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TGF-? induced EMT and stemness characteristics are associated with epigenetic regulation in lung cancer.

ABSTRACT: Transforming growth factor-? (TGF-?) promotes tumor invasion and metastasis by inducing epithelial-mesenchymal transition (EMT). EMT is often related with acquisition of stemness characteristics. The objective of this study was to determine whether EMT and stemness characteristics induced by TGF-? might be associated with epigenetic regulation in lung cancer. A human normal lung epithelial cell line and four lung cancer cell lines were treated with TGF-?. Transcriptome analysis of BEAS-2B and A549 cells incubated with TGF-? were analyzed through next-generation sequencing (NGS). Western blotting was carried out to investigate expression levels of epithelial and mesenchymal markers. Wound healing and Matrigel invasion assay, sphere formation assay, and in vivo mice tumor model were performed to evaluate functional characteristics of EMT and stemness acquisition. To investigate whether activation of EMT and stem cell markers might be involved in epigenetic regulation of lung cancer, experiment using a DNA methyltransferase inhibitor (5-azacytidine, AZA), methylation-specific PCR (MSP) and bisulfite sequencing were performed. NGS revealed changes in expression levels of EMT markers (E-cadherin, N-cadherin, fibronectin, vimentin, slug and snail) and stem cell markers (CD44 and CD87) in both BEAS-2B and A549 cells. Functional analysis revealed increased migration, invasion, sphere formation, and tumor development in mice after TGF-? treatment. Expression of slug and CD87 genes was activated following treatment with AZA and TGF-?. MSP and bisulfite sequencing indicated DNA demethylation of slug and CD87 genes. These results suggest that TGF-? induced EMT and cancer stemness acquisition could be associated with activation of slug and CD87 gene by their promoter demethylation.

SUBMITTER: Kim BN

PROVIDER: S-EPMC7326979 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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TGF-β induced EMT and stemness characteristics are associated with epigenetic regulation in lung cancer.

Kim Bit Na BN Ahn Dong Hyuck DH Kang Nahyeon N Yeo Chang Dong CD Kim Young Kyoon YK Lee Kyo Young KY Kim Tae-Jung TJ Lee Sug Hyung SH Park Mi Sun MS Yim Hyeon Woo HW Park Jong Y JY Park Chan Kwon CK Kim Seung Joon SJ

Scientific reports 20200630 1

Transforming growth factor-β (TGF-β) promotes tumor invasion and metastasis by inducing epithelial-mesenchymal transition (EMT). EMT is often related with acquisition of stemness characteristics. The objective of this study was to determine whether EMT and stemness characteristics induced by TGF-β might be associated with epigenetic regulation in lung cancer. A human normal lung epithelial cell line and four lung cancer cell lines were treated with TGF-β. Transcriptome analysis of BEAS-2B and A5 ...[more]

PMID: 32606331

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Project description:Our group is interested in epithelial-to-mesenchymal transition (EMT), in particular, TGF-beta induced EMT. TGF-beta signalling has been shown to be an important factor in the induction of EMT and it has been demonstrated that adding TGF-beta to epithelial cells in culture is a convenient way to study the process of EMT. In response to TGF-beta, Smad2 and 3 are activated, and form complexes with Smad4, which then regulate transcription of target genes through interactions with other DNA binding transcription factors. In the induction of EMT, the activated Smads mediate transcriptional regulation through three families of transcription factors, resulting in repression of epithelial marker gene expression and activation of mesenchymal gene expression (Xu J, et al. 2009) Also investigated in this study is the role of H2A.Z in EMT. H2A.Z is an evolutionary conserved and a metazoan essential histone variant of the H2A class. Mice deficient in H2A.Z die during early development but the reason for this is unknown (Faast et al. 2001). Previously, our laboratory showed that the loss of H2A.Z in Xenpous laevis impaired cell movement required for the formation of the mesoderm and neural crest (Ridgway et al. 2004). Given that mesoderm formation is critically dependent upon EMT, we therefore wondered whether H2A.Z might be a chromatin regulator of EMT. We transfected MDCK cells with a lentiviral vector to express a construct encoding an shRNA targeting canine H2A.Z as we wanted to test the hypothesis that H2A.Z is involved in the maintenance of cellular identity and that its loss might trigger de-differentiation. In order to investigate changes in histone variant H2A.Z occupancy associated with TGF-beta induced epithelial-to-mesenchymal transition (EMT) we performed H2A.Z ChIP-Seq in untreated and TGFb-treated MDCK cells. The MDCK cell line has been extensively used as a model system for EMT because they convert fully from the epithelial to the mesenchymal state in response to TGF-beta. Please note that RNA-seq data generated in conjunction to this ChIP-seq data set were also deposited at ArrayExpress under accession number E-MTAB-5628 ( https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5628 ).

