Fine-tuning of Epithelial EGFR signals Supports Coordinated Mammary Gland Development

Abstract

SummaryDuring puberty, robust morphogenesis occurs in the mammary gland; stem- and progenitor-cells develop into mature basal- and luminal-cells to form the ductal tree. The receptor signals that govern this process in mammary epithelial cells (MECs) are incompletely understood. The EGFR has been implicated and here we focused on EGFR’s downstream pathway component Rasgrp1. We find that Rasgrp1 dampens EGF-triggered signals in MECs. Biochemically and in vitro, Rasgrp1 perturbation results in increased EGFR-Ras-PI3K-AKT and mTORC1-S6 kinase signals, increased EGF-induced proliferation, and aberrant branching-capacity in 3D cultures. However, in vivo, Rasgrp1 perturbation results in delayed ductal tree maturation with shortened branches and reduced cellularity. Rasgrp1-deficient MEC organoids revealed lower frequencies of basal cells, the compartment that incorporates stem cells. Molecularly, EGF effectively counteracts Wnt signal-driven stem cell gene signature in organoids. Collectively, these studies demonstrate the need for fine-tuning of EGFR signals to properly instruct mammary epithelium during puberty.

Single-cell and spatial analyses reveal a tradeoff between murine mammary proliferation and lineage programs associated with endocrine cues

“…Similarly, Wnt pathway activation is strongly and specifically activated by PR signaling, 90 , 115 while EGF signaling may be induced by either ER or PR. 87 , 91 , 116 In bulk RNA-seq datasets from mammary organoids treated with EGF, a synthetic Wnt agonist, or both, 111 the LE AP signature was enhanced 30% by Wnt stimulation irrespective of EGF co-treatment ( Figure 7G ), but, perhaps unexpectedly, suppression of the HE AP signature was reversed by EGF co-treatment ( Figure S7G ). The LE BA signature was enhanced only in the combined treatment ( Figure S7H ), possibly indicating EGF and Wnt signaling cooperate to induce BA proliferation.…”

“… (G) Plot showing the LE AP signature score in C57BL/6J mammary epithelial organoids under Wnt-stimulated, EGF-stimulated, or dual-stimulated conditions (n = 6 each). 111 Data presentation as in (D). Significance determined via one-way ANOVA followed by Tukey’s multiple comparisons test.…”