Non-small Cell Lung Cancer Tumorigenesis Research Articles

WTAP-Mediated m6A Modification of circSMOC1 Accelerates the Tumorigenesis of Non-Small Cell Lung Cancer by Regulating miR-612/CCL28 Axis.

Accumulating evidence reveals that deregulated N6-methyladenosine (m6A) RNA methylation and circular RNAs (circRNAs) are required for the tumorigenesis of non-small cell lung cancer (NSCLC). We aimed to uncover the underlying mechanisms by which WTAP-mediated m6A modification of circRNA contributes to NSCLC. The differentially-expressed circRNAs were identified by a circRNA profiling microarray. The association of circSMOC1 with clinicopathological features and prognosis in patients with NSCLC was estimated by fluorescence insitu hybridization. WTAP-mediated m6A modification of circRNA was validated by RNA immunoprecipitation (RIP) and methylated RIP (MeRIP) assays. The role of circSMOC1 in NSCLC cells was validated by invitro functional experiments and invivo tumorigenesis models. CircSMOC1-specific binding with miR-612 was verified by RIP, luciferase gene report and RT-qPCR assays. The effect of circSMOC1 and/or miR-612 on CCL28 expression was detected by RT-qPCR and Western blotting analysis. We found that the expression levels of circSMOC1 were elevated in NSCLC tissues and associated with TNM stage and poor survival in patients with NSCLC. Knockdown of circSMOC1 impaired the tumorigenesis of NSCLC invitro and invivo, whereas restored expression of circSMOC1 displayed the opposite effect. Furthermore, WTAP was upregulated in NSCLC and mediated m6A modification of circSMOC1 and circSMOC1 abolished WTAP knockdown-caused tumour-suppressive effects. Then, circSMOC1 acted as a sponge of miR-612 to upregulate CCL28 and miR-612 inhibitors abrogated circSMOC1 knockdown-caused anti-proliferation effects and CCL28 downregulation in NSCLC cells. Knockdown of CCL28 inhibited cell proliferation and invasion and counteracted miR-612 inhibitor-caused tumour-promoting effects. Our findings unveil that WTAP-mediated m6A modification of circSMOC1 facilitates the tumorigenesis of NSCLC by regulating the miR-612/CCL28 axis.

Circular RNA SPECC1 promoted tumorigenesis and osimertinib resistance in lung adenocarcinoma via a circular RNA-microRNA network.

Tyrosine kinase inhibitors (TKIs) are the first-line therapy for patients with non-small cell lung cancer (NSCLC) with sensitized mutations in the epidermal growth factor receptor (EGFR). However, resistance to TKIs is a major clinical issue that affects the survival and prognosis of the patients, with the mechanisms underlying this resistance remaining elusive. Circular RNAs (circRNAs) are a class of single-stranded, covalently closed RNA molecules, which are generated from pre-messenger RNAs (mRNAs) through back splicing. The aim of this study was to investigate the role of cRNA SPECC1 in promoting resistance to TKIs in NSCLC and to explore its potential involvement in tumorigenesis and metastasis of lung adenocarcinoma (LUAD). In this study, we identified differentially expressed genes through RNA sequencing from three tumor samples obtained from patients with poor postoperative TKI treatment outcomes. Validation was performed using quantitative real-time polymerase chain reaction (qRT-PCR) and cell function experiments. We further constructed a competing endogenous RNA (ceRNA) network and performed Gene Ontology (GO) analysis to explore the underlying mechanisms of circRNA. SPECC1 circular RNA (circSPECC1) was found to be significantly upregulated in tumors as compared to adjacent tissues. Knockdown of circSPECC1 in NSCLC cell lines resulted in decreased proliferation, migration, and invasion. Additionally, apoptosis was increased in cell lines with TKI-sensitive EGFR mutations when treated with osimertinib. circSPECC1 may promote TKI resistance and contribute to the tumorigenesis and metastasis of NSCLC. This study offers a novel perspective on TKI resistance research at the RNA level.

