Mammary stem cells (MaSC) are a heterogeneous population that give rise to progenitor cells subsequently developing into differentiated cells. The exact mammary epithelial cell (MEC) hierarchy and particularly the signals that govern this hierarchy are largely unknown. Several studies have implied a role for EGFR signals in the mammary epithelium but the exact role that of EGFR (Epidermal Growth Factor Receptor) has remained rather elusive. Insights from a 3-D ex vivo model derived from human breast tissue and studies using a hypomorphic EGFR allele have put forth a model that the strength of EGFR signals may impact cell fate decisions in the mammary gland. We obtained recent evidence indicating that the Ras guanine nucleotide exchange factor (RasGEF) Rasgrp1 can dampen EGFR signals in epithelial cells.
We demonstrate that RasGEF Rasgrp1 is expressed in mammary epithelium and use cleared fat pad approaches to reveal a previously unrecognized functional role for RasGRP1 in the mammary gland. These approaches involved the transplantation of mammary epithelial cells (MECs) from Rasgrp1-deficient animals and from mice with a single point mutation in Rasgrp1, the Rasgrp1Anaef model. Biochemically, loss of Rasgrp1 as well as the Rasgrp1Anaef point mutation resulted in increased EGF-induced Ras-effector kinase signaling. MECs showed increase activation of the ERK-, Akt-, and mTORC1-S6- kinase pathways upon EGF stimulation when the MECs were isolated from Rasgrp1-/- or the Rasgrp1Anaef females. Using a model cell line, Eph4, we demonstrated that Rasgrp1’s suppressive action depends on its catalytic activity.
Functionally, Rasgrp1 perturbation led to shortened mammary ductal trees in mammary whole mounts, increases in numbers of proliferative terminal end buds (TEBs), and sustained proliferation of mammary ductal cells (with high phospho-Akt) in areas where proliferation has normally halted. Organoid assays to reveal that Rasgrp1 perturbation led to a gain-of- function EGF-driven phenotype. With WT MECs, EGF drove the formation of spheres in organoid assays. By contrast, EGF induced robust branching of Rasgrp1-/- and Rasgrp1Anaef MECs that could be prevented when EGFR inhibitors are added. Thus, Rasgrp1 critically controls the mammary morphogenetic program as indicated by elevated branching morphogenesis upon EGF-EGFR signaling in the absence of Rasgrp1 function.
MEC colony assays using Rasgrp1-/- and Rasgrp1Anaef cells revealed that progenitors form more and larger colonies than WT counterparts and with increased proliferation (Ki67) in response to EGF and strong staining for pAkt, all of which were reduced with EGFR inhibitors in the assays. Lastly, capitalizing on FACS profiling of mammary epithelial cells we established that Rasgrp1 perturbation led to alterations in cell fate of stem- and progenitor-cells in the mammary gland. Rasgrp1-/- and Rasgrp1Anaef mammary glands revealed decreased total cellularity of CD31-/CD45-/Ter119- lineage-negative cells but increased luminal CD49fmedium/EpCAMhigh and basal CD49fhigh/EpCAMmedium cell numbers that encompasses the MaSCs.
In sum, we provide mechanistic insights on the biochemical signals that impact MACSs and progenitor cells. We show that Rasgrp1 is required to balance MEC EGFR signals and regulate mammary stem and progenitor cell fate to dampen proliferation so that cells can exit mitosis and maturate and differentiate.