Project description:TGFβ signaling plays crucial role during development and cancer, however the role for TGFβ signaling in regulating the noncoding part of the human genome in triple negative breast cancer (TNBC) is still being unraveled. Herein, we provide the transcriptional landscape of TNBC in response to TGFβ activation and subsequent inhibition employing SB431542, selective TGFβ1 Receptor ALK5 Inhibitor. Our data revealed 72 commonly upregulated [fold change (FC) ≥ 2.0], including PLAU, TPM1, TAGLN, COL1A1, TGFBI, and SNAI1, and 53 downregulated (FC ≤ 2.0) protein coding genes in BT-549 and MDA-MB-231 models in response to TGFβ1 activation. Alignment to the geocode (V33) identified 41 upregulated (FC ≥ 2.0) and 22 downregulated (FC ≤ 2.0) long non-coding RNA (lncRNA) in response to TGFβ1 activation, which were inhibited by concurrent treatment with SB431542. To place our data from the in vitro models into their clinical context, we identified AC015909.1, AC013451.1, CYP1B1-AS1, AC004862.1, LINC01824, AL138828.1, B4GALT1-AS1, AL353751.1, AC090826.3, AC104695.4, ADORA2A-AS1, PTPRG-AS1, LINC01943, AC026954.3, TPM1-AS, ZFPM2-AS1, AC007362.1, AC112721.2, MALAT1, AL513314.2, AC112721.1, AC010343.3, LINC01711, and MAP3K2-DT lncRNA expression to positively correlate with TGFβ1 expression in a cohort of 360 TNBC patients. To provide mechanistic insight into lncRNA regulation by TGFβ signaling, SMAD2/3 ChIp-Seq data from BT-549 TNBC model retrieved from Gene Expression Omnibus (GEO) revealed direct binding of SMAD2/SMAD3 to the promoter of AC112721.1, AC112721.2, MALAT1, HHIP-AS1, LINC00472, and SLC7A11, suggesting their direct regulation by TGFβ1/SMAD2/SMAD3 pathway. Interestingly, AC112721.1, AC112721.2 exhibited higher expression in TNBC compared to normal breast tissue suggesting a possible role for those lncRNA in TNBC biology. Our miRNA analysis in the BT-549 model in response to exogenous TGFB1 revealed several affected miRNAs (2.0 ≤ FC ≤ 2.0), whose expression pattern was reversed in the presence of SB431542, suggesting those miRNA as plausible targets for TGFβ regulation. In particular, we observed hsa-miR-1275 to be downregulated in response to TGFB1 which was highly predicted to regulate PCDH1, FIBCD1, FXYD7, GDNF, STC1, EDN1, ZSWIM4, FGF1, PPP1R9B, NUAK1, PALM2AKAP2, IGFL3, and SPOCK1 whose expression were upregulated in response to TGFβ1 stimulus. On the other hand, hsa-miR-181b-5p was among the top upregulated miRNAs in response to TGFB1, which is also predicted to regulate CDKN1B, TNFRSF11B, SIM1, and ARSJ in the BT-549 model. Taken together, our data is the first to provide such in depth analysis of lncRNA and miRNA epigenetic changes in response to TGFβ signaling in TNBC.

Dataset Information

TGF-? induced EMT and stemness characteristics are associated with epigenetic regulation in lung cancer.

Publications

TGF-β induced EMT and stemness characteristics are associated with epigenetic regulation in lung cancer.

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