RNA binding protein RBM22 suppresses non-small cell lung cancer tumorigenesis by stabilizing LATS1 mRNA.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related mortality worldwide. Despite advancements in diagnostics and therapeutics, the prognosis for NSCLC remains poor, highlighting the urgent need for novel treatment options. RNA binding proteins, particularly RBM22, have emerged as significant contributors to cancer progression by influencing RNA splicing and gene expression. This study investigates the role of RBM22 in NSCLC and its potential as a therapeutic target. We focus on the effects of RBM22 on cell proliferation, invasion, stemness, and its interaction with LATS1 mRNA. RBM22 expression was assessed in samples and cell lines of NSCLC through techniques such as real-time PCR and western blot analysis. To modify RBM22 levels, overexpression and knockdown methods were employed utilizing vectors and siRNAs. We conducted assays for cell proliferation, invasion, and stemness to evaluate the effects of altering RBM22. The interaction between RBM22 and LATS1 mRNA was investigated using RNA immunoprecipitation. In addition, in vivo studies involving subdermal tumor and lung metastasis models in athymic mice were carried out to evaluate how changes in RBM22 influence the tumorigenic and metastatic characteristics of NSCLC. Our analysis revealed a significant underexpression of RBM22 in NSCLC tissues compared to adjacent healthy tissues. Increasing RBM22 expression in NSCLC cell lines led to a marked decrease in cellular proliferation, invasiveness, and stemness, while silencing RBM22 produced opposing effects. Further investigations confirmed that RBM22 directly interacts with LATS1 mRNA, thereby stabilizing and enhancing its expression. In vivo studies validated that elevated RBM22 expression substantially reduced tumor formation and pulmonary metastases, as evidenced by decreased tumor size, mass, and Ki-67 proliferation marker expression, along with a significant reduction in the number of metastatic nodules in the lungs. Our study demonstrates that RBM22 suppresses NSCLC by stabilizing LATS1 mRNA, which in turn reduces tumor growth and metastasis. Consequently, RBM22 emerges as a valuable therapeutic target for NSCLC, offering new strategies for addressing this challenging condition.

USP8-mediated PTK7 promotes PIK3CB-related pathway to accelerate the malignant progression of non-small cell lung cancer.

Protein tyrosine kinase 7 (PTK7) has been found to be highly expressed in non-small cell lung cancer (NSCLC), but its specific molecular mechanism needs to be further explored. PTK7 mRNA expression in NSCLC tumor tissues was examined by quantitative real-time PCR. The protein levels of PTK7, ubiquitin-specific peptidase 8 (USP8), PIK3CB, and PI3K/AKT were determined by western blot. Human monocytes (THP-1) were induced into macrophages and then co-cultured with the conditioned medium of NSCLC cells. Macrophage M2 polarization was assessed by detecting CD206+ cells using flow cytometry. The interaction between PTK7 and USP8 or PIK3CB was assessed by Co-IP assay. Animal study was performed to evaluate the effects of PTK7 knockdown and PIK3CB on NSCLC tumorigenesis in vivo. PTK7 expression was higher in NSCLC tumor tissues and cells. After silencing of PTK7, NSCLC cell proliferation, invasion, and macrophage M2 polarization were inhibited, while cell apoptosis was promoted. USP8 enhanced PTK7 protein expression by deubiquitination, and the repressing effects of USP8 knockdown on NSCLC cell growth, invasion, and macrophage M2 polarization were reversed by PTK7 overexpression. PTK7 interacted with PIK3CB, and PIK3CB overexpression could abolish the regulation of PTK7 silencing on NSCLC cell progression. USP8 positively regulated PIK3CB expression by PTK7, thus activating PI3K/AKT pathway. Downregulation of PTK7 reduced NSCLC tumorigenesis by decreasing PIK3CB expression. USP8-deubiquitinated PTK7 facilitated NSCLC malignant behavior via activating the PIK3CB/PI3K/AKT pathway, providing new idea for NSCLC treatment.

