Abstract

Abstract Cancer is driven by a complex and heterogeneous combination of genes and pathways, many of which are not explicitly oncogenic on their own. These so-called non-oncogenes are required for maintenance of the tumorigenic state, but they are not mutated themselves. One way to identify “non-oncogene” pathways is to evaluate how proliferating cells adjust to variable and evolving environments. By examining heterogeneous transcriptional regulatory states of single cells in a 3D culture model of breast epithelial organization, we uncovered NRF2 as a candidate transcription factor upstream of an important regulatory state for morphogenesis. NRF2 is a known oncogene in lung cancer, but mutations in the transcription factor are uncommon in breast cancer. To examine the functional impact of NRF2 signaling, we engineered several triple-negative breast cancer cell lines with inducible short hairpin RNA to knock down NRF2. In claudin-low MDA-MB-231 cells, knockdown of NRF2 caused dramatic reduction in stellate invasion compared with shGFP control. In basal-like HCC-1806 cells, knockdown of NRF2 caused a reduction in “ruptured” tumorspheres, in which cells disseminate from their originating site. In a non-transformed breast epithelial cell line, overexpression of a non-degradable form of NRF2 caused greater variation in the size of acini, while knockdown of NRF2 narrowed the size distribution of acini. Therefore, knockdown of NRF2 substantially normalizes the size and shape of both non-transformed and cancerous 3D structures. We also analyzed expression levels of the genes in the provisional NRF2-regulated cluster after NRF2 knockdown or overexpression. Upon NRF2 knockdown, the p53-regulated transcripts CDKN1A (p21) and RPS27L were strongly induced, suggesting that p53 may be activated after NRF2 loss. Our results suggest a functional importance for NRF2 in multiple breast-epithelial and TNBC contexts, and raise the possibility of interplay between NRF2 and p53. A detailed understanding of NRF2 regulation and dynamics could enable new strategies to disrupt this non-oncogene addiction and re-sensitize cancer cells to their environment and to chemotherapy. Citation Format: Elizabeth Pereira, Kevin Janes. Single-cell misregulation of NRF2 in basal-like breast cancer [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 3954. doi:10.1158/1538-7445.AM2017-3954

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