Abstract 3349: Nanog-expressing breast cancer tumor initiating cells (TICs) are highly metastatic and are resistant to the effects of proteasome inhibition

Abstract

Abstract Functional heterogeneity of transformed cells exists within a single tumor mass and between distinct tumor nodules. Many tumors have also been demonstrated to contain a subpopulation of tumor initiating cells (TICs) whose epigenetic modifications convey resistance to conventional therapies. It has been hypothesized that TICs represent the terminal product of epithelial-mesenchymal transition which is linked to metastatic potential. Therefore we examined the metastatic potential of TICs in the 4T1 murine breast carcinoma model, and evaluated Nanog expression as a biomarker for highly metastatic 4T1 sub-populations. Traditionally TICs have been identified and isolated through expression of selected cell surface markers and functional assays, however cell surface phenotype alone fails to identify CSC in all tumors from the same organ or in all patients. We hypothesized that expression of transcription factors that are required for induction and maintenance of pluripotency (Oct-4, Sox-2 and Nanog) could be superior to cell surface phenotype for identification of TICs. Thus we adapted a commercial promoter reporter system where the Nanog promoter drives copepod GFP which has been destabilized to better reflect rapid changes in transcriptional activity. I ntroduction of a Nanog promoter reporter construct into 4T1.2 cells resulted in stable expression of GFP in about 0.5% of the cells. In vivo tumor transfer studies confirmed that Nanog promoter activity (4T1.2-GFPpos cells) was associated with a significantly enhanced ability to form experimental lung metastases. In addition, in vitro TIC surrogate assays showed that 4T1.2 GFPpos cells produced increased numbers of spheroids in both soft agar and under non-adherent growth conditions. Finally Nanog-GFPpos cells were more resistant to killing by Paclitaxel than Nanog-GFPneg cells, but were more sensitive to Salinomycin. Together these findings suggest that Nanog promoter activity is a robust marker of metastatic 4T1.2 TICs. We have previously observed that administration of the proteasome inhibitor Bortezomib (Bzb) has moderate anti-4T1.2 tumor activity in vivo therefore we examined the sensitivity of the various 4T1.2 populations to Bzb treatment in vitro. 4T1.2 Nanog-GFPpos cells were resistant to the cytostatic effects of Bzb while 4T1.2 Nanog-GFPneg cells were sensitive. The molecular basis of this difference is under investigation, but is not due to differences in accumulation of ubiquitinated proteins. Taken together, these findings show that Nanog promoter activity is a marker of highly metastatic TICs, and suggest that eradication of TICs may be required for curative anti-cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3349. doi:10.1158/1538-7445.AM2011-3349