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/ß-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 ß-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.
Subject(s)
Cell Transformation, Neoplastic/genetics , Epithelial Cells/metabolism , Gastrins/genetics , Gene Expression , Phenotype , Protein Precursors/genetics , Stem Cells/metabolism , Animals , Annexin A2/genetics , Annexin A2/metabolism , Biomarkers/metabolism , Cell Line , Cell Transformation, Neoplastic/metabolism , Doublecortin-Like Kinases , Epithelial Cells/pathology , Gastrins/metabolism , Gene Expression Regulation , Humans , Hyaluronan Receptors/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Nude , Mice, SCID , Neoplasm Metastasis/genetics , Protein Precursors/metabolism , Protein Serine-Threonine Kinases/metabolism , Spheroids, Cellular , Stem Cells/pathology , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
BACKGROUND AND AIMS: Obesity is increasingly a health problem and a risk factor for diabetes in young Mexican-American populations. Genetic association studies in older, mostly non-Hispanic populations have reported that polymorphisms in the candidate genes HSD11B1, CRP, ADIPOQ, PPARG, ANKK1, ABCC8 and SERPINF1 are associated with obesity or diabetes. We analyzed the polymorphisms rs846910, rs1205, rs1501299, rs1801282, rs1800497, rs757110 and rs1136287 in these candidate genes, for association with obesity and metabolic traits in a young Mexican-American population from south Texas. METHODS: Genotyping of the seven common SNPs were performed by allelic discrimination assays in 448 unrelated Mexican Americans (median age = 16 years) from south Texas. χ(2) tests and regression analyses using additive models were used for genetic association analyses adjusting for covariates; p values were corrected for multiple testing by permutation analyses. RESULTS: rs1800497 (ANKK1) shows association with waist circumference (p = 0.009) and retains the association (p = 0.03) after permutation testing. Analysis of metabolic quantitative traits shows that rs846910 (HSD11B1) was associated with HOMA-IR (p = 0.04) and triglycerides (p = 0.03), and rs1205 (CRP) with HOMA-IR (p = 0.03) and fasting glucose levels (p = 0.007). However, the quantitative traits associations are not maintained after permutation analysis. None of the other SNPs in this study showed associations with obesity or metabolic traits in this young Mexican-American population. CONCLUSIONS: We report a potential association between rs1800497 (linked to changes in brain dopamine receptor levels) and central obesity in a young Mexican-American population.