Expression of Five Important Biomarkers in Non-small Cell Lung Cancer: MicroRNAs-128, miR-335-5p, miR-1254, VEGF, and K-RAS

Background: Lung cancers, such as non-small cell lung cancer (NSCLC), are the most common malignancy and leading cause of cancer-related death worldwide. MicroRNAs (miRNAs) play a role in the occurrence of lung cancer by targeting both tumor suppressor genes and oncogenes. Among them, miR-128, miR-335-5p, and miR-1254 are mainly reported to function in regulating lung cancer cell behavior. Objectives: The aim of the study was to investigate the existence and expression levels of miRNA-128, miR-335-5p, and miR-1254 as well as mRNA and protein expression levels of VEGF and mRNA expression levels of K-RAS in both peripheral blood and cancerous tissue of NSCLC patients compared to the healthy subjects. Methods: Blood and tissue samples were collected from 50 healthy donors and 50 NSCLS patients. The serum level of VEGF was measured using the commercial enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of K-RAS and VEGF, as well as the expression of miRNA-128, miR-335-5p, and miR-1254, were determined, using real-time PCR. Results: Lower mRNA expression levels of miRNA-128, miR-335-5p, and miR-1254, as well as higher mRNA expression levels of VEGF and K-RAS, were detected in both peripheral blood and tissue samples from NSCLC patients compared to those of the healthy subjects. The VEGF protein levels in the peripheral blood of the patients were also found to be significantly lower than those in the healthy subjects. Conclusions: The findings from this study suggest that miR-335-5p, miR-1254, and miR-128, may contribute to the pathogenesis and tumorigenesis of NSCLC at least in part by modulation of proliferation, migration, and angiogenesis via targeting VEGF and K-RAS signaling pathways. K-RAS and VEGF may be target genes for miR-335-5p, miR-1254, and miR-128 in NSCLC patients.

SIRT1 silencing ameliorates malignancy of non-small cell lung cancer via activating FOXO1

Non-small cell lung cancer (NSCLC), being the most prevalent and lethal malignancy affecting the lungs, poses a significant threat to human health. This research aims at illustrating the precise role and related mechanisms of silent information regulator type-1 (SIRT1) in NSCLC progression. The expression pattern of SIRT1 in NSCLC cell lines was examined using quantitative real-time polymerase chain reaction and western blotting. Functional assays in NSCLC cell lines validated the biological capabilities of SIRT1 on malignant phenotypes, and its impact on tumorigenicity was further evaluated in vivo. In addition, the FOXO1 inhibitor AS1842856 was applied to verify the role of SIRT1 on FOXO pathway in vitro. SIRT1 expression was prominently elevated in NSCLC cell lines. The depletion of SIRT1 retarded the capabilities of proliferation, migration and invasion, while enhancing apoptosis in NSCLC cells. Furthermore, SIRT1 silencing restricted the tumorigenesis of NSCLC in vivo. Additionally, AS1842856 treatment ameliorated the inhibitory effect of SIRT1 deficiency on malignant phenotypes in NSCLC cells. SIRT1 deletion exerted an anti-oncogenic role in NSCLC via activation of FOXO1.

MAGE-A4-Responsive Plasma Cells Promote Non-Small Cell Lung Cancer.

Adaptive immunity is critical to eliminate malignant cells, while multiple tumor-intrinsic factors can alter this protective function. Melanoma antigen-A4 (MAGE-A4), a cancer-testis antigen, is expressed in several solid tumors and correlates with poor survival in non-small cell lung cancer (NSCLC), but its role in altering antitumor immunity remains unclear. We found that expression of MAGE-A4 was highly associated with the loss of PTEN , a tumor suppressor, in human NSCLC. Here we show that constitutive expression of human MAGE-A4 combined with the loss of Pten in mouse airway epithelial cells results in metastatic adenocarcinoma enriched in CD138 + CXCR4 + plasma cells, predominantly expressing IgA. Consistently, human NSCLC expressing MAGE-A4 showed increased CD138 + IgA + plasma cell density surrounding tumors. The abrogation of MAGE-A4-responsive plasma cells (MARPs) decreased tumor burden, increased T cell infiltration and activation, and reduced CD163 + CD206 + macrophages in mouse lungs. These findings suggest MAGE-A4 promotes NSCLC tumorigenesis, in part, through the recruitment and retention of IgA + MARPs in the lungs.

