Epithelial-to-mesenchymal Transition In Lung Cancer Cells Research Articles

Partial Activation of PPAR-γ by Synthesized Quercetin Derivatives Modulates TGF-β1-Induced EMT in Lung Cancer Cells.

Non-small cell lung cancer (NSCLC) has a very low survival rate due to poor response to chemotherapy and late detection. Epithelial to mesenchymal transition (EMT) is regarded as a major contributor to drive metastasis during NSCLC progression. Towards this, transforming growth factor-beta 1 (TGF-β1) is the key driver that endows cancer cells with increased aggressiveness. Recently, this group synthesized a series of Schiff base quercetin derivatives (QDs) and ascertained their effectiveness on EMT markers of A549 cell line. This study evidenced that the EMT process is counteracted via the partial activation of a nuclear hormone receptor, Peroxisome proliferator-activated receptor (PPAR)-γ through QDs. Here, that work is extended to investigate the interplay between PPAR-γ partial activation and TGF-β1-induced EMT in human lung cancer A549 cells. The results reveal that TGF-β1 plays a critical role in suppressing PPAR-γ, which is markedly reversed and increased by partial agonists: QUE2FH and QUESH at both protein and transcriptional levels. The partial agonists not only stimulate PPAR-γ in a balanced manner but also prevent the loss of E-cadherin and acquisition of TGF-β1-induced mesenchymal markers (Snail, Slug, Vimentin, and Zeb-1). Subsequently, the effects are accompanied by attenuation of TGF-β1-induced migratory ability of A549 cells.

MCM5 Aggravates the HDAC1-Mediated Malignant Progression of Lung Cancer.

BackgroundHistone deacetylase 1 (HDAC1) is essential in the malignant progression of tumors. However, there is no obvious relationship between the expression of HDAC1 and the survival of lung cancer patients. Herein, we explored the involvement of minichromosome maintenance complex component 5 (MCM5) and HDAC1 interaction in the epithelial-to-mesenchymal transition (EMT)-dependent malignant progression of lung cancer.MethodsWe analyzed the expression of MCM5 and HDAC1 in The Cancer Genome Atlas database and clinical samples, as well as their impact on patient survival. Cell and animal experiments were performed to verify the promotion of EMT in lung cancer cells mediated by MCM5 and HDAC1.ResultsWe found that lung adenocarcinoma patients with high expression of MCM5 and HDAC1 had poor survival time. Overexpression of MCM5 and HDAC1 in A549 and H1975 cells can promote proliferation and invasion in vitro and tumor growth and metastasis in vivo. Moreover, astragaloside IV can block the interaction between HDAC1 and MCM5, which can then inhibit the malignant progression of lung cancer in vivo and in vitro.ConclusionThe interaction between MCM5 and HDAC1 aggravated the EMT-dependent malignant progression of lung cancer. Astragaloside IV can block the interaction between MCM5 and HDAC1 to inhibit the progression of lung cancer.

Silent information regulator 1 suppresses epithelial-to-mesenchymal transition in lung cancer cells via its regulation of mitochondria status

AimsSilent information regulator 1 (SIRT1) is a NAD+-dependent protein-modifying enzyme involved in regulating gene expression, DNA damage repair, cell metabolism, and mitochondrial functions. Given that it acts as both a tumor promoter and suppressor, the complex mechanisms underlying SIRT1 signaling in cancer remain controversial. Epithelial-to-mesenchymal transition (EMT) plays a key role in the progression of carcinogenesis and tumors metastasis. Studies have shown that mitochondrial defects are critical in EMT process, and SIRT1 is found to regulate the generation and energy metabolism of mitochondria. Here, we elucidate a novel mechanism by which SIRT1 affects EMT in lung cancer cells via its regulation on mitochondria. Main methodsSIRT1 signaling was detected in TGF-β1-induced EMT and was found to regulate mitochondria status, including mitochondrial biogenesis-related protein levels as detected by western blotting, mitochondrial structure observed by transmission electron microscopy, and respiratory functions analyzed by a respiration capacity assay. The effects of modulating SIRT1 expression on EMT and migration of lung cancer cells or normal cells were evaluated by in vitro and in vivo models. Key findingsWe found that the regulation of SIRT1 signaling on the biogenesis or functions of mitochondria was critical to EMT. Overexpression of SIRT1 reduced EMT or metastasis potential of lung cancer cells by improving the quantity and quality of mitochondria, whereas silencing SIRT1 promote EMT in cancer cells, even in normal cells by disturbing mitochondria status. SignificanceConsequently, SIRT1 is an attractive therapeutic target for reversing EMT or tumor metastasis.

