvironment directly induced ETS1 to regulate the expression of ITGB6, and then the highly expressed ITGB6 further mediate EMT and activates the downstream focal adhesion pathways, eventually promotes the invasion and migration in NSCLC progression and metastasis.

Obese Mouse Fat Cell-derived Extracellular Vesicles Transport miR-99a-5p to Mitigate the Proliferation and Migration of Non-small Cell Lung Cancer Cells.

Fat cells-derived extracellular vesicles (FC-EVs) play a role in regulating the tumor microenvironment in cancers by transporting RNAs. MicroRNAs (miRNAs) are vital regulators of cancer development. This study was conducted to explore the role of FC-EVs in the proliferation and migration of non-small cell lung cancer (NSCLC) cells, providing targets for NSCLC treatment. The obese mouse model was established via high-fat diet (HFD), followed by separation and characterization of FC-EVs (HFD-EVs). The levels of miR-99a-5p, precursor-miR-99a-5p, and heparan sulfate-glucosamine 3-sulfotransferase 3B1 (HS3ST3B1) were measured by RT-qPCR or Western blot assay. Cell proliferation and migration were evaluated by 3-(4, 5-dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium bromide and wound healing assays. The expression of Cy3-labeled miR-99a-5p in A549 cells (one NSCLC cell line) was observed via confocal microscopy. The binding of miR-99a-5p to HS3ST3B1 was analyzed by the dual luciferase assay. Rescue experiments were performed to confirm the role of HS3ST3B1 in NSCLC cells. miR-99a-5p was upregulated in adipose tissues, FCs, and HFD-EVs. HFD-EVs mitigated the proliferation and migration of NSCLC cells. HFD-EVs transported miR-99a-5p into A549 cells, which upregulated miR-99a-5p expression and inhibited HS3ST3B1 expression in A549 cells. HS3ST3B1 overexpression reversed the inhibition of HFD-EVs on the proliferation and migration of NSCLC cells. HFD-EVs transported miR-99a-5p into NSCLC cells and inhibited HS3ST3B1, thereby inhibiting proliferation and migration of NSCLC cells.

Mebendazole induces apoptosis and inhibits migration via the reactive oxygen species-mediated STAT3 signaling downregulation in non-small cell lung cancer.

The incidence and mortality of non-small cell lung cancer (NSCLC) are extremely high. Previous research has confirmed that the signal transducer and activator of the transcription 3 (STAT3) protein critically participate in the tumorigenesis of NSCLC. Mebendazole (MBZ) has exerts a larger number of pharmacological activities and has anticancer effects in lung cancer, but its mechanism of action remains unclear. This study thus aimed to clarify the impacts of MBZ on NSCLC cell. Cell proliferation, migration, and apoptosis were investigated via cell counting kit 8 (CCK-8) assay, Transwell assay, colony formation assay, wound-healing assay, and flow cytometry. Reactive oxygen species (ROS) were detected with a multifunctional microplate reader. Markers of cell migration and apoptosis were detected with Western blotting. The transcriptional activity of STAT3 was detected via luciferase assay. ROS scavenger N-acetylcysteine (NAC) was used to determine the effect of MBZ on NSCLC via ROS-regulated STAT3 inactivation and apoptosis. A xenograft model was constructed in vivo to investigate the role of MBZ in NSCLC tumor growth. The findings demonstrated that MBZ inhibited NSCLC cell proliferation and migration while promoting apoptosis through triggering ROS generation. In addition, the Janus kinase 2 (JAK2)-STAT3 signaling pathway was abrogated with the treatment of MBZ. NAC could distinctly weaken MBZ-induced apoptosis and STAT3 inactivation. Moreover, MBZ inhibited the tumor growth of NSCLC in vivo. In summary, MBZ inhibited NSCLC cell viability and migration by inducing cell apoptosis via the ROS-JAK2-STAT3 signaling pathway. These data provide a theoretical basis for the use of MBZ in treating NSCLC.