Epithelial-mesenchymal Transition In Non-small Cell Lung Cancer Research Articles

Expression and potential molecular mechanism of TOP2A in metastasis of non-small cell lung cancer

DNA topoisomerase II alpha (TOP2A) expression, gene alterations, and enzyme activity have been studied in various malignant tumors. Abnormal elevation of TOP2A expression is considered to be related to the development of non-small cell lung cancer (NSCLC). However, its association with tumor metastasis and its mode of action remains unclear. Bioinformatics, real-time quantitative PCR, immunohistochemistry and immunoblotting were used to detect TOP2A expression in NSCLC tissues and cells. Cell migration and invasion assays as well as cytoskeletal staining were performed to analyze the effects of TOP2A on the motility, migration and invasion ability of NSCLC cells. Cell cycle and apoptosis assays were used to verify the effects of TOP2A on apoptosis as well as cycle distribution in NSCLC. TOP2A expression was considerably upregulated in NSCLC and significantly correlated with tumor metastasis and the occurrence of epithelial–mesenchymal transition (EMT) in NSCLC. Additionally, by interacting with the classical ligand Wnt3a, TOP2A may trigger the canonical Wnt signaling pathway in NSCLC. These observations suggest that TOP2A promotes EMT in NSCLC by activating the Wnt/β-catenin signaling pathway and positively regulates malignant events in NSCLC, in addition to its significant association with tumor metastasis. TOP2A promotes the metastasis of NSCLC by stimulating the canonical Wnt signaling pathway and inducing EMT. This study further elucidates the mechanism of action of TOP2A, suggesting that it might be a potential therapeutic target for anti-metastatic therapy.

Evaluation of drug resistance for EGFR-TKIs in lung cancer via multicellular lung-on-a-chip

Drug resistance to irreversible epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a primary factor affecting their therapeutic efficacy in human non-small cell lung cancer (NSCLC). NSCLC cells can undergo epithelial-mesenchymal transition (EMT) induced by many factors in the tumour microenvironment (TME), which plays a crucial role in tumour drug resistance. In this study, a multicellular lung-on-a-chip that can realise the cell co-culture of the human non-small cell lung cancer cell line HCC827, human foetal lung fibroblasts (HFL-1), and human umbilical vein endothelial cells (HUVECs) is prepared. The TME was simulated on the chip combined with perfusion and other factors, and the drug evaluation of osimertinib was performed to explore the drug resistance mechanism of EGFR-TKIs. In the early stages, a two-dimensional static cell co-culture was achieved by microchip, and the results showed that HFL-1 cells could be transformed into cancer-associated fibroblasts (CAFs), and HCC827 cells could undergo EMT, both of which were mediated by Interleukin-6 (IL-6). Vimentin (VIM) and Alpha Skeletal Muscle Actin (a-SMA) expression of HFL-1 was upregulated, whereas E-cadherin (E-cad) expression of HCC827 was down-regulated. Further, N-cadherin (N-cad) expression of HCC827 was upregulated. In both the static cell co-culture and multicellular lung-on-a-chip, HCC827 cells with CAFs co-culture or IL-6 treatment developed resistance to osimertinib. Further use of the IL-6 antibody inhibitor tocilizumab could reverse EGFR-TKI resistance to a certain extent. Combination therapy with tocilizumab and EGFR-TKIs may provide a novel therapeutic strategy for overcoming EGFR-TKI resistance caused by EMT in NSCLC. Furthermore, the lung-on-a-chip can simulate complex TME and can be used for evaluating tumour resistance and exploring mechanisms, with the potential to become an important tool for personalised diagnosis, treatment, and biomedical research.

Circ-10720 as a ceRNA adsorbs microRNA-1238 and modulates ZEB2 to boost NSCLC development by activating EMT

