Abstract 4248: NANOG depletion decreases cell proliferation rate in colorectal carcinoma cells and tumor growth in nude mice

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Nanog is a critical transcription factor involved in maintaining embryonic stem cells in a pluripotent, dedifferentiated state and expression of NANOG or its nearly identical retrogene NANOGP8 is increased in human gastric, hepatocellular, and colon cancer. Our postulate is that NANOG/NANOGP8 is critical for the functions of putative CSC in colorectal carcinoma (CRC): self-renewal, pluripotency, spherogenicity, tumorigenicity and resistance to chemotherapy. Five human CRC lines are being used in this study (HCC 2998, KM-12c, Clone A, MIP-101, CX-1). CX-1 cells are more aggressive than the other CRC because they form more spheroids from single cells and are highly metastatic after intrasplenic compared to the other CRC that are tumorigenic but weakly metastatic. qRT-PCR analysis of spheroids of CX-1 and Clone A cultured in suspension in serum free medium indicated that NANOG transcript levels were increased by 43- and 12-fold, respectively, relative to monolayer cultures while the relative increase in transcript levels of several cancer stem cell markers such as EPCAM, ALDH1A1, ALDH3A1, CD44, CD133, and CD166 was less than 5-fold except for POU5F1 and SOX2 in Clone A, which were 5- and10-fold respectively. NANOG promoter activity was low in monolayer culture in CRC stable transfectants with a GFP NANOG promoter, but consistently increased as CRC detached and formed spheroids in serum free media. Stable transfection with lentiviral NANOG shRNA decreased NANOG transcript levels and spherogenicity of Clone A and CX1 cells compared to vector controls in a single cell spherogenicity assay in serum-free medium. NANOG downregulation also induced a S phase cell cycle arrest with inhibition of ATM and WEE1 expression with associated reduction in phosphorylation of other key cell-cycle checkpoint proteins. Finally, marked tumor growth retardation was observed in NOD/SCID mice injected subcutaneously with NANOG shRNA transfected cells compared to parental or vector controls. NANOG shRNA transfected cells were also more resistant to treatment with Topotecan, a camptothecin. In summary, our data suggest that expression of NANOG/NANOGP8 occurs in human CRC and may be critical for such functions of putative CSC as spherogenicity, cell proliferation, tumor formation and resistance to chemotherapy 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 4248.