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.