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Conditioned medium of primary lung cancer cells induces EMT in A549 lung cancer cell line by TGF-ß1 and miRNA21 cooperation.

ABSTRACT: The epithelial-mesenchymal transition (EMT) plays a key role in tumor progression, drug resistance and metastasis. Recently, numerous microRNA (miRNA) have been described to regulate EMT in tumor progression. In this study, we found that conditioned medium from the LC212 non-small-cell lung cancer (NSCLC) cell line (LC212-CM) induces morphological changes and overexpression of Vimentin, CD90, SMAD 2/3, SLUG and TWIST in A549 NSCLC cells, consistent with a mesenchymal phenotype. To identify the soluble mediators in LC212-CM involved in this phenomenon, we performed miRNA profiling and TGF-?1 quantification. We found that LC212-CM contains high levels of TGF-?1 as well as different secreted miRNAs. We focused our attention on Homo sapiens-microRNA21 (hsa-miR21), one of most relevant miRNA associated with lung cancer progression, metastasis and EMT. An hsa-miR21 antagomiR was able to prevent the LC212-CM-induced EMT phenotype in A549 cells. Furthermore, we found that TGF-?1 and hsa-miR21 cooperate in the induction of EMT in A549 cells. Intriguingly, TGF-?1 was found to induce hsa-miR21 expression in A549 cell, thus suggesting that the hsa-miR21 mediates at least in part the pro-EMT effects of TGF-?1. In conclusion, hsa-miR21 and TGF-?1 are involved in autocrine and paracrine circuits that regulate the EMT status of lung cancer cells.

SUBMITTER: Camerlingo R

PROVIDER: S-EPMC6657837 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Conditioned medium of primary lung cancer cells induces EMT in A549 lung cancer cell line by TGF-ß1 and miRNA21 cooperation.

Camerlingo Rosa R Miceli Roberta R Marra Laura L Rea Giuseppina G D'Agnano Igea I Nardella Marta M Montella Roberta R Morabito Alessandro A Normanno Nicola N Tirino Virginia V Rocco Gaetano G

PloS one 20190725 7

The epithelial-mesenchymal transition (EMT) plays a key role in tumor progression, drug resistance and metastasis. Recently, numerous microRNA (miRNA) have been described to regulate EMT in tumor progression. In this study, we found that conditioned medium from the LC212 non-small-cell lung cancer (NSCLC) cell line (LC212-CM) induces morphological changes and overexpression of Vimentin, CD90, SMAD 2/3, SLUG and TWIST in A549 NSCLC cells, consistent with a mesenchymal phenotype. To identify the s ...[more]

PMID: 31344049

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Project description:Xanthohumol (XN), the main prenylated flavonoid from hop cones, has been recently reported to exert significant proapoptotic, anti-proliferative, and growth inhibitory effects against lung cancer in both in vitro and in vivo studies. However, its anti-metastatic potential towards this malignancy is still unrevealed. Previously, we indicated that the human lung adenocarcinoma A549 cell line was sensitive to XN treatment. Therefore, using the same tumour cell model, we have studied the influence of XN on the phorbol-12-myristate-13-acetate (PMA)-induced cell migration and invasion. The effects of XN on the expression/activity of pro-invasive MMP-9 and MMP-2 and the expression of MMP inhibitors, i.e., TIMP-1 and TIMP-2 (anti-angiogenic factors), were evaluated. Additionally, the influence of XN on the production of the key pro-angiogenic cytokine, i.e., VEGF, and the release of TGF-β, which is both a pro-angiogenic cytokine and an epithelial-mesenchymal transition (EMT) stimulator, was studied. Furthermore, the influence of XN on the expression of EMT-associated proteins such as E-cadherin and α-E-catenin (epithelial markers), vimentin and N-cadherin (mesenchymal markers), and Snail-1 (transcriptional repressor of E-cadherin) was studied. To elucidate the molecular mechanism underpinning the XN-mediated inhibition of metastatic progression in PMA-activated cells, the phosphorylation levels of AKT, FAK, and ERK1/2 kinases, which are signalling molecules involved in EMT program activation, were assayed. The results showed that XN in non-cytotoxic concentrations impaired the PMA-driven migratory and invasive capacity of A549 cells by decreasing the level of expression of MMP-9 and concomitantly increasing the expression of the TIMP-1 protein, i.e., a specific blocker of pro-MMP-9 activation. Moreover, XN decreased the PMA-induced production of VEGF and TGF-β. Furthermore, the XN-treatment counteracted the PMA-induced EMT of the A549 cells by the upregulation of E-cadherin and α-E-catenin and the downregulation of N-cadherin, vimentin, and Snail-1 expression. The proposed mechanism underlying the anti-invasive XN activity involved the inhibition of the ERK/MAPK pathway and suppression of FAK and PI3/AKT signalling. Our results suggesting migrastatic properties of XN against lung cancer cells require further verification in in vivo assays.

Project description:BackgroundSupport for early detection of lung cancer has emerged from the National Lung Screening Trial (NLST), in which low-dose computed tomography (LDCT) screening reduced lung cancer mortality by 20 % relative to chest x-ray. The US Preventive Services Task Force (USPSTF) recently recommended annual screening for the high-risk population, concluding that the benefits (life years gained) outweighed harms (false positive findings, abortive biopsy/surgery, radiation exposure). In making their recommendation, the USPSTF noted that the moderate net benefit of screening was dependent on the resolution of most false-positive results without invasive procedures. Circulating biomarkers may serve as a valuable adjunctive tool to imaging.ResultsWe developed a broad-based proteomics discovery program, integrating liquid chromatography/mass spectrometry (LC/MS) analyses of freshly resected lung tumor specimens (n = 13), lung cancer cell lines (n = 17), and conditioned media collected from tumor cell lines (n = 7). To enrich for biomarkers likely to be found at elevated levels in the peripheral circulation of lung cancer patients, proteins were prioritized based on predicted subcellular localization (secreted, cell-membrane associated) and differential expression in disease samples. 179 candidate biomarkers were identified. Several markers selected for further validation showed elevated levels in serum collected from subjects with stage I NSCLC (n = 94), relative to healthy smoker controls (n = 189). An 8-marker model was developed (TFPI, MDK, OPN, MMP2, TIMP1, CEA, CYFRA 21-1, SCC) which accurately distinguished subjects with lung cancer (n = 50) from high risk smokers (n = 50) in an independent validation study (AUC = 0.775).ConclusionsIntegrating biomarker discovery from multiple sample types (fresh tissue, cell lines and conditioned medium) has resulted in a diverse repertoire of candidate biomarkers. This unique collection of biomarkers may have clinical utility in lung cancer detection and diagnoses.

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Conditioned medium of primary lung cancer cells induces EMT in A549 lung cancer cell line by TGF-ß1 and miRNA21 cooperation.

Publications

Conditioned medium of primary lung cancer cells induces EMT in A549 lung cancer cell line by TGF-ß1 and miRNA21 cooperation.

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