Wilms' Tumor 1-Associating Protein Promotes Nonsmall-Cell Lung Cancer Through the Expression of Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5.

This study aimed to analyze the functional roles and molecular mechanism of Wilms' tumor 1-associating protein (WTAP) in the tumorigenesis of nonsmall-cell lung cancer (NSCLC). Retrospective analysis was used. Tumor tissues and surrounding nontumor tissues of 150 patients with NSCLS who were surgically resected in the Fourth Hospital of Hebei Medical University from January 2016 to January 2018 were selected. The expression of WTAP in NSCLC tissues was detected by immunohistochemistry. Clinicopathologic parameters were then subjected to univariate and multivariate Cox regression analysis in purpose of uncovering the independent risk factors for overall survival time. MTS (3-[4,5-dimethylthiazol-zyl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazoliuzolium, inner salt) assay, colony formation assay, and transwell assays were performed to estimate cell proliferation, migration, and invasion. Meanwhile, the relationship between WTAP and the cell migration and invasion marker-related proteins were evaluated by Western blot analysis and RT-qPCR. WTAP expression was knocked-down in cell lines by shRNA, and RNA-Seq was performed to investigate the pathways regulated by WTAP. In NSCLC patients, WTAP was highly expressed in tumor tissues and the higher expression was significantly associated with poor overall survival (OS) ( P <0.01). Compared with the control group in vitro, the overexpression of WTAP could significantly promote cell proliferation, migration, and invasion ( P <0.01), while knock-down WTAP significantly reduces the above effects ( P <0.01). In a mouse orthotopic implantation model, higher WTAP abundance could significantly promote tumor enlargement compared with the control group ( P <0.01). Compared with the control group, the knock-down of WTAP significantly inhibit the expression of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) in cell lines ( P <0.01). Besides, in NSCLC, knocked-down CEACAM5 significantly reduced the impact of WTAP on cell proliferation, migration, and invasion compared with the control group ( P <0.05). This study suggests that high expression of WTAP was associated with poor clinical outcomes. CEACAM5 may play a synergistic role with WTAP to jointly promote NSCLC progression by enhancing cell proliferation, invasion, and migration.

Comprehensive analysis and validation reveal DEPDC1 as a potential diagnostic biomarker associated with tumor immunity in non-small-cell lung cancer.