Ephemeranthol A Suppresses Epithelial to Mesenchymal Transition and FAK-Akt Signaling in Lung Cancer Cells

Epithelial to mesenchymal transition (EMT) is a cellular process that facilitates cancer metastasis. Therefore, therapeutic approaches that target EMT have garnered increasing attention. The present study aimed to examine the in vitro effects of ephemeranthol A on cell death, migration, and EMT of lung cancer cells. Ephemeranthol A was isolated from Dendrobium infundibulum. Non-small cell lung cancer cells H460 were treated with ephemeranthol A and apoptosis was evaluated by Hoechst 33342 staining. Anoikis resistance was determined by soft agar assay. Wound healing assay was performed to test the migration. The regulatory proteins of apoptosis and cell motility were determined by western blot. Treatment with ephemeranthol A resulted in a concentration-dependent cell apoptosis. At non-toxic concentrations, the compound could inhibit anchorage-independent growth of the cancer cells, as indicated by the decreased colony size and number. Ephemeranthol A also exhibited an inhibitory effect on migration. We further found that ephemeranthol A exerts its antimetastatic effects via inhibition of EMT, as indicated by the markedly decrease of N-cadherin, vimentin, and Slug. Furthermore, the compound suppressed the activation of focal adhesion kinase (FAK) and protein kinase B (Akt) proteins, which are key regulators of cell migration. As for the anticancer activity, ephemeranthol A induced apoptosis by decreasing Bcl-2 followed by the activation of caspase 3 and caspase 9. The pro-apoptotic and anti-migratory effects of ephemeranthol A on human lung cancer cells support its use for the development of novel anticancer therapies.

Abstract B011: Inhibition of G9a reestablishes the MHC class I loss due to EMT in lung cancer cells

Abstract The epithelial-to-mesenchymal transition (EMT) is an important phenotype for cancer cells to invade and metastasizes. In addition, cancer cells should escape from the immune system during malignant progression. Here, we aimed to demonstrate the potential of G9a inhibition of EMT in cancer cells and most importantly, the reestablishment of MHC class I expression (MHC-I), one of the important factors associated with the loss of antigenicity of cancer cells. The lung epithelial cancer cell line A549 was cultured under controlled conditions in RPMI culture media supplemented with 10% of bovine fetal serum (BFS), 1% of antibiotics (penicillin and streptomycin), 2% of glutamine in incubator at 37oC and air atmosphere containing 5% of CO2. The tumor growth factor beta (TGFb) exposure for 5 days was used to induce EMT. In addition, another group of A549 cells received TGFb plus the UNC0368 an G9a (a histone methyl transferase) inhibitor for 5 days. Then, cells were evaluated for acquiring mesenchymal cell morphology and MHC-I gene expression (relative quantification). After 5 days, A549 cells exposed to TGFb clearly have gone through EMT by changing their morphology to a fibroblast-like phenotype. Also, the MHC-I gene expression decreased significantly (p=0.0036) after EMT. The G9a inhibitor partially impeded the EMT induced changes in morphology of A549 cells and reestablished the MHC-I gene expression to the control level. In conclusion, G9a inhibition seemed a promising therapeutic target to improve the efficacy of immunotherapies depending on neoantigen expression by cancer cells. However, more studies are necessary and are under way in our laboratory. Citation Format: Heidge Fukumasu, Pedro R.L. Pires, Pedro L.P. Xavier. Inhibition of G9a reestablishes the MHC class I loss due to EMT in lung cancer cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B011.

Schwann Cells Augment Cell Spreading and Metastasis of Lung Cancer.

Although lungs are densely innervated by the peripheral nervous system (PNS), the role of the PNS in the progression of lung cancer is unknown. In this study, we report that mouse adult Schwann cells (SC), the principal glial cells of the PNS, can regulate the motility of lung cancer cells in vitro and the formation of metastases in vivo SCs promoted epithelial-to-mesenchymal transition (EMT) and the motility of two lung cancer cell lines by increasing expression of Snail and Twist in tumor cells; blocking of Snail and Twist expression abolished SC-induced motility of tumor cells. SC-derived CXCL5 was responsible for EMT in lung cancer cells, as the inhibition of CXCL5 or its receptor CXCR2 reduced SC-induced expression of Snail and Twist and reduced motility in tumor cells. CXCL5/CXCR2 binding activated the PI3K/AKT/GSK-3β/Snail-Twist signaling pathway in lung cancer cells, and the PI3K inhibitor blocked CXCL5-dependent phosphorylation of AKT and GSK-3β, reduced expression of Snail/Twist, and limited tumor cell invasiveness. SC conditioning of tumor cells prior to their injection into mice significantly increased the formation of metastases in the regional lymph nodes. In summary, SCs can regulate the CXCL5/CXCR2/PI3K/AKT/GSK-3β/Snail-Twist pathway to promote EMT, invasiveness, and metastatic potential of lung cancer cells. Our results reveal a new role of the PNS in the functional organization of the tumor microenvironment and tumor progression.Significance: This study increases our understanding of how nerves and, in particular, specific glial cells, Schwann cells, in the peripheral nervous system, may help promote tumor growth and metastasis. Cancer Res; 78(20); 5927-39. ©2018 AACR.