BackgroundCircular RNAs (circRNAs) are critical regulators in the progression of tumors. This experimental design aimed to explore the mechanism of circ-10720 in non-small cell lung cancer (NSCLC).MethodsWe used RT-qPCR to measure circ-10720 expression in clinical samples and analyzed its relationship with the clinicopathological characteristics of NSCLC patients. The expression levels of microRNA-1238 (miR-1238) and Zinc Finger E-box-binding Homeobox 2 (ZEB2) in clinical samples were detected by RT-qPCR. NSCLC cells were transfected with relevant plasmids or sequences. Circ-10720, miR-1238, and ZEB2 expressions in cells were analyzed via RT-qPCR or western blot. Cell proliferation, apoptosis, migration, and invasion were assessed with CCK-8, flow cytometry, and transwell assay, respectively. The protein expression of ZEB2 and epithelial–mesenchymal transition (EMT)-related markers (E-cadherin, Vimentin, N-cadherin) were detected via western blot. Xenograft assay was used to determine the effect of circ-10720 on NSCLC in vivo. Circ-10720 and ZEB2 expressions in tumors were detected using RT-qPCR or Western blot. Immunohistochemistry was used to evaluate E-cadherin and N-cadherin expression in tumors. Finally, the binding relationship between miR-1238 with circ-10720 or ZEB2 was verified by the bioinformatics website, dual luciferase reporter assay, RNA pull-down assay, and RIP assay.ResultsCirc-10720 was upregulated in NSCLC and correlated with TNM stage of NSCLC patients. MiR-1238 was lowly expressed but ZEB2 was highly expressed in NSCLC. Circ-10720 silencing suppressed the proliferation, metastasis, and EMT of NSCLC cells. Mechanically, circ-10720 was a competitive endogenous RNA (ceRNA) for miR-1238, and ZEB2 was a target of miR-1238. circ-10720-modulated ZEB2 via competitively binding with miR-1238 to control NSCLC progression. In addition, circ-10720 knockdown suppressed tumor growth in vivo.ConclusionsCirc-10720 acts as a ceRNA to adsorb miR-1238 and modulate ZEB2 to facilitate the proliferation, migration, invasion, and EMT of NSCLC cells.Graphical

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.

CCL2 promotes metastasis and epithelial-mesenchymal transition of non-small cell lung cancer via PI3K/Akt/mTOR and autophagy pathways.

In non-small cell lung cancer (NSCLC), metastasis is the most common phenotype, and autophagy plays a vital role in its regulation. However, there are limited data on how autophagy-related genes and metastasis-related genes affect NSCLC progression. Our goal was to identify the genes that regulate autophagy and metastasis in NSCLC, and to assess the underlying mechanisms in this current study. RNA sequencing data from public databases were used to screen differentially expressed autophagy- and metastasis-associated genes. Enrichment analyses and immune correlations were conducted to identify hub genes and potential regulating pathways in NSCLC. In this study, we found that CCL2 expression was highly expressed in NSCLC tissues and high CCL2 level was correlated with strong infiltration in lung tissues from NSCLC patients. Overexpression of CCL2 can enhance the metastasis of NSCLC cells in nude mice. Furthermore, CCL2 activated the PI3K/Akt/mTOR signalling pathway axis, promoted epithelial-mesenchymal transition (EMT), and blocked the autophagic flux in NSCLC cells. Therefore, our results indicate that CCL2 promotes metastasis and EMT of NSCLC via PI3K/Akt/mTOR axis and autophagy signalling pathways. We believe that CCL2 could be a probable target for the diagnosis and therapeutics of NSCLC, and this study may expand our understanding of lung cancer.

Abstract 3600: MET-mediated STING activation regulates EMT and NF-ΚB pathway in non-small cell lung cancer

Abstract The cyclic GAMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is known to have anti-tumor activity by activating the innate immune response against cytosolic double-stranded DNA (dsDNA). The regulating mechanism of STING remains unclear, and a pro-tumorigenic effect of STING pathway was recently reported. In this study, we investigated the expression of STING by MET and a novel role of the STING pathway in non-small cell lung cancer (NSCLC). The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) analysis showed that cGAS/STING pathway is enriched and STING expression is higher in MET-overexpressing tumors. Western blot for NSCLC cells and immunohistochemistry for NSCLC tumor tissues showed positive correlation between MET and STING expression in NSCLC cells. MET-overexpression and a MET agonist (HGF) treatment increased STING expression and CXCL10 and IFNβ expression and secretion, whereas MET-knockdown and a MET inhibitor (capmatinib) treatment decreased STING expression in NSCLC cells. In MET-positive and STING-positive cells, a STING agonist (poly-dAdT and diABZI) treatment led to increased activation of STING signaling pathway. In EBC-1 cells, MET knockdown decreased STING expression and STING-agonist-induced STING-pathway activation and cytokine production. To further investigate the role STING in NSCLC, EBC-1 cells were transfected with STING siRNA and submitted to RNS-seq. Gene set enrichment analysis revealed that several pathways were affected by STING expression. Epithelial-mesenchymal transition (EMT) and TNFα-signaling via NF-κB signaling were significantly downregulated in STING-knockdowned cells. In vitro experiments using EBC-1 cells, STING overexpression increased SLUG and TWIST1 expression and migration of cells, whereas STING knockdown decreased SLUG and TWIST1 expression and migration of cells. In summary, this study demonstrated that MET and STING expression were positively correlated with each other in NSCLC and MET overexpression increased STING expression and subsequent activation upon STING agonist stimulation. In addition, STING contributed to EMT in NSCLC. Citation Format: Seungchan Mun, Bogyeong Han, HyunKyung Ahn, Jaemoon Koh, Sehui Kim, DooHyun Chung, YoonKyung Jeon. MET-mediated STING activation regulates EMT and NF-ΚB pathway in non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3600.

