title: Progastrin overexpression imparts tumorigenic/metastatic potential to embryonic epithelial cells: phenotypic differences between transformed and nontransformed stem cells.
abstract: We recently reported that overexpression of progastrin (PG) in embryonic epithelial cells (HEKmGAS cells) increased proliferation of the cells compared to that of control HEKC cells. Here, we report the novel finding that tumorigenic and metastatic potential of HEKmGAS cells is also increased significantly compared to that of HEKC cells. Cell surface-associated annexinA2 (CS-ANXA2) binds PG and is overexpressed on cancer cells, allowing us to successfully use fluorescently labeled PG peptide for enumerating metastatic lesions of transformed/cancer cells in vivo. Next, we examined the hypothesis that increased tumorigenic/metastatic potential of isogenic HEKmGAS versus HEKC cells maybe due to transformed phenotype of stem cells. FACSorting/FACScanning of cells demonstrated significant increases in percent doublecortin-CAM-kinase-like1 (DCLK1)/Lgr5-positive stem cells, coexpressing cluster of differentiation44 (CD44)/CS-ANXA2, in HEKmGAS versus HEKC cells. Distinct differences were noted in the morphology of HEKC versus HEKmGAS spheroidal growths on nonadherent cultures (selective for stem cells). HEKC spheroids were rounded with distinct perimeters (e.g., basement membranes), whereas HEKmGAS spheroids were amorphous with no perimeters. Relative levels of DCLK1/Lgr5/CD44 and ANXA2/β-catenin/pNFκBp65/metalloproteinases were significantly increased in HEKmGAS versus HEKC cells, growing as monolayer cultures, 3D spheroids (in vitro), or xenografts (in vivo). Interestingly, HEKC cells enriched for CS-ANXA2 developed amorphous spheroids, whereas downregulation of ANXA2 in HEKmGAS clones resulted in loss of matrixmetalloproteinases (MMPs) and re-formation of rounded spheroids, suggesting that high levels of CS-ANXA2/MMPs may impact spheroid morphology. Downregulation of DCLK1 significantly attenuated activation of β-catenin, with loss of proliferation of HEKmGAS and HEKC cells, suggesting that DCLK1 is required for maintaining proliferation of cells. Our results suggest the novel possibility that transformed stem cells, unlike nontransformed stem cells, coexpress stem cell markers DCLK1 and CD44 with CS-ANXA2.
authors.affiliation: Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555-1043, USA.
authors.forename: Shubhashish
authors.initials: S
authors.lastname: Sarkar
authors.forename: Carla
authors.initials: C
authors.lastname: Kantara
authors.forename: Ixiu
authors.initials: I
authors.lastname: Ortiz
authors.forename: Rafal
authors.initials: R
authors.lastname: Swiercz
authors.forename: Joyce
authors.initials: J
authors.lastname: Kuo
authors.forename: Robert
authors.initials: R
authors.lastname: Davey
authors.forename: Kenneth
authors.initials: K
authors.lastname: Escobar
authors.forename: Robert
authors.initials: R
authors.lastname: Ullrich
authors.forename: Pomila
authors.initials: P
authors.lastname: Singh
mesh_terms: D000818:Animals; D017306:Annexin A2 / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics* / Q000378:metabolism; D000089742:Doublecortin-Like Kinases; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D005755:Gastrins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D016513:Mice, SCID; D009362:Neoplasm Metastasis / Q000235:genetics; D010641:Phenotype; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D018874:Spheroids, Cellular; D013234:Stem Cells / Q000378:metabolism* / Q000473:pathology; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism
chemical_list: D017306:Annexin A2; D015415:Biomarkers; D005755:Gastrins; D018960:Hyaluronan Receptors; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; D051176:beta Catenin; C013706:big gastrin; C117347:DCLK1 protein, human; D000089742:Doublecortin-Like Kinases; D017346:Protein Serine-Threonine Kinases