Abstract P6-11-08: Non-transformed breast epithelial cells show neural-like gene signature after lipid exposure
Authors: Cottone Gannon , Eduardo Mariana Bustamante , Yadav Shivangi , Khan Seema , Clare Susan 
Published: March 2023

Abstract
Introduction: The identification of women specifically at risk for estrogen receptor negative breast cancer (ER-BC) and the targeted treatment of this disease are significantly unmet clinical needs. To that end, we analyzed the gene expression profiles of epithelial cells from the contralateral unaffected breasts (CUBs) of BC patients and identified a lipid metabolism gene signature, which was enriched in the CUBs of women with ER-negative BC (PMID: 28263391). Subsequent experiment revealed that exposure of non-transformed breast epithelial cells to lipid results in significant changes gene expression, chromatin accessibility and histone posttranslational modifications (PMID: 35508495). Several of the upregulated genes are hallmarks of the various fates of vagal neural crest: Neural, neurogenic and mesenchymal lineages. We hypothesize that lipid exposure imparts a survival advantage of stem-like cells, that lipid-induced epigenetic changes lead to a neural crest-like transcription signature and that these genes are not expressed normally in the breast. Methods: MCF10A cells were exposed to vehicle or octanoic acid (OA) for 24 hours. Gene expression was assayed by RNA-seq and OA responsive genes were identified (PMID: 35508495). Single-cell RNA sequencing (scRNA-seq) data from 14 human reduction mammoplasties (RM) was obtained from a publicly accessible data set (PMID: 34031589). The scRNA data was clustered and identified by unsupervised clustering (Seurat, v3.4.1) using cell-type markers curated using Supplementary table 2 from (PMID: 34031589). The bulk RNA-seq data from the OA treated cells was deconvoluted to cell-lineages using Bisque. The most significant upregulated VNC neural/neuronal/mesenchymal genes from the gene expression analysis were then plotted on the lineage clusters using FeaturePlot to determine if these markers are found in the normal breast epithelium, or other cell lineages. The plots were then filtered and re-clustered to look at basal-luminal cell types only. We utilized a second resource, a web-application for snATAC-seq data from various stages of mouse mammary development developed by the Wahl lab (https://wahl-lab-salk.shinyapps.io/Mammary_snATAC/), to query these same genes. Results: Deconvolution of the bulk RNA-sequencing data revealed a transition to a pericyte transcription program following exposure to OA. Nerve growth factor (NGF) was found to be expressed in pericytes while nerve growth factor receptor (NGFR) was found within the basal epithelial cell lineage. Genes overexpressed in the VNC neural cluster and overexpressed in the OA-exposed MCF10 cells, PPP1R1C (2.39x, adj p=1.6E-5), FOXD3 (6.7x, adj p=6.7E-10), DIO3 (5.9x, adj p=3.9E-6) and MOXD3 (4.2x, adj p=1.5E-23) all evidence little to no expression in the normal breast but are observed in murine fetal mammary stem cells. Schwann cell precursor (SCP) markers, CDH19 and ROPN1, significantly upregulated in OA treated cells: 5.4x and 1.6x, respectively, exhibited low expression in luminal progenitors but in the mouse were observed in the mammary stroma. CDH19, a gene exclusive to SCPs, is expressed in stroma following murine birth. PRRX1, a key regulator of the VNC mesenchymal cell fate cluster, is 9.3-fold (p adj=4.9E-49) overexpressed in the OA treated cells, expressed strongly in pericytes and stroma and to a lesser extent in basal epithelial cells of the normal human breast and stroma of the murine mammary gland. Conclusions: Treatment of non-transformed mammary epithelial cells with lipid, specifically OA, shows significant upregulation of multiple VNC genes associated with both neural and mesenchymal fate. scRNA-seq from RM patients reveals that many of these same markers are either found in non-epithelial cell clusters or are found with low expression in luminal mammary lineages (both progenitors and mature).
Citation Format: Gannon Cottone, Mariana Bustamante Eduardo, Shivangi Yadav, Seema Khan, Susan Clare. Non-transformed breast epithelial cells show neural-like gene signature after lipid exposure [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-08.