Pharmacological targeting of MTHFD2 suppresses NSCLC via the regulation of ILK signaling pathway

Lung cancer is the most common cause of cancer related deaths worldwide with the highest mortality rate. Non-small cell lung cancer (NSCLC) accounts for about 85 % of lung cancers. Mitochondrial methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is a bifunctional enzyme and is the most differentially expressed metabolic enzyme in various tumors including lung cancer. However, little is known about how MTHFD2 functions in NSCLC. Integrin-linked kinase (ILK) signaling plays key a role in tumor progression including metastasis, proliferation and migration. Here, we show that MTHFD2 inhibition results in suppression of cell growth, migration, invasion and epithelial-mesenchymal transition (EMT) in NSCLC. Microarray analysis suggests that MTHFD2 is positively associated with ILK signaling based on western blotting results. In addition, the phosphorylation of AMPKα plays an essential role in MTHFD2 regulation of ILK signaling. Further, the small-molecule compound C18 inhibits MTHFD2 with great efficiency. C18 blocks MTHFD2/ILK signaling pathway and restrains cell growth, migration, invasion, and EMT of NSCLC and induces apoptosis. In brief, our study found that the positive impact of MTHFD2 is mediated via ILK signaling pathway in NSCLC. Thus, blocking MTHFD2 represents a promising therapeutic strategy against NSCLC clinically.

PLK1-mediated phosphorylation of β-catenin enhances its stability and transcriptional activity for extracellular matrix remodeling in metastatic NSCLC.

Rationale: β-catenin is a component for cell adhesion and a transcriptional coactivator in epithelial-mesenchymal transition (EMT). Previously we found that catalytically active PLK1 drives EMT in non-small cell lung cancer (NSCLC), upregulating extracellular matrix factors including TSG6, laminin γ2, and CD44. To understand the underlying mechanism and clinical significance of PLK1 and β-catenin in NSCLC, their relationship and function in metastatic regulation were investigated. Methods: The clinical relevance between the survival rate of NSCLC patients and the expression of PLK1 and β-catenin was analyzed by a KM plot. Immunoprecipitation, kinase assay, LC-MS/MS spectrometry, and site-directed mutagenesis were performed to reveal their interaction and phosphorylation. A lentiviral doxycycline-inducible system, Transwell-based 3D culture, tail-vein injection model, confocal microscopy, and chromatin immunoprecipitation assays were used to elucidate the function of phosphorylated β-catenin in the EMT of NSCLC. Results: Clinical analysis revealed that the high expression of CTNNB1/PLK1 was inversely correlated with the survival rates of 1,292 NSCLC patients, especially in metastatic NSCLC. In TGF-β-induced or active PLK1-driven EMT, β-catenin, PLK1, TSG6, laminin γ2, and CD44 were concurrently upregulated. β-catenin is a binding partner of PLK1 in TGF-β-induced EMT and is phosphorylated at S311. Phosphomimetic β-catenin promotes cell motility, invasiveness of NSCLC cells, and metastasis in a tail-vein injection mouse model. Its upregulated stability by phosphorylation enhances transcriptional activity through nuclear translocation for the expression of laminin γ2, CD44, and c-Jun, therefore enhancing PLK1 expression by AP-1. Conclusions: Our findings provide evidence for the critical role of the PLK1/β-catenin/AP-1 axis in metastatic NSCLC, implying that β-catenin and PLK1 may serve as a molecular target and prognostic indicator of the therapeutic response in metastatic NSCLC patients.

Circular RNA hsa_circ_0004689 (circSWT1) promotes NSCLC progression via the miR-370-3p/SNAIL axis by inducing cell epithelial-mesenchymal transition (EMT).

