big-gastrin and Cell-Transformation--Neoplastic

The role of the gastrin peptide hormones (G17, G34) and their precursors (progastrins, PG; gly-extended gastrin, G-gly), in gastrointestinal (GI) cancers has been extensively reviewed in recent years [W. Rengifo-Cam, P. Singh, Role of progastrins and gastrins and their receptors in GI and pancreatic cancers: targets for treatment, Curr. Pharm. Des. 10 (19) (2004) 2345-2358; M. Dufresne, C. Seva, D. Fourmy, Cholecystokinin and gastrin receptors, Physiol. Rev. 86 (3) (2006) 805-847; A. Ferrand, T.C. Wang, Gastrin and cancer: a review, Cancer Lett. 238 (1) (2006) 15-29]. A possible important role of progastrin peptides in colon carcinogenesis has become evident from experiments with transgenic mouse models [W. Rengifo-Cam, P. Singh, (2004); A. Ferrand, T.C. Wang, (2006)]. It is now known that growth stimulatory and co-carcinogenic effects of gastrin/PG peptides are mediated by both proliferative and anti-apoptotic effects of the peptides on target cells [H. Wu, G.N. Rao, B. Dai, P. Singh, Autocrine gastrins in colon cancer cells Up-regulate cytochrome c oxidase Vb and down-regulate efflux of cytochrome c and activation of caspase-3, J. Biol. Chem. 275 (42) (2000) 32491-32498; H. Wu, A. Owlia, P. Singh, Precursor peptide progastrin(1-80) reduces apoptosis of intestinal epithelial cells and upregulates cytochrome c oxidase Vb levels and synthesis of ATP, Am. J. Physiol. Gastrointest. Liver Physiol. 285 (6) (2003) G1097-G1110]. Several receptor subtypes have been described that mediate growth effects of gastrin peptides [W. Rengifo-Cam, P. Singh (2004); M. Dufresne, C. Seva, D. Fourmy, (2006)]. Recently, we identified Annexin II as a high affinity binding protein for gastrin/PG peptides [P. Singh, H. Wu, C. Clark, A. Owlia, Annexin II binds progastrin and gastrin-like peptides, and mediates growth factor effects of autocrine and exogenous gastrins on colon cancer and intestinal epithelial cells, Oncogene (2006), doi:10.1038/sj.onc.1209798]. Importantly, the expression of Annexin II was required for mediating growth stimulatory effects of gastrin and PG peptides on intestinal epithelial and colon cancer cells [P. Singh, H. Wu, C. Clark, A. Owlia, Annexin II binds progastrin and gastrin-like peptides, and mediates growth factor effects of autocrine and exogenous gastrins on colon cancer and intestinal epithelial cells, Oncogene (2006), doi:10.1038/sj.onc.1209798], suggesting that Annexin-II may represent the elusive novel receptor for gastrin/PG peptides. The imp

Topics: Animals; Annexin A2; Cell Membrane; Cell Transformation, Neoplastic; Gastrins; Gastrointestinal Neoplasms; Humans; Protein Kinases; Protein Precursors; Protein Processing, Post-Translational

Transgenic mice overexpressing human progastrin (hGAS) show colonic crypt hyper-proliferation and elevated susceptibility to colon carcinogenesis. We aimed to investigate effects of p53 mutation on colon carcinogenesis in hGAS mice. We show that introducing a p53 gene mutation further increases progastrin dependent BrdU labeling and results in markedly elevated number of aberrant crypt foci (ACF) and colonic tumors. We demonstrate that hGAS/Lgr5-GFP mice have higher number of Lgr5+ colonic stem cells per crypt when compared to Lgr5-GFP mice indicating that progastrin changes crypt biology through increased stem cell numbers and additional p53 mutation leads to more aggressive phenotype in this murine colon cancer model.

Topics: Aberrant Crypt Foci; Animals; Azoxymethane; Carcinogens; Cell Proliferation; Cell Transformation, Neoplastic; Colonic Neoplasms; Disease Models, Animal; Female; Gastrins; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Protein Precursors; Tumor Suppressor Protein p53

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.

Topics: Animals; Annexin A2; beta Catenin; Biomarkers; Cell Line; Cell Transformation, Neoplastic; Doublecortin-Like Kinases; Epithelial Cells; Gastrins; Gene Expression; Gene Expression Regulation; Humans; Hyaluronan Receptors; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Nude; Mice, SCID; Neoplasm Metastasis; Phenotype; Protein Precursors; Protein Serine-Threonine Kinases; Spheroids, Cellular; Stem Cells

Aberrant activation of the beta-catenin/Tcf-4 transcriptional complex represents an initiating event for colorectal carcinogenesis, shifting the balance from differentiation toward proliferation in colonic crypts. Here, we assessed whether endogenous progastrin, encoded by a target gene of this complex, was in turn able to regulate beta-catenin/Tcf-4 activity in adenomatous polyposis coli (APC)-mutated cells, and we analyzed the impact of topical progastrin depletion on intestinal tumor growth in vivo.. Stable or transient RNA silencing of the GAST gene was induced in human tumor cells and in mice carrying a heterozygous Apc mutation (APCDelta14), which overexpress progastrin but not amidated or glycine-extended gastrin.. Depletion of endogenous progastrin production strongly decreased intestinal tumor growth in vivo through a marked inhibition of constitutive beta-catenin/Tcf-4 activity in tumor cells. This effect was mediated by the de novo expression of the inhibitor of beta-catenin and Tcf-4 (ICAT), resulting from a down-regulation of integrin-linked kinase in progastrin-depleted cells. Accordingly, ICAT down-regulation was correlated with progastrin overexpression and Tcf-4 target gene activation in human colorectal tumors, and ICAT repression was detected in the colon epithelium of tumor-prone, progastrin-overexpressing mice. In APCDelta14 mice, small interfering RNA-mediated progastrin depletion not only reduced intestinal tumor size and numbers, but also increased goblet cell lineage differentiation and cell apoptosis in the remaining adenomas.. Thus, depletion of endogenous progastrin inhibits the tumorigenicity of APC-mutated colorectal cancer cells in vivo by promoting ICAT expression, thereby counteracting Tcf-4 activity. Progastrin targeting strategies should provide an exciting prospect for the differentiation therapy of colorectal cancer.

Topics: Adaptor Proteins, Signal Transducing; Adenoma; Adenomatous Polyposis Coli; Animals; Apoptosis; beta Catenin; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Colorectal Neoplasms; Gastrins; Gene Expression Regulation, Neoplastic; Genes, APC; Humans; Mice; Mice, Nude; Phosphatidylinositol 3-Kinases; Protein Precursors; Random Allocation; Repressor Proteins; RNA, Small Interfering; Signal Transduction; TCF Transcription Factors; Transcription Factor 7-Like 2 Protein; Transcription Factors; Transcriptional Activation; Transplantation, Heterologous