Aspirin-triggered resolvin D1 inhibits TGF-β1-induced EMT through the inhibition of the mTOR pathway by reducing the expression of PKM2 and is closely linked to oxidative stress.

Transforming growth factor-β1(TGF-β1) is a potent stimulator of the epithelial-to-mesenchymal transition(EMT), which is a key event in the initiation of tumor cell metastasis. Aspirin-triggered resolvin D1(AT-RvD1) is known to be involved in the resolution of inflammation; however, whether AT-RvD1 exerts effects on TGF-β1-induced EMT remains unclear. Thus, we first explored the effects of AT-RvD1 on the EMT of lung cancer cells. Treatment with TGF-β1 increased the level of reactive oxygen species(ROS) and reduced the expression of nuclear factor (erythroid-derived2)-like2(Nrf2). The expression of E-cadherin in A549 lung cancer cells was reduced, and the expression of vimentin was enhanced. AT-RvD1 enhanced the expression of E-cadherin in a concentration‑dependent manner and suppressed the expression of Nrf2 and vimentin. AT-RvD1 did not affect the proliferation of A549 lung cancer cells whereas it suppressed the TGF-β1‑induced migration and invasion of A549 cells. The expression of pyruvate kinase M2(Pkm2), which is associated with TGF-β-induced factor homeobox2(TGIF2), was significantly increased during the TGF-β1-induced EMT of A549 lung cancer cells. The mTOR pathway is known to regulate the expression of various glycolytic enzymes including Pkm2. We examined the involvement of the mTOR pathway in the suppressive effects of AT-RvD1 on Pkm2 expression in A549 cells. The mTOR activator restored the expression of Pkm2 and partially downregulated the expression of E-cadherin. However, the mTOR activator had no clear effect on the TGF-β1-induced EMT. These results suggest that AT-RvD1 is closely linked to oxidative stress and partially inhibits TGF-β1-induced EMT through the inhibition of the mTOR pathway by reducing the expression of Pkm2.

Ginkgolic Acid Inhibits Invasion and Migration and TGF-β-Induced EMT of Lung Cancer Cells Through PI3K/Akt/mTOR Inactivation.

Epithelial-to-mesenchymal transition (EMT) is a critical cellular phenomenon regulating tumor metastases. In the present study, we investigated whether ginkgolic acid can affect EMT in lung cancer cells and the related underlying mechanism(s) of its actions. We found that ginkgolic acid C15:1 (GA C15:1) inhibited cell proliferation, invasion, and migration in both A549 and H1299 lung cancer cells. GA C15:1 also suppressed the expression of EMT related genes (Fibronectin, Vimentin, N-cadherin, MMP-9, MMP-2, Twist and Snail) and suppressed TGF-β-induced EMT as assessed by reduced expression of mesenchymal markers (Fibronectin, Vimentin, N-cadherin), MMP-9, MMP-2, Twist and Snail. However, GA C15:1 did not affect the expression of various epithelial marker proteins (Occludin and E-cadherin) in both A549 and H1299 cells. TGF-β-induced morphologic changes from epithelial to mesenchymal cells and induction of invasion and migration were reversed by GA C15:1. Finally, GA C15:1 not only abrogated basal PI3K/Akt/mTOR signaling cascade, but also reduced TGF-β-induced phosphorylation of PI3K/Akt/mTOR pathway in lung cancer cells. Overall, these findings suggest that GA C15:1 suppresses lung cancer invasion and migration through the inhibition of PI3K/Akt/mTOR signaling pathway and provide a source of potential therapeutic compounds to control the metastatic dissemination of tumor cells. J. Cell. Physiol. 232: 346-354, 2017. © 2016 Wiley Periodicals, Inc.

MiR-93 promotes TGF-β-induced epithelial-to-mesenchymal transition through downregulation of NEDD4L in lung cancer cells.

