Elevated EGFR signals in epithelial cells delay mammary gland development and skew organoids away from stem cell signatures

During puberty, robust morphogenesis occurs in the mammary gland and mammary 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. Here we focused on the EGF receptor and its downstream pathway component Rasgrp1, expressed in MECs. Rasgrp1-/- or point-mutated Rasgrp1Anaef MECs demonstrated increased activation of ERK-, AKT-, and S6- kinase pathways. Rasgrp1 perturbation delayed ductal tree maturation, accompanied by aberrant MEC proliferation and AKT signals. In colony formation- and 3D Matrigel- assays, loss of Rasgrp1 function leads to elevated progenitor cell proliferation and gain-of-function branching, both in EGFR-dependent manners. Our studies revealed that unbalanced EGFR signals in Rasgrp1-/- or Rasgrp1Anaef females did not impact the intrinsic ability to form basal and luminal cells, but that a Wnt-driven stem cell gene signature disappears when EGF is added to organoid assays. mRNA libraries were generated from wildtype (WT) and RasGRP1-/- mouse mammary epithelial cell organoids that were grown in media containing epidermal growth factor (EGF), EGF + (next generation surrogate Wnt) NGS-Wnt + R-spondin 2 (Rspo2), or NGS-Wnt + Rspo2. Three biological replicates were performed for each condition. mRNA libraries were sequenced using HiSeq4000 (Illumina).