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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE149275 | GEO | 2020/08/01

REPOSITORIES: GEO

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Project description:CCAAT/enhancer binding protein beta (C/EBPb) is a member of a family of highly conserved transcription factors that regulates numerous genes involved in proliferation and differentiation in a variety of tissues. C/EBPb is deregulated in human breast cancer and germline deletion of this gene results in multiple defects in mammary gland development. We hypothesized that C/EBPb regulates mammary stem cell self-renewal, maintenance and/or differentiation through the regulation of multiple target genes that coordinate mammary gland development. Utilizing both a germline knockout mouse model and a conditional knockout strategy, we demonstrated that mammosphere formation was significantly decreased in C/EBPb-deficient mammary epithelial cells (MECs). The ability of C/EBPb-deleted MECs to regenerate the mammary gland in vivo was severely impaired when transplanted at limiting dilution. Furthermore, serial transplantation of C/EBPb-null mammary tissue resulted in decreased outgrowth potential when compared to wildtype, and an early senescence phenotype. Flow cytometric analysis revealed that C/EBPb-null MECs contain a lower frequency of repopulating stem cells accompanied by an increase in committed, differentiated luminal cells as compared to wildtype. Microarray analysis of stem/progenitor cell populations was performed and revealed an alteration in cell fate specification in C/EBPb-null glands, exemplified by the aberrant expression of basal markers in the luminal cell compartment. Collectively, our studies demonstrate that C/EBPb is a critical regulator of mammary stem cell differentiation, and an important determinant of luminal cell fate specification. Experiment Overall Design: To identify potential signaling pathways regulated by C/EBPb in stem/progenitor cells, microarray analysis was performed on two stem/progenitor cell subpopulations. For this analysis, subpopulations defined by LIN-CD24+CD29hi and LIN-CD24hiCD29lo were FACS sorted from wildtype and germline C/EBPb-/- glands, and RNA was isolated from each group.

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

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