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 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 progastrin and is over-expressed on cancer-cells, allowing us to successfully use fluorescently-labeled progastrin-peptide for enumerating metastatic lesions of transformed/cancer cells in vivo. Next, we examined the hypothesis that increased tumorigenic/metastatic potential of isogenic HEKmGAS vs HEKC cells maybe due to transformed-phenotype of stem-cells. FACSorting/FACScanning of cells demonstrated significant increases in percent DCLK1/Lgr5 positive stem-cells, co-expressing CD44/CS-ANXA2, in HEKmGAS vs HEKC-cells. Distinct differences were noted in morphology of HEKC vs HEKmGAS spheroidal growths on non-adherent cultures (selective for stem cells). HEKC-spheroids were rounded with distinct perimeters (basement membranes?), while HEKmGAS-spheroids were amorphous, with no perimeters. Relative levels of DCLK1/Lgr5/CD44 and AnnexinA2/β-catenin/pNFκBp65/metalloproteinases were significantly increased in HEKmGAS vs HEKC-cells, growing either as mono-layer cultures, 3D-spheroids (in vitro), or xenografts (in vivo). Interestingly, HEKC-cells enriched for CS-ANXA2, developed amorphous spheroids, while down-regulation of ANXA2 in HEKmGAS-clones, resulted in loss of matrixmetalloproteinases and re-formation of rounded spheroids, suggesting high levels of CS-ANXA2/matrixmetalloproteinases may impact spheroid morphology. Down-regulation of DCLK1 significantly attenuated activation of β-catenin, with loss of proliferation of HEKmGAS and HEKC-cells, suggesting DCLK1 is required for maintaining proliferation of cells. Conclusions Our results suggest the novel possibility that transformed stem-cells, unlike non-transformed stem-cells, co-express stem-cell-markers DCLK1 and CD44 with CS-ANXA2.

Epigenetic regulation of human DCLK-1 gene during colon-carcinogenesis: clinical and mechanistic implications

“…While recent literature demonstrates hypermethylation and epigenetic silencing of 5' promoter of hDCLK1 gene in several human cancers, we and others have reported high levels of DCLK1 protein in hCRCs and human colon cancer cell lines (hCCCs) (23,31,(41)(42)(43)(44). High levels of DCLK1 staining in hCRC samples was reported to be associated with an increase in cancer specific mortality (43), suggesting possible prognostic value of measuring expression levels of DCLK1 in CRCs.…”

“…The role of DCLK1 in transformed cells of human origin was examined using isogenic clones of human embryonic epithelial cells (HEK293) (23), which either expressed the empty vector (HEKC) or expressed the full-length progastrin (PG) peptide (HEKmGAS). PG peptide induces hyper proliferation of colonic crypts and increases colon-carcinogenesis by many fold, in response to AOM±DSS (19,20,(24)(25)(26)(27).…”

“…Besides, PG is expressed by >80% of human colon cancer cell lines (hCCCs) and CRCs (28,29). Surprisingly, overexpression of PG, resulted in imparting tumorigenic/metastatic phenotype to HEK293 cells, associated with significant up-regulation of DCLK1+ cell populations in HEKmGAS vs. HEKC cells (23). Down-regulation of DCLK1 expression in HEKmGAS cells caused loss of tumorigenic/metastatic potential of the cells, which led us to conclude that DCLK1 is required for sustaining tumorigenic/metastatic potential of transformed HEKmGAS cells (23), and likely marks both normal stem cells (NSCs) and cancer stem cells (CSCs) in humans.…”

“…Dclk1+NSCs, from mouse colons, form enteroids, positive for all differentiated lineages (22). Embryonic hNSCs (in HEK293 cells) formed wellrounded spheroids in vitro (23); presence of differentiated lineages was not examined. The transformed CSCs from HEKmGAS and hCCCs, on the other hand, formed illdefined spheroids with amorphous structures, which correlated with the metastatic potential of the cells (23,31).…”

“…Embryonic hNSCs (in HEK293 cells) formed wellrounded spheroids in vitro (23); presence of differentiated lineages was not examined. The transformed CSCs from HEKmGAS and hCCCs, on the other hand, formed illdefined spheroids with amorphous structures, which correlated with the metastatic potential of the cells (23,31). Thus morphologically, the nature of spheroidal growths may reflect metastatic potential of CSCs in tumorous growths, which needs further investigation.…”