Abstract 4259: Over-expression of autocrine progastrin up-regulates DCAMKL+1/CD44 positive stem/progenitor cells, associated with surprising transformation of HEK-293 cells, in vitro and in vivo (2010)

Abstract Background and Aims. In recent years we have demonstrated that progastrin peptide (PG) exerts anti-apoptotic and proliferative effects on normal and cancerous epithelial cells, in vitro and in vivo, and increases risk of colon carcinogenesis in transgenic mice overexpressing PG (Cancer 2004; Oncogene 2007; CanRes 2007; Oncogene 2008; JBC 2009). In vivo studies with PG overexpressing hGAS mice suggest that there is an increase in the census of DCAMKL+1 and CD44 positive colonic crypt cells (Jin, et al, JCI 2009). It is, however, not known if PG can directly induce an increase in the number of stem/progenitor cells, in vitro, and if this increase translates into transformation of the cells. To address this novel possibility, we used PG responsive HEK-293 cells, which are incompletely transformed with negligible clonogenic/tumorigenic potential. Methods and Results. Mutant-hGAS-HEK293 clones (HEK-PG) were generated and confirmed to overexpress full-length (9KDa) PG. HEK293 clones transfected with empty vector (HEK-C) served as controls. A significant increase in proliferation of HEK-PG vs HEK-C cells was measured in vitro. Cells, stained for either the putative stem cell marker (DCAMKL+1) or progenitor cells marker (CD44), were examined by fluorescense confocal microscopy and quantified by Western Blot analysis. Expression of DCAMKL+1/CD44 increased 2-3 fold in HEK-PG clones vs HEK-C clones, suggesting for the first time, that PG directly enhances the number of stem/progenitor cells. Significant increase in DCAMKL+1/CD44 cells was confirmed in colonic crypts of Fabp-PG mice, overexpressing PG in colons. The most surprising finding was that overexpression of PG in HEK-PG clones imparted a clonogenic/tumorigenic/metastatic potential to the cells, in vitro and in vivo (using SCID mouse model). However, IEC-18-PG clones were not rendered clonogenic/tumorigenic, suggesting that overexpression of PG in the background of HEK-293 cells, represented the second hit required for transformation. IEC-18 cells are immortalized but apparently lack the first hit (initiating events) and thus remain non-transformed in the presence of PG over-expression. In associated studies we have measured activation of two powerful transcription factors, NFκB/β-catenin, in response to PG, which may represent the second hit for transformation of cells, in response to autocrine PG, in pre-initiated cells (such as HEK-293). Since a large % of GI cancers express autocrine PG, down-regulation of PG and/or its receptor (Annexin2), and/or its mediatory signaling pathways (NFκB/β-catenin) should prove to be a powerful method for reducing stem cell/progenitor cell populations and attenuating tumorigenic/metastatic potential of PG-expressing cancer cells. Supported by NIH grants R01CA114264 and R01CA097959 and a Sealy Center grant to P. Singh Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4259.