Previous studies have reported the role of circular RNAs (circRNAs) in the progression of non-small-cell lung cancer (NSCLC). SWT1-derived circRNAs were confirmed to affect the apoptosis of cardiomyocytes; however, the biological functions of SWT1-derived circRNAs in cancers are still unknown. Here, we investigated the potential role of SWT1-derived circRNAs in NSCLC. We used quantitative real-time polymerase chain reaction (qRT-PCR) to measure the expression of circSWT1 in NSCLC tissues and paired normal tissues. The potential functions of circSWT1 in tumor progression were assessed by CCK-8, colony formation, wound healing, and matrigel transwell assays in vitro and by xenograft tumor models in vivo. Next, epithelial-mesenchymal transition (EMT) was evaluated by western blotting, immunofluorescence, and immunohistochemistry (IHC). Moreover, circRIP, RNA pulldown assays, luciferase reporter gene assays, and FISH were conducted to illuminate the molecular mechanisms of circSWT1 via the miR-370-3p/SNAIL signal pathway. Then, we knocked out SNAIL in A549 and H1299 cells to identify the roles of circSWT1 in the progression and EMT of NSCLC through SNAIL. Finally, circSWT1 functions were confirmed in vivo using xenograft tumor models. CircSWT1 expression was significantly upregulated in NSCLC tissues, and high expression of circSWT1 predicted poor prognosis in NSCLC via survival analysis. In addition, overexpression of circSWT1 promoted the invasion and migration of NSCLC cells. Subsequently, we found that overexpression of circSWT1 induced EMT and that knockdown of circSWT1 inhibited EMT in NSCLC cells. Mechanistically, circSWT1 relieved the inhibition of downstream SNAIL by sponging miR-370-3p. Moreover, we found that these effects could be reversed by knocking out SNAIL. Finally, we verified that circSWT1 promoted NSCLC progression and EMT in xenograft tumor models. CircSWT1 promoted the invasion, migration, and EMT of NSCLC. CircSWT1 could serve as a potential biomarker and a potential therapeutic target for NSCLC.

LMNA Reduced Acquired Resistance to Erlotinib in NSCLC by Reversing the Epithelial-Mesenchymal Transition via the FGFR/MAPK/c-fos Signaling Pathway.

For patients exhibiting non-small-cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutations, epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are a first-line treatment. However, most patients who initially responded to EGFR-TKIs eventually developed acquired resistance, limiting the effectiveness of therapy. It has long been known that epithelial-mesenchymal transition (EMT) leads to acquired resistance to EGFR-TKIs in NSCLC. However, the mechanisms underlying the resistance dependent on EMT are unknown. This research aimed to reveal the effects of LMNA in the regulation of acquired resistance to erlotinib by EMT in NSCLC. The acquired erlotinib-resistant cells (HCC827/ER) were induced by gradual increase of concentrations of erlotinib in erlotinib-sensitive HCC827 cells. RNA sequencing and bioinformatics analysis were performed to uncover the involvement of LMNA in the EMT process that induced acquired resistance to erlotinib. The effect of LMNA on cell proliferation and migration was measured by clone-formation, wound-healing, and transwell assays, respectively. The EMT-related protein, nuclear shape and volume, and cytoskeleton changes were examined by immunofluorescence. Western blot was used to identify the underlying molecular mechanism of LMNA regulation of EMT. HCC827/ER cells with acquired resistance to erlotinib underwent EMT and exhibited lower LMNA expression compared to parental sensitive cells. LMNA negatively regulated the expression of EMT markers; HCC827/ER cells showed a significant up-regulation of mesenchymal markers, such as CDH2, SNAI2, VIM, ZEB1, and TWIST1. The overexpression of LMNA in HCC827/ER cells significantly inhibited EMT and cell proliferation, and this inhibitory effect of LMNA was enhanced in the presence of 2.5 μM erlotinib. Furthermore, a decrease in LMNA expression resulted in a higher nuclear deformability and cytoskeletal changes. In HCC827/ER cells, AKT, FGFR, ERK1/2, and c-fos phosphorylation levels were higher than those in HCC827 cells; Furthermore, overexpression of LMNA in HCC827/ER cells reduced the phosphorylation of AKT, ERK1/2, c-fos, and FGFR. In conclusion, our findings first demonstrated that downregulation of LMNA promotes acquired EGFR-TKI resistance in NSCLC with EGFR mutations by EMT. LMNA inhibits cell proliferation and migration of erlotinib-resistant cells via inhibition of the FGFR/MAPK/c-fos signaling pathway. These findings indicated LMNA as a driver of acquired resistance to erlotinib and provided important information about the development of resistance to erlotinib treatment in NSCLC patients with EGFR mutations.