Current evidence suggests that DEP domain containing 1 (DEPDC1) has an important effect on non-small-cell lung cancer (NSCLC). However, the diagnostic value and the regulatory function within NSCLC are largely unclear. This work utilized publicly available databases and in vitro experiments for exploring, DEPDC1 expression, clinical features, diagnostic significance and latent molecular mechanism within NSCLC. According to our results, DEPDC1 was remarkably upregulated in the tissues of NSCLC patients compared with non-carcinoma tissues, linked with gender, stage, T classification and N classification based on TCGA data and associated with smoking status and stage according to GEO datasets. Meanwhile, the summary receiver operating characteristic (sROC) curve analysis result showed that DEPDC1 had a high diagnostic value in NSCLC (AUC = 0.96, 95% CI: 0.94-0.98; diagnostic odds ratio = 99.08, 95%CI: 31.91-307.65; sensitivity = 0.89, 95%CI: 0.81-0.94; specificity = 0.92, 95%CI: 0.86-0.96; positive predictive value = 0.94, 95%CI: 0.89-0.98; negative predictive value = 0.78, 95%CI: 0.67-0.90; positive likelihood ratio = 11.77, 95%CI: 6.11-22.68; and negative likelihood ratio = 0.12, 95%CI: 0.06-0.22). Subsequently, quantitative real-time PCR (qRT-PCR) and western blotting indicated that DEPDC1 was high expressed in NSCLC cells. According to the in vitro MTS and apoptotic assays, downregulated DEPDC1 expression targeting P53 signaling pathway inhibited the proliferation of NSCLC cells while promoting apoptosis of NSCLC cells. Moreover, DEPDC1 was significantly correlated with immune cell infiltrating levels in NSCLC based on TCGA data, which were primarily associated with T cells CD4 memory activated, macrophages M1, B cells memory, mast cells resting, T cells regulatory, monocytes, and T cells CD4 memory resting. Compared with the group with high expression of DEPDC1, the group with low expression level had higher scores for immune checkpoint inhibitors (ICIs) treatment. GSEA confirmed that DEPDC1 was involved in gene expression and tumor-related signaling pathways. Finally, DEPDC1 and its associated immune-related genes were shown to be enriched in 'receptor ligand activity', 'external side of plasma membrane', 'regulation of innate immune response', and 'Epstein-Barr virus infection' pathways. The present study demonstrates that DEPDC1 may contribute to NSCLC tumorigenesis and can be applied as the biomarker for diagnosis and immunology.

Exosomal circ_0000735 contributes to non-small lung cancer malignant progression.

Circular RNA is an important regulator for non-small cell lung cancer (NSCLC). Circ_0000735 has been found to be significantly overexpressed in NSCLC tissues. Therefore, its role and mechanism in NSCLC progression need to be further explored. The expression levels of circ_0000735, miR-345-5p and A disintegrin and metalloprotease 19 (ADAM19) were determined using quantitative real-time PCR. EdU staining, wound healing and transwell assays were utilized to detect cell proliferation and metastasis. The protein levels of metastasis markers, exosome markers and ADAM19 were determined using western blot. Animal experiments were performed to confirm the role of circ_0000735 in NSCLC tumorigenesis. The exosomes from cells and serum were identified using transmission electron microscopy and nanoparticle tracking analysis. We found that circ_0000735 was upregulated in NSCLC, and its knockdown repressed NSCLC cell proliferation and metastasis. In terms of mechanism, circ_0000735 targeted miR-345-5p to regulate ADAM19. MiR-345-5p inhibitor reversed the suppressive effect of circ_0000735 knockdown on NSCLC progression, and ADAM19 overexpression abolished the inhibition effect of miR-345-5p on NSCLC progression. Also, animal experiments showed that silencing of circ_0000735 reduced NSCLC tumorigenesis. In addition, exosomes mediated the intercellular transmission of circ_0000735, and serum exosomal circ_0000735 might be an important indicator for the diagnosis of NSCLC. In conclusion, circ_0000735 facilitated NSCLC progression via miR-345-5p/ADAM19 pathway, and serum exosomal circ_0000735 might be a potential biomarker for NSCLC diagnosis.

Abstract 4675: Reduction in NSCLC tumorigenesis by targeting sphingosine kinase 2

