Overexpression Of Nrf1 Leads To The Generation Of Cancer Stem-Like Cells And Resistance To Anoikis _ Pathways To Anchorage-Independent Growth During Estrogen-Induced Malignant Transformation

The actual role nuclear respiratory factor-1 (NRF-1) plays in breast cancer remains the least studied of all transcription factors. A growth role for the NRF1 through regulation of cell cycle genes during estrogen-induced malignant transformation has recently been reported by us; however, the underlying mechanism of its contribution to estrogen-induced breast carcinogenesis is undefined. In this study we provide new mechanistic insight into the mode by which NRF1 may contribute in estrogen-induced neoplastic conversion of cells by suppressing anoikis; identified BNIP3 as a key transcriptional target of NRF1; and survival signals conferred by NRF1 seems to impinge on the hypoxia-inducible death factor BNIP3. ChIP, qPCR, mass spectrometry, redox Western blot, colony formation, cell proliferation, ROS assay, immunofluorescence microscopy were used to study the role of NRF-1. Our data showed that higher expresion of NRF-1 in MCF10A produced large sizes of spheroids with increased level of stem cell markers -CD44+CD24+CD49f+ compared to cells transfected with vector alone. E2-induced increase of spheroid formation, suppression of cell death and cell survival were amplified by expression of NRF1 and these effects were diminished by loss of NRF1 function through expression of dominant negative NRF1. Breast cancer cell lines - MCF7 and MDA-MB231 overexpressed both NRF1 and BNIP3, and formed larger tumor spheroids compared to the MCF10A or vector-transformed breast cancer cells. E2 treatment as well as overexpression of NRF1 increased mRNA and protein levels of BNIP3. In addition, we found the increased transcriptional interaction of NRF-1 transcription factor in the BNIP3 promoter region by ChIP-qPCR assay in cells treated with E2. We also found the increased functional activity of the BNIP3 promoter in cells treated with E2 and this was decreased with co-treatment of ROS scavenger-Ebselen. NRF1 spheroids have the ability to micro-invasion. In summary, overexpression of NRF1 altered the cell morphology towards mesenchymal stem-like shape and promoted cancer stem cell-like phenotype. Findings of this study unveiled the underlying mechanism of NRF1 action through the activation of BNIP3 which is regulated by NRF1 and is presumably responsible for the estrogen-induced malignant transformation of MCF10A cells, including anoikis resistance, anchorage-independent cell growth, and increased cell migration and invasion. This work was in part supported by a VA MERIT Review (VA BX001463) grant to DR. Citation Format: Jayanta Das, Deodutta Roy. Overexpression of NRF1 leads to the generation of cancer stem-like cells and resistance to anoikis _ pathways to anchorage-independent growth during estrogen-induced malignant transformation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 803. doi:10.1158/1538-7445.AM2015-803