Exosomal epidermal growth factor receptor is involved in HPV-16 E7-induced epithelial-mesenchymal transition of non-small cell lung cancer cells: A driver of signaling in vivo?

ABSTRACT Our previous studies have demonstrated that human papillomavirus (HPV)-16 E7 oncoprotein promoted epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) cells. Moreover, recent studies have found that exosomes can mediate EMT of NSCLC cells and epidermal growth factor receptor (EGFR) is related to the progression of NSCLC. Here, we further investigated the role of exosomal EGFR in HPV-16 E7-induced EMT of NSCLC cells. Our results showed that the exosomes derived from the stable HPV-16 E7-overexpressing A549 and NCI-H460 NSCLC cells (E7 Exo) significantly increased migration, invasion, and proliferation abilities of NSCLC cells as compared with the exosomes derived from empty vector-infected NSCLC cells (ev Exo). Moreover, both in vitro and in vivo results demonstrated that E7 Exo dramatically enhanced EMT of NSCLC cells and promoted the growth of subcutaneous NSCLC xenografts. Additionally, HPV-16 E7 enhanced the expression of EGFR and p-EGFR in both NSCLC cells and exosomes. Furthermore, the inhibition of EGFR activation or exosome secretion suppressed E7 Exo-induced migration, invasion, and EMT of NSCLC. Moreover, 12 kinds of differentially expressed miRNAs between E7 Exo and ev Exo (fold change≥2, P ≤ .05) were screened out, of which 7 miRNAs were up-regulated while 5 miRNAs were down-regulated in A549 E7 Exo. Taken together, our findings suggest that exosomal EGFR is involved in HPV-16 E7-induced EMT of NSCLC cells, which may play a key role in the progression of HPV-related NSCLC.

Overexpression of 14-3-3ζ primes disease recurrence, metastasis and resistance to chemotherapy by inducing epithelial-mesenchymal transition in NSCLC.

The prognosis of non-small cell lung cancer (NSCLC) is disappointing because disease recurrence and distant metastasis inevitably occurred. The aim of the present study is to identify novel biomarkers predicting tumor recurrence and metastasis. The 14-3-3ζ protein has been extensively described as a tumor promoter in a panel of solid tumors, including NSCLC. However, there is a big gap regarding the knowledge between 14-3-3ζ and NSCLC recurrence. In this study, we found that overexpression of 14-3-3ζ was more frequent in NSCLC tumor tissues with tumor recurrence. By using scratch healing assay and transwell assay, we demonstrated that NSCLC cells with high expression of 14-3-3ζ gained increased motile and invasive capacity, whereas siRNA-mediated knockdown of endogenous 14-3-3ζ abrogated cancer cell dissemination. Intriguingly, we found that NSCLC cells underwent epithelial-mesenchymal transition (EMT) after the induction of 14-3-3ζ in vitro and in vivo. These findings could be readily recaptured in clinical setting since we showed that NSCLC tumor specimen with high expression of 14-3-3ζ revealed biological features of EMT. Overexpression of 14-3-3ζ also enhanced the phosphorylation of Akt and promoted the proliferation of NSCLC cell lines. In agreement with this notion, we reported that NSCLC cells with high expression of 14-3-3ζ became resistant to chemotherapy-induced apoptosis. These findings strongly suggested that 14-3-3ζ as a novel biomarker predicting risks of disease recurrence and screening 14-3-3ζ status may be a promising approach to select patients who experienced high risks of cancer recurrence and resistance to chemotherapy.

RNA demethylase ALKBH5 inhibits TGF-β-induced EMT by regulating TGF-β/SMAD signaling in non-small cell lungcancer.