Abstract Lung cancer will account for 21% of US cancer deaths in 2023 with 238,340 cases and 127,070 deaths. Treatment with EGFR-TKI (epidermal growth factor receptor tyrosine kinase) inhibitors initially decrease tumor growth, but often leads to TKI resistance. According to studies, non-small cell lung cancer (NSCLC) tumors express more SphK2 (Sphingosine kinase 2) than normal lung tissue and patients with overexpression have worse survival chances. However, SphK2's role in TKI resistance is unclear. We hypothesize that inhibiting/downregulating SphK2 could reduce tumorigenicity and overcome ER (Erlotinib) and OR (Osimertinib) resistance. We studied the expression of SphK2 in both drug-resistant (OR/ER) and drug sensitive (parental) H358, H2170, H3255, and PC9 NSCLC cell lines using Western blotting, qRT-PCR, and immunofluorescence. H3255OR cells were also bioprinted with GelMA hydrogel using an R-Gen 200 bioprinter to mimic the physiological tumor environment and treated with SphK2 siRNA or ABC294640 (SphK2i). Immunoblotting was done to analyze changes in the expression of SphK2 in H358 (WT-EGFR) and H3255 (mutant EGFR- L858R) parental and OR-resistant cell lines. Results indicated a 1.5-1.8-fold upregulation of SphK2. Increased SphK2 gene expression was observed using qPCR in TKI-resistant H3255OR (4.3-fold), PC9ER/OR with mutant EGFR (E746-A750del) (1.8-3.3-fold), and H2170ER/OR with WT EGFR (1.3-1.5-fold) compared to parental cells. Immunofluorescence studies showed increased fluorescence of SphK2 in H3255OR (2.6-fold), H358 ER (2.4-fold) and OR cells (2.3-fold) compared to parental cells. Spheroids formed from single cancer cells can "re-create" the growth of a tumor. The spheroid count of H3255OR was reduced by 73.98% and 56%, respectively, when SphK2i was added to the 3D-cell culture with Matrigel, compared to the diluent and OR alone (p≤0.01). We used 3D bioprinting to precisely stack cells on biological scaffolds to recreate lung tumors from patients and boost the therapeutic efficacy of these findings. When bioprinted spheroids of H3255OR were treated with SphK2i in combination with OR, the number of spheroids per microscopic field were reduced by 87.3% compared to OR alone. Bioprinted H3255OR spheroids treated with SphK2 siRNA and OR revealed a 75.8% and 84% reduction of spheroid count, respectively, compared to treatments with siRNA alone or OR alone (p≤0.05). This data supports the hypothesis that SphK2 suppression or downregulation can assist in mitigating oncogenesis. We also used H3255OR transwell migration assays to examine how SphK2 inhibition affects cell migration. Combining OR with SphK2i resulted in a 94.7% and 91.4% decrease in cell migration compared to diluent and OR alone (p≤0.01). In H358OR-resistant cells, combining OR with SphK2i resulted in a 94.97% inhibition compared to diluent (p≤0.01). These results suggest that targeting SphK2 could be a valuable approach for treating lung cancer. Citation Format: Ayesha Khan, Bamby Dieng, Namrata Dube, Mohammad Fazle Alam, Neelu Puri. Reduction in NSCLC tumorigenesis by targeting sphingosine kinase 2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4675.

GABPB1 plays a cancer-promoting role in non-small cell lung cancer

BackgroundGABPB1, the gene that encodes two isoforms of the beta subunit of GABP, has been identified as an oncogene in multiple malignant tumors. However, the role and mode of action of GABPB1 in malignant tumors, especially in lung cancer, are not well understood and need further research.MethodsOur research focused on examining the biological function of GABPB1 in NSCLC (Non-Small Cell Lung Cancer). We analysed tumor data from public databases to assess the expression of GABPB1 in NSCLC and its correlation with patient prognosis and investigated GABPB1 expression and methylation patterns in relation to the tumor microenvironment. In parallel, experiments were conducted using short hairpin RNA (shRNA) to suppress the GABPB1 gene in human lung cancer cells to evaluate the effects on cell proliferation, viability, and apoptosis.ResultsGABPB1 was widely expressed in various tissues of the human body. Compared to that in normal tissues, the expression of this gene was different in multiple tumor tissues. GABPB1 was highly expressed in lung cancer tissues and cell lines. Its expression was associated with molecular subtype and cellular signalling pathways, and a high level of GABPB1 expression was related to a poor prognosis in lung adenocarcinoma patients. The expression and methylation of GABPB1 affect the tumor microenvironment. After suppressing the expression of GABPB1 in both A549 and H1299 cells, we found a decrease in cell growth and expression, the formation of clones and an increase in the apoptosis rate.ConclusionsOur research verified that GABPB1 promotes the tumorigenesis of NSCLC and has an inhibitory effect on tumor immunity. The specific role of GABPB1 may vary among different pathological types of NSCLC. This molecule can serve as a prognostic indicator for lung adenocarcinoma, and its methylation may represent a potential breakthrough in treatment by altering the tumor immune microenvironment in lung squamous cell carcinoma. The role and mechanism of action of GABPB1 in NSCLC should be further explored.

