title: Evidence for embryonic stem-like signature and epithelial-mesenchymal transition features in the spheroid cells derived from lung adenocarcinoma.
abstract: Identification of the cellular and molecular aspects of lung cancer stem cells (LCSCs) that are suggested to be the main culprit of tumor initiation, maintenance, drug resistance, and relapse is a prerequisite for targeted therapy of lung cancer. In the current study, LCSCs subpopulation of A549 cells was enriched, and after characterization of the spheroid cells, complementary DNA (cDNA) microarray analysis was applied to identify differentially expressed genes (DEGs) between the spheroid and parental cells. Microarray results were validated using quantitative real-time reverse transcription-PCR (qRT-PCR), flow cytometry, and western blotting. Our results showed that spheroid cells had higher clonogenic potential, up-regulation of stemness gene Sox2, loss of CD44 expression, and gain of CD24 expression compared to parental cells. Among a total of 160 genes that were differentially expressed between the spheroid cells and the parental cells, 104 genes were up-regulated and 56 genes were down-regulated. Analysis of cDNA microarray revealed an embryonic stem cell-like signature and over-expression of epithelial-mesenchymal transition (EMT)-associated genes in the spheroid cells. cDNA microarray results were validated at the gene expression level using qRT-PCR, and further validation was performed at the protein level by flow cytometry and western blotting. The embryonic stem cell-like signature in the spheroid cells supports two important notions: maintenance of CSCs phenotype by dedifferentiating mechanisms activated through oncogenic pathways and the origination of CSCs from embryonic stem cells (ESCs). PI3/AKT3, as the most common up-regulated pathway, and other pathways related to aggressive tumor behavior and EMT process can confer to the spheroid cells' high potential for metastasis and distant seeding.
authors.affiliation: Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran.|Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran.|Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran.
authors.forename: Raheleh
authors.initials: R
authors.lastname: Roudi
authors.affiliation: Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran. Zahra.madjd@yahoo.com.|Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran. Zahra.madjd@yahoo.com.|Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran. Zahra.madjd@yahoo.com.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Madjd
authors.affiliation: Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. Mebrahimi@royaninstitute.org.
authors.forename: Marzieh
authors.initials: M
authors.lastname: Ebrahimi
authors.affiliation: Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Najafi
authors.affiliation: Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran.
authors.forename: Alireza
authors.initials: A
authors.lastname: Korourian
authors.affiliation: Department of Nuclear Medicine and Molecular Imaging, State University of New York at Buffalo, Buffalo, NY, 14214, USA.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Shariftabrizi
authors.affiliation: Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Samadikuchaksaraei
mesh_terms: D000072283:A549 Cells; D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D015153:Blotting, Western; D053595:Embryonic Stem Cells / Q000378:metabolism; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D053263:Gene Regulatory Networks; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D000071317:Nanog Homeobox Protein / Q000235:genetics / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D018874:Spheroids, Cellular / Q000378:metabolism*
chemical_list: D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C489427:MYC protein, human; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D016271:Proto-Oncogene Proteins c-myc; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors
keywords: Cancer stem cells; Embryonic stem cell-like signature; Lung cancer; Sphere formation assay; cDNA microarray