AlkB homolog 5 (ALKBH5) has been revealed as a key RNA N6 -methyladenosine (m6 A) demethylase that is implicated in development and diseases. However, the function of ALKBH5 in TGF-β-induced epithelial-mesenchymal transition (EMT) and tumor metastasis of non-small-cell lung cancer (NSCLC) remains unknown. Here, we firstly show that ALKBH5 expression is significantly reduced in metastatic NSCLC. ALKBH5 overexpression inhibits TGF-β-induced EMT and invasion of NSCLC cells, whereas ALKBH5knockdown promotes the corresponding phenotypes. ALKBH5 overexpression suppresses TGF-β-stimulated NSCLC cell metastasis in vivo. ALKBH5 overexpression decreases the expression and mRNA stability of TGFβR2 and SMAD3 but increases those of SMAD6, while ALKBH5knockdown causes the opposite results. Importantly, ALKBH5 overexpression or knockdown leads respectively to an attenuated or augmented phosphorylation of SMAD3, an indispensable downstream effector that activates TGF-β/SMAD signaling. Moreover, m6 A-binding proteins YTHDF1/3 promotes TGFβR2 and SMAD3 expression, and YTHDF2 inhibits SMAD6 expression. YTHDF1/2/3 facilitates TGF-β-stimulated EMT and invasion of NSCLC cells. Mechanistically, ALKBH5 affects TGFβR2, SMAD3 and SMAD6 expression and mRNA stability by erasing m6 A modification in NSCLC cells. ALKBH5 weakens YTHDF1/3-mediated TGFβR2 and SMAD3mRNA stabilization, and abolishes YTHDF2-mediated SMAD6mRNA degradation, supporting the notion that ALKBH5 inhibits TGF-β-induced EMT and invasion of NSCLC cells via YTHD1/2/3-mediated mechanism. Taken together, our findings highlight an important role of ALKBH5 in regulating TGF-β/SMAD signaling, and establish a mechanistic interaction of ALKBH5 with TGFβR2/SMAD3/SMAD6 for controlling TGF-β-induced EMT in NSCLCs.

Circular RNA circZCCHC6 contributes to tumorigenesis by regulating LPCAT1 via miR-433-3p in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-associated mortality worldwide. Circular RNA (circRNA) circZCCHC6 has been reported to be upregulated in the plasma from NSCLC patients. This study is designed to explore the role and mechanism of circZCCHC6 in NSCLC. CircZCCHC6, microRNA-433-3p (miR-433-3p), and lysophosphatidylcholine acyltransferase 1 (LPCAT1) level were determined by real-time quantitative polymerase chain reaction. Cell viability, cell cycle progression, migration, and invasion were assessed by 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry, wound healing, and transwell assays, severally. The binding relationship between miR-433-3p and circZCCHC6 or LPCAT1 was predicted by Circinteractome or Starbase, and then verified by a dual-luciferase reporter, RNA pull-down, or RNA Immunoprecipitation (RIP) assays. Protein levels of LPCAT1, Cyclin D1, E-cadherin, and Vimentin were examined by western blot assay. The biological role of circZCCHC6 on NSCLC tumor growth and epithelial-mesenchymal transition (EMT) was examined by the xenograft tumor model in vivo. CircZCCHC6 was highly expressed in NSCLC serum, tissues, and cells. Moreover, circZCCHC6 knockdown could repress cell viability, cell cycle progression, migration, invasion, and EMT in NSCLC cells in vitro. The mechanical analysis suggested that circZCCHC6 acted as a sponge of miR-433-3p to regulate LPCAT1 expression. CircZCCHC6 silencing hindered cell growth and EMT of NSCLC in vivo. CircZCCHC6 inhibited the progression of NSCLC cells partly by regulating the miR-433-3p/LPCAT1 axis, implying a promising therapeutic target for the NSCLC treatment.

Activation of the Hedgehog pathway mediates resistance to epidermal growth factor receptor inhibitors in non-small cell lung cancer.

The current study aimed to investigate the function of the Hedgehog pathway and its association with epithelial-mesenchymal transition (EMT) in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance in non-small cell lung cancer (NSCLC). Lung tumor tissue specimens from EGFR TKI-resistant patients, including those with brain metastases, had hyperactive Hedgehog signaling compared with those from TKI-sensitive patients. SHH stimulation promoted GLI1 activation as well as cell motility in parental PC9 cells while suppressing gefitinib-induced apoptosis in gefitinib-resistant cells. SHH also promoted EMT in parental PC9 cells via E-cadherin suppression and N-cadherin and vimentin upregulation. The knockdown of GLI1 exhibited the opposite effects. Besides, SHH induced, whereas GLI1 knockdown reversed gefitinib resistance in xenograft tumors. The Hedgehog pathway inhibitor GDC-0449 synergized with gefitinib to increase xenograft tumor sensitivity to chemotherapy and extend survival in tumor-bearing animals. These results suggest the Hedgehog pathway mediates EGFR TKI resistance and induces EMT in NSCLC, representing a potential therapeutic target to defeat TKI resistance.