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.

N6-methyladenosine demethyltransferase FTO mediated m6A modification of estrogen receptor alpha in non-small cell lung cancer tumorigenesis.

Fat mass and obesity-associated protein (FTO), which is closely linked with obesity and dietary intake, plays an important role in diet-related metabolic diseases. However, the underlying mechanism of the N6-methyladenosine (m6A) demethyltransferase FTO in tumor development and progression remains largely unexplored. Here, we demonstrated that FTO expression was largely lower in non-small cell lung cancer (NSCLC) samples than in adjacent healthy tissues, and its expression negatively correlated with poor prognosis. Gain- and loss-of-function assays revealed that FTO inhibited NSCLC tumor cell growth and metastasis in vitro and in vivo. Mechanistically, estrogen receptor alpha (ESR1) is a target of FTO, and increased FTO expression significantly impaired the m6A levels of ESR1 mRNA. There were two clear m6A modification sites (5247A and 5409A) in the 3' untranslated region (3'UTR) of ESR1, and FTO could decrease their methylation. Moreover, the m6A readers YTHDF1 and IGF2BP3 recognized and bound the m6A sites in ESR1 mRNA, thereby enhancing its stability and facilitating tumor growth. We also showed that ESR1 has good diagnostic value for NSCLC. In conclusion, we uncovered an important mechanism of epitranscriptomic regulation by the FTO-YTHDF1-IGF2BP3-ESR1 axis and identified the potential of m6A-dependent therapeutic strategies for NSCLC.

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.

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.

Promotion of non-small cell lung cancer tumor growth by FHL2 via inducing angiogenesis and vascular permeability.

Antiangiogenetic therapy is one of the effective strategies for non-small cell lung cancer (NSCLC) treatment. Four-and-a-half LIM-domain protein 2 (FHL2) serves as a key function in cell growth and metastasis of multiple cancers, but the role of FHL2 in NSCLC angiogenesis has not been intensely examined. FHL2 expression in NSCLC tissues and cell lines and its correlation with patients prognosis were investigated by using The Cancer Genome Atlas (TCGA) database and quantitative polymerase chain reaction (qPCR). Cell Counting Kit-8 (CCK-8) assay, EdU (5-ethynyl-2'-deoxyuridine) assay, and a xenograft model were used to investigate the effects of FHL2 on NSCLC progression in vitro and in vivo. CCK-8, wound-healing, Transwell invasion, tube formation, and permeability assays were performed to determine the roles of FHL2 in angiogenesis and vascular permeability. Vascular endothelial growth factor A (VEGFA) enzyme-linked immunosorbent assay (ELISA) assay, Western blot analysis, and MK-2206 were used to investigate the specific mechanism mediated by FHL2. We demonstrated that FHL2 was significantly upregulated in NSCLC tissues and cell lines and was associated with poor prognosis. FHL2 overexpression enhanced the cell viability of NSCLC cells, as well as the proliferation, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs). In addition, we determined that FHL2 activated the AKT-mTOR signaling pathway in HUVECs by promoting VEGFA secretion from NSCLC cells, thereby inducing angiogenesis and vascular leakiness. We further confirmed that FHL2 also promoted NSCLC tumor growth in vivo. Our study revealed the role of FHL2 in NSCLC and the mechanism by which FHL2 promotes NSCLC tumorigenesis, providing novel insights into targeted therapy for NSCLC.

