Life time exposure to elevated levels of estrogen (E2) is a major risk factor for breast cancer. Among women with ER/PR positive tumors, only 50 to 60% of women respond to endocrine therapy. Intra-tumoral heterogeneity of breast cancer cells may be one of the reason for unresponsiveness to endocrine therapy in more than 40% of ER-positive breast cancer cases. In this study we examined that heterogeneous breast cancer stem cells (BCSCs) are produced by exposure to E2 and/or ectopic expression of molecular risk factor - nuclear respiratory factor 1 (NRF1). To elucidate this, we used single cell confocal imaging, flow sorting, ChIP and qRT-PCR approaches. We also measured the key regulators of pluripotency, epithelial-mesenchymal transition (EMT), stemness, cell apoptosis and cell cycle regulation. The functional assay for cancer stem cells enrichment employed were selection of cells in B27 medium as spheroids, anchorage-independent growth and mammosphere formation assays. Carcinogenic treatments of E2 to MCF10 A CD24- cells induced formation of ~1.46% CD44+/CD24+ and 26.02% of CD44+/CD24- subpopulations, which are a typical phenotypes associated with human breast tumor-initiating cells (BTICs) or BCSCs. These subpopulations are capable of forming self-renewing mammospheres. In contrast, CD24+ or CD24-/CD44- cells did not form mammospheres. Stable NRF1 overexpression induced formation of 2.36% CD44+/CD24+ and of 64% CD44+/CD24- subpopulations. Carcinogenic treatments of E2 to stable NRF1 overexpressing MCF10 A cells, induced to acquire 21.54% CD44+/CD24+ and 44.54% CD44+/CD24- subpopulations. E2 treatment to NRF1 overexpressing BSCSs markers significantly increased mammosphere forming capability, compared to NRF1 alone. The dominant negative form of NRF1 diminished the effects of E2 and/or NRF1 induced acquiring of BCSCs antigen markers and their capability of forming mammospheres. E2 induced BTICs/BCSCs were heterogeneous. Each subpopulation was characterized by a different transcriptional and biomarker profiles of CD24, CD44, CD49f, CD133, ALDH1A1, CXCR4, and NRF1. The expression of different markers for pluripotency (OCT4, SOX2 and Nanog), EMT (E-cadherin, N-cadherin and Vimentin), stemness (CD24, CD44, CD39f, CD133, ALDH1A1), cell cycle (p16,CDC2,CDC25C, cyclinB1) and metastasis (CXCR4), were associated with BTICs produced by E2 and/or NRF1. In summary, we observed new roles of NRF1 in contributing to acquire breast tumor initiating stem-like cells and in regulating EMT and invasiveness of BCSCs, thus opens a new direction of NRF1’s role in breast cancer research. A better understanding of how E2 dependent breast neoplasm heterogeneity depends on NRF1 network may open new avenues for therapeutic strategy against breast cancer. This work was supported partly by VA MERIT Review (VA BX001463) grant to DR.

Citation Format: Jayanta Kumar Das, Deodutta Roy. Estrogen induced NRF1 signaling is a molecular mechanism underlying the generation of different breast cancer stem cell subpopulations leading to intratumoral heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 938. doi:10.1158/1538-7445.AM2017-938