STYK1/NOK Promotes Metastasis and Epithelial-Mesenchymal Transition in Non-small Cell Lung Cancer by Suppressing FoxO1 Signaling.

AimsSerine/threonine/tyrosine kinase 1 (STYK1) has been previously shown to have oncogenic properties, and emerging evidence suggests that STYK1 expression correlates with epithelial-mesenchymal transition (EMT). However, the mechanism of STYK1 involvement in oncogenesis remains unknown. The present study aimed to elucidate how STYK1 expression level relates to the metastasis, migration, invasion, and EMT in non-small cell lung cancer (NSCLC) and to determine the molecular mechanism of STYK1 effects.MethodsSerine/threonine/tyrosine kinase 1 (STYK1) expression level and its relationship with the prognosis of NSCLC were determined using the ONCOMINE database and clinical cases. Non-small cell lung cancer cell lines with the overexpression or knockdown of STYK1 were established to determine whether STYK1 promotes cell migration, invasion, and EMT in vitro and in vivo. In addition, a constitutively active FoxO1 mutant (FoxO1AAA) was used to examine the role of FoxO1 in the STYK1-mediated upregulation of metastasis and EMT in NSCLC.ResultsSerine/threonine/tyrosine kinase 1 (STYK1) was upregulated in NSCLC tissues and cell lines, and its overexpression correlated with poor prognosis in patients with NSCLC after surgery. Enhanced expression of STYK1 potentiated the migration, invasion, and EMT in SW900 cells, thereby promoting metastasis, whereas knockdown of STYK1 inhibited these cellular phenomena in Calu-1 cells. Furthermore, STYK1 expression was positively related to the level of phosphorylated-FoxO1, whereas the constitutively active FoxO1 mutant protected against the positive effect of STYK1 overexpression on cell migration, invasion, and EMT.ConclusionSerine/threonine/tyrosine kinase 1 (STYK1) was upregulated in NSCLC and correlated with poor clinical outcomes. In addition, STYK1 suppressed FoxO1 functions, thereby promoting metastasis and EMT in NSCLC.

Repression of RhoJ expression promotes TGF-β-mediated EMT in human non-small-cell lung cancer A549cells

Non-small-cell lung cancer (NSCLC) accounts for most cancer-related deaths because of its strong metastatic ability. It is important to understand NSCLC's molecular mechanisms of metastasis. RhoJ, a protein that belongs to the Rho family of small GTPases, regulates endothelial motility, angiogenesis, and adipogenesis. Recently, bioinformatics analysis showed that NSCLC patients with lower RhoJ expression had a worse survival outcome than those with high RhoJ expression. However, little is known about RhoJ's role in NSCLC.In the present study, we demonstrated that RhoJ knockdown accelerated TGF-βmediated epithelial-to-mesenchymal transition (EMT), an important cancer metastasis process, in A549 and PC-9 cells. Furthermore, using Matrigel-coated transwell chambers, we showed that RhoJ knockdown enhanced the invasion capacity of A549 cells that had undergone EMT. Also, reduced RhoJ expression increased Smad3 phosphorylation and Snail expression during the EMT process. Our results provide the first evidence of a potential novel role for RhoJ in the inhibition of EMT via modulation of the TGF-β–Smad signaling pathway, and shed new light on the mechanisms underlying EMT in NSCLC.

SATB1 protein is associated with the epithelial‑mesenchymal transition process in non‑small cell lung cancers.

Lung cancer is one of the most frequently diagnosed neoplasms and the leading cause of cancer-related mortality worldwide. Its predominant subtype is non-small cell lung cancer (NSCLC), which accounts for over 80% of the cases. Surprisingly, the majority of lung cancer-related deaths are caused not by a primary tumour itself, but by its metastasis to distant organs. Therefore, it becomes especially important to identify the factors involved in lung cancer metastatic spread. Special AT-rich binding protein 1 (SATB1) is a nuclear matrix protein that mediates chromatin looping and plays the role of global transcriptional regulator. During the past decade, it has received much attention as a factor promoting tumour invasion. In breast, colorectal and prostate cancers, SATB1 has been shown to influence the epithelial-mesenchymal transition (EMT) process, which is thought to be crucial for cancer metastasis. The aim of this study was to analyse the possible correlations between the expression of SATB1 and major EMT-associated proteins in NSCLC clinical samples. Additionally, the impact of EMT induction in NSCLC cell lines on SATB1 mRNA expression was also investigated. Immunohistochemistry was used to assess the expression of SATB1, SNAIL, SLUG, Twist1, E-cadherin, and N-cadherin in 242 lung cancer clinical samples. EMT was induced by TGF-β1 treatment in the A549 and NCI-H1703 lung cancer cell lines. Changes in gene expression profiles were analyzed using real-time PCR and Droplet Digital PCR. SATB1 expression was positively correlated with the expression of SNAIL (R=0.129; P=0.045), SLUG (R=0.449; P<0.0001), and Twist1 (R=0.264; P<0.0001). Moreover, SATB1 expression significantly increased after in vitro EMT induction in A549 and NCI-H1703 cell lines. The results obtained may point to the role of SATB1 as one of the regulators of EMT in NSCLC.

