Epithelial to Mesenchymal Transition by TGFβ-1 Induction Increases Stemness Characteristics in Primary Non Small Cell Lung Cancer Cell Line

Abstract

BackgroundCancer Stem Cells (CSCs) hypothesis asserts that only a small subset of cells within a tumour is capable of both tumour initiation and sustainment. The Epithelial-Mesenchymal Transition (EMT) is an embryonic developmental program that is often activated during cancer invasion and metastasis. The aim of this study is to shed light on the relationship between EMT and CSCs by using LC31 lung cancer primary cell line.Materials and MethodsA549 and LC31 cell lines were treated with 2 ng/ml TGFβ-1 for 30 days, and 80 days, respectively. To evaluate EMT, morphological changes were assessed by light microscopy, immunofluorescence and cytometry for following markers: cytokeratins, e-cadherin, CD326 (epithelial markers) and CD90, and vimentin (mesenchymal markers). Moreover, RT-PCR for Slug, Twist and β-catenin genes were performed. On TGFβ-1 treated and untreated LC31 cell lines, we performed stemness tests such as pneumospheres growth and stem markers expression such as Oct4, Nanog, Sox2, c-kit and CD133. Western Blot for CD133 and tumorigenicity assays using NOD/SCID mice were performed.ResultsTGFβ-1 treated LC31 cell line lost its epithelial morphology assuming a fibroblast-like appearance. The same results were obtained for the A549 cell line (as control). Immunofluorescence and cytometry showed up-regulation of vimentin and CD90 and down-regulation of cytocheratin, e-cadherin and CD326 in TGFβ-1 treated LC31 and A549 cell lines. Slug, Twist and β-catenin m-RNA transcripts were up-regulated in TGFβ-1 treated LC31 cell line confirming EMT. This cell line showed also over-expression of Oct4, Nanog, Sox2 and CD133, all genes of stemness. In addition, in TGFβ-1 treated LC31 cell line, an increased pneumosphere-forming capacity and tumours-forming ability in NOD/SCID mice were detectable.ConclusionsThe induction of EMT by TGFβ-1 exposure, in primary lung cancer cell line results in the acquisition of mesenchymal profile and in the expression of stem cell markers.

Identification of CD90 as Putative Cancer Stem Cell Marker and Therapeutic Target in Insulinomas

“…The difference in progenitor marker expression can be explained by the fact that the CM cell line has a b-cell phenotype [35], whereas BON1 cells originate from a pancreatic carcinoid, lacking b-cell characteristics [36]. Regarding the upregulation of EMT markers, previously it has been described that chemoresistance to doxorubicin induces EMT in colon cancer and lung cancer cells [37,38]. Furthermore, in several tumor types, including pancreatic adenocarcinoma (PDAC), a link has been described between CSCs and EMT [39].…”

“…Although it is difficult to determine which event occurs first, a likely explanation is that EMT contributes to the establishment of CSCs. EMT has been described to upregulate the expression of CD90 on breast cancer and lung cancer cells [38,40]. Furthermore, EMT has been demonstrated to occur in pNETs, since positive immunohistochemical staining of human INS and INS of Rip1tag2 mice was found for SNAI1 and TWIST [11].…”

Pituitary tumors contain a side population with tumor stem cell-associated characteristics

“…Compared with the other cells in the tumor, the CSC subpopulation is considered to be more tumorigenic and more resistant to therapy, thereby surviving treatment and finally regrowing the (heterogeneous) tumor (Vankelecom & Gremeaux 2010, Clevers 2011, Vankelecom 2012, Vankelecom & Chen 2014. Recently, CSCs have been reported to display characteristics of epithelial-mesenchymal transition (EMT), which may drive these cells in their tumor expansion and invasive activity (Mani et al 2008, Morel et al 2008, Kong et al 2010, Pirozzi et al 2011, Yoon et al 2012. EMT represents a multi-step cell-conversion process during which epithelial cells gradually acquire mesenchymal features.…”

“…In general, EMT is known to play a key role in cancer pathogenesis, particularly during the growth, maintenance (including resistance to hostile conditions), and progression of the tumor with local invasion and/or metastasis (De Craene & Berx 2013). In several types of cancer, it has been shown that EMT markers are enriched in the CSC fraction, that the induction of EMT causes the upregulated expression of 'stemness' genes, and that EMT acts as a key driver in the generation and activity of the CSCs (Mani et al 2008, Morel et al 2008, Kong et al 2010, Pirozzi et al 2011, Yoon et al 2012.…”

“…In recent studies, CSCs and EMT have been linked, in that EMT has been shown to act as a major driver of CSC generation and activity (Mani et al 2008, Morel et al 2008, Kong et al 2010, Pirozzi et al 2011, Yoon et al 2012, De Craene & Berx 2013. In addition to being an argument in favor of the CSC(-like) phenotype of the pSP, the detection of EMT may also have implications for understanding of pituitary tumor behavior.…”