The level of microRNA-93 (miR-93) in tumors has been recently reported to be negatively correlated with survival of lung cancer patients. Considering that the most devastating aspect of lung cancer is metastasis, which can be promoted by transforming growth factor-β (TGF-β)-induced epithelial-to-mesenchymal transition (EMT), we sought to determine whether miR-93 is involved in this process. Here, we report that a previously unidentified target of miR-93, neural precursor cell expressed developmentally downregulated gene 4-like (NEDD4L), is able to mediate TGF-β-mediated EMT in lung cancer cells. miR-93 binds directly to the 3'-UTR of the NEDD4L messenger RNA (mRNA), leading to a downregulation of NEDD4L expression at the protein level. We next demonstrated that the downregulation of NEDD4L enhanced, while overexpression of NEDD4L reduced TGF-β signaling, reflected by increased phosphorylation of SMAD2 in the lung cancer cell line after TGF-β treatment. Furthermore, overexpression of miR-93 in lung cancer cells promoted TGF-β-induced EMT through downregulation of NEDD4L. The analysis of publicly available gene expression array datasets indicates that low NEDD4L expression correlates with poor outcomes among patients with lung cancer, further supporting the oncogenic role of miR-93 in lung tumorigenesis and metastasis.

Protein kinase Cα mediates erlotinib resistance in lung cancer cells.

Overexpression and mutational activation of the epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of non-small cell lung cancer (NSCLC). EGFR tyrosine-kinase inhibitors (TKIs) are given as a primary therapy for advanced patients with EGFR-activating mutations; however, the majority of these tumors relapse and patients eventually develop resistance to TKIs. To address a potential role of protein kinase C (PKC) isozymes in the resistance to TKIs, we used the isogenic NSCLC H1650 cell line and its erlotinib-resistant derivative H1650-M3, a cell line that displays a mesenchymal-like morphology driven by transforming growth factor-β signaling. We found that H1650-M3 cells display remarkable PKCα upregulation and PKCδ downregulation. Notably, silencing PKCα from H1650-M3 cells using RNA interference caused a significant reduction in the expression of epithelial-to-mesenchymal transition (EMT) markers vimentin, Zeb2, Snail, and Twist. Moreover, pharmacological inhibition or PKCα RNA interference depletion and PKCδ restoring sensitized H1650-M3 cells to erlotinib. Whereas ectopic overexpression of PKCα in parental H1650 cells was not sufficient to alter the expression of EMT genes or to confer resistance to erlotinib, it caused downregulation of PKCδ expression, suggesting a unidirectional crosstalk. Finally, mechanistic studies revealed that PKCα upregulation in H1650-M3 cells is driven by transforming growth factor-β. Our results identified important roles for specific PKC isozymes in erlotinib resistance and EMT in lung cancer cells, and highlight PKCα as a potential target for lung cancer treatment.

6-OH-BDE-47 promotes human lung cancer cells epithelial mesenchymal transition via the AKT/Snail signal pathway

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) have been detected in the various human tissues. The OH-PBDEs are suggested to be stronger endocrine-disrupting compounds than PBDEs, therefore the toxicological effects of OH-PBDEs had received lots of attention. However, there is no study about the carcinogenic effect of OH-PBDEs and their estrogen potencies on the tumorigenesis and development of cancer. In the present study, we found that 6-hydroxy-2,2′,4′,4′-tetrabromodiphenyl ether (6-OH-BDE-47), the most abundant OH-PBDE congeners in human serum, promoted the in vitro migration of lung cancer A549 and H358 cells by induction of epithelial to mesenchymal transition (EMT). This was confirmed by that 6-OH-BDE-47 significantly down regulated the expression of epithelial markers E-cadherin (E-Cad) and zona occludin-1 (ZO-1) while up regulated the mesenchymal markers vimentin (Vim) and N-cadherin (N-Cad). 6-OH-BDE-47 up regulated the protein while not mRNA levels of Snail, which was the key transcription factor of EMT. Silencing of Snail by use of siRNA attenuated the 6-OH-BDE-47 induced EMT. This suggested that the stabilization of Snail was essential for 6-OH-BDE-47 induced EMT. Further, the treatment of 6-OH-BDE-47 increased the phosphorylation of AKT and ERK in A549 cells. Only PI3K/AKT inhibitor (LY294002), but not ERK inhibitor (PD98059), completely blocked the 6-OH-BDE-47 induced up regulation of Snail and down regulation of E-Cad, suggesting that PI3K/AKT pathway is important for 6-OH-BDE-47-mediated Snail stabilization and EMT in A549 cells. Generally, our results revealed for the first time that 6-OH-BDE-47 promoted the EMT of lung cancer cells via AKT/Snail signals. This suggested that more attention should be paid to the effects of OH-PBDEs on tumorigenesis and development of lung cancer.