Tumor-associated macrophages secret exosomal miR-155 and miR-196a-5p to promote metastasis of non-small-cell lung cancer.

BackgroundUnderstanding the molecular basis underlying metastasis of non-small cell lung cancer (NSCLC) may provide a new therapeutic modality for the treatment of NSCLC. However, the mechanisms by which tumor-associated macrophages (TAMs) affect NSCLC metastasis remain undefined. In this study, we aimed to discover a novel regulatory pathway involved in NSCLC metastasis.MethodsCell Counting Kit-8 (CCK-8), Transwell, western blot assays were used to assess cell viability, migration, invasion and epithelial-mesenchymal transition (EMT). Exosomes from macrophages medium were characterized, and in vitro cell coculture was further conducted to investigate M2 derived exosomes mediated crosstalk between TAMs and tumor cells. Besides, miRNA microarray was used to analyze miRNA expression profiles of M0 and M2 derived exosomes. Luciferase reporter assay was used to verify the potential binding between miRNA and mRNA. Moreover, 6-week-old male BALB/c nude mice were performed to establish transplantation tumor model using tail vein injection. Hematoxylin & eosin staining was used to detect the metastasis of tumor tissues.ResultsWe found that M2 TAMs were the main TAMs in metastatic tissues of NSCLC patients and exosomes derived from M2 TAMs were able to promote cell viability, cell migration, cell invasion and EMT in NSCLC. We demonstrated that miR-155 and miR-196a-5p were abundant in M2 TAMs and exosomes secreted by M2 TAMs. Functional experiments demonstrated that the deletion of miR-155 and miR-196a-5p in M2 TAMs significantly prevented NSCLC metastasis in vitro and in vivo. To clarify the mechanism governing miR-155 and miR-196a-5p from M2 TAMs, we carried out bioinformatics analysis to predict potential target genes. Mechanistically, miR-155 and miR-196a-5p directly bound to the 3'-UTR of Ras association domain family member 4 (RASSF4), and negatively regulating RASSF4 expression. At last, rescue assays demonstrated that miR-155 and miR-196a-5p exerted its performance by RASSF4.ConclusionsOverall, we revealed a new regulatory pathway that was M2 TAMs secreted exosomal miR-155 and miR-196a-5p to promote NSCLC metastasis. This dynamic and reciprocal cross-talk between NSCLC and macrophages innovatively provided a potential opportunity for diagnosis and treatment of NSCLC.

Nogo-B receptor is required for stabilizing TGF-β type I receptor and promotes the TGF-β1-induced epithelial-to-mesenchymal transition of non-small cell lung cancer.

Background and Objective: Metastasis is the leading cause of death in patients with advanced non-small cell lung cancer (NSCLC), and epithelial-mesenchymal transition (EMT) is a crucial event in the metastasis of NSCLC. Our previous works demonstrated that NgBR promoted EMT in NSCLC. However, the molecular mechanism was unclear.Methods: TGF-β1 was used to induce EMT process of NSCLC cells. The biological functions of NgBR in promoting TGF-β1-induced NSCLC metastasis were studied by gain- and loss-of-function assays both in vitro and in vivo. The underlying mechanisms were studied using molecular biology assays.Results: We found that knockdown of NgBR inhibited TGF-β1-induced cell migration and invasion in NSCLC cells. In contrast, NgBR overexpression promoted TGF-β1-induced EMT of A549 cells. Mechanically, we found that knockdown of NgBR facilitated ubiquitination and degradation of TβRI, leading to downregulation of TβRI expression in NSCLC cells. Moreover, we confirmed a positive correlation between NgBR and TβRI in NSCLC tissues.Conclusions: Our findings provide a novel role of NgBR in modulating TGF-β1-induced EMT and propose NgBR as a new therapeutic target for treating NSCLC patients.