Comprehensive functional interrogation of susceptibility loci in GWASs identified KIAA0391 as a novel oncogenic driver via regulating pyroptosis in NSCLC

Approximately 51 non-small-cell lung cancer (NSCLC) risk loci have been identified by genome-wide association studies (GWASs). We conducted a high throughput RNA-interference (RNAi) screening to identify the candidate causal genes in NSCLC risk loci. KIAA0391 at 14q13.1 had the highest score and could promote proliferation and metastasis of NSCLC in vitro and in vivo. We next prioritized rs3783313 as a causal variant at 14q13.1, by integrating a large-scale population study consisting of 27,120 lung cancer cases and 27,355 controls, functional annotation, and expression quantitative trait locus (eQTL) analysis. Then we found that rs3783313 could facilitate a promoter-enhancer interaction to upregulate KIAA0391 expression by affecting the affinity of transcription factor NFYA. Mechanistically, KIAA0391 knockdown dramatically influenced pyroptosis-related pathways and increased the expression of CASP1. And KIAA0391 transcriptionally repressed CASP1 by binding to SMAD2 and induced an anti-pyroptosis phenotype, promoting tumorigenesis of NSCLC, which provides new insights and potential target for NSCLC.

UCP2 promotes NSCLC proliferation and glycolysis via the mTOR/HIF-1α signaling.

Metabolic disturbance is a hallmark of cancers. Targeting key metabolic pathways and metabolism-related molecular could be a potential therapeutic approach. Uncoupling protein 2 (UCP2) plays a pivotal part in the malignancy of cancer and its capacity to develop resistance to pharmaceutical interventions. However, it is unclear about the mechanism of how UCP2 acts in the tumor growth and metabolic reprogramming process in non-small cell lung cancer (NSCLC). Here, we conducted qRT-PCR to investigate the expression of UCP2 in both NSCLC tissues and cell lines. Subsequent functional studies including colony formation assay, CCK-8 assay, and glycolysis assay were conducted to investigate the functions of UCP2 in NSCLC. The regulatory mechanism of UCP2 toward the mammalian target of rapamycin (mTOR) and hypoxia-inducible factor-1 alpha (HIF-1α) signaling in NSCLC was confirmed through western blotting. We observed a significant upregulation of UCP2 in both NSCLC tissues and cell lines. The increased expression of UCP2 has a strong association with a worse outlook. Silencing UCP2 remarkably dampened NSCLC cell proliferation and glycolysis capacities. Mechanically, UCP2 promoted NSCLC tumorigenesis partially via regulating the mTOR/HIF-1α axis. Taken together, we explored the functions as well as the mechanisms of the UCP2/mTOR/HIF-1α axis in NSCLC progression, uncovering potential biological signatures and targets for NSCLC treatment.

Proline Dehydrogenase (PRODH) Is Expressed in Lung Adenocarcinoma and Modulates Cell Survival and 3D Growth by Inducing Cellular Senescence.

The identification of markers for early diagnosis, prognosis, and improvement of therapeutic options represents an unmet clinical need to increase survival in Non-Small Cell Lung Cancer (NSCLC), a neoplasm still characterized by very high incidence and mortality. Here, we investigated whether proline dehydrogenase (PRODH), a mitochondrial flavoenzyme catalyzing the key step in proline degradation, played a role in NSCLC tumorigenesis. PRODH expression was investigated by immunohistochemistry; digital PCR, quantitative PCR, immunoblotting, measurement of reactive oxygen species (ROS), and functional cellular assays were carried out. PRODH expression was found in the majority of lung adenocarcinomas (ADCs). Patients with PRODH-positive tumors had better cancer-free specific and overall survival compared to those with negative tumors. Ectopic modulation of PRODH expression in NCI-H1299 and the other tested lung ADC cell lines decreased cell survival. Moreover, cell proliferation curves showed delayed growth in NCI-H1299, Calu-6 and A549 cell lines when PRODH-expressing clones were compared to control clones. The 3D growth in soft agar was also impaired in the presence of PRODH. PRODH increased reactive oxygen species production and induced cellular senescence in the NCI-H1299 cell line. This study supports a role of PRODH in decreasing survival and growth of lung ADC cells by inducing cellular senescence.