A SUMOylation-Dependent Pathway Regulates SIRT1 Transcription and Lung Cancer Metastasis

Epithelial-to-mesenchymal transition (EMT) plays a pivotal role in lung cancer metastasis. The class III deacetylase sirtuin 1 (SIRT1) possesses both pro- and anticarcinogenic properties. The role of SIRT1 in lung cancer EMT is largely undefined. The effect of SIRT1 on migration of lung cancer cells was evaluated by wound healing assay in vitro and metastasis assay in nude mice in vivo. Protein expression in human lung cancers and cultured lung cancer cells was assessed by western blotting and immunohistochemistry. Interaction between protein and DNA was measured by chromatin immunoprecipitation assay. SIRT1 promoter activity was determined by reporter assay. SIRT1 activation antagonized migration of lung cancer cells by suppressing EMT in vitro. Activation of SIRT1 by resveratrol also statistically significantly hampered (by 68.33%; P < .001, two-sided test) lung cancer cell metastasis in vivo. Hypoxia repressed SIRT1 transcription through promoting the competition between Sp1 and HIC1 on the SIRT1 proximal promoter in a SUMOylation-dependent manner. Disruption of SUMOylation by targeting either Ubc9 or PIASy restored SIRT1 expression in and favored an epithelial-like phenotype of cancer cells, thereby preventing metastasis. Decreased SIRT1 combined with elevated PIASy expression was implicated in more-invasive types of lung cancers in humans. We have identified a novel pathway that links SIRT1 down-regulation to hypoxia-induced EMT in lung cancer cells and may shed light on the development of novel antitumor therapeutics.

Midkine and Hypoxia Potentiate Epithelial‐to‐Mesenchymal Transition in Human Lung Cancer Cells

Epithelial‐to‐mesenchymal transition (EMT) plays an important role during progression of lung cancer. Recently, hypoxia and midkine (MK) have been shown to be key regulators of EMT in various types of cancer cells. Signal transducer and activator of transcription 3 (STAT3) also plays a critical role in this process. Therefore, we hypothesize that MK will enhance EMT in lung cancer cells grown in a hypoxic microenvironment through STAT3 activation. Human lung cancer cells (A549) were exposed to 1% oxygen in the presence of MK to test our hypothesis. EMT was evaluated by assessment of cell morphology as well as expression of marker proteins. Cell morphology was altered from cuboidal to elongated shape after 48 hours of treatments. Levels of E‐cadherin, marker of epithelial cells, were decreased. However, expression of alpha smooth muscle actin (αSMA) and vimentin, markers of mesenchymal cells, were augmented. Hypoxia/MK treatment stimulated STAT3 phosphorylation at Ser727 as well as Tyr705 residues. Interestingly, the two phosphospecies had unique subcellular localization patterns. PhophoSTAT3(Tyr705) was restricted to focal points within the cytoplasm, whereas Ser727 phosphoSTAT3 was present predominantly in the nuclear compartment. Therefore, our data strongly suggest that hypoxia/MK potentiates EMT process and this process might be mediated through STAT3 activation in human lung cancer cells.Funded by HL64917 (MD); P20RR016474 (NCRR &amp; Wyoming INBRE)

Collagen I Promotes Epithelial-to-Mesenchymal Transition in Lung Cancer Cells via Transforming Growth Factor–β Signaling

Epithelial-to-mesenchymal transition (EMT) is a fundamental biological process whereby epithelial cells lose their polarity and undergo a transition to a mesenchymal phenotype. When cancer cells invade adjacent tissues, they use a mechanism akin to EMT, and understanding the molecular mechanisms that drive this transition will facilitate studies into new targets for prevention of metastasis. Extracellular stimuli, such as growth factors, and their cytosolic effectors cooperate to promote EMT. In highly fibrotic cancers like lung cancer, it is thought that extracellular matrix molecules, including collagen, can initiate signals that promote EMT. Here, we present data showing that collagen I induces EMT in non-small cell lung cancer cell lines, which is prevented by blocking transforming growth factor (TGF)-beta3 signaling. In addition, we show that collagen I-induced EMT is prevented by inhibitors of phosphoinositide 3-kinase and extracellular signal-related kinase signaling, which promotes transcription of TGF-beta3 mRNA in these cells. Thus, our data are consistent with the hypothesis that collagen I induces EMT in lung cancer cells by activating autocrine TGF-beta3 signaling. Epidermal growth factor also seems to initiate EMT via a TGF-dependent mechanism.