Inducible knockout of ∆Np63 alters cell polarity and metabolism during pubertal mammary gland development

Abstract

The ∆Np63 isoform of the p53‐family transcription factor Trp63 is a key regulator of mammary epithelial stem cells that is involved in breast cancer development. To investigate the role of ∆Np63 at different stages of normal mammary gland development, we generated a ∆Np63‐inducible conditional knockout (cKO) mouse model. We demonstrate that the deletion of ∆Np63 at puberty results in depletion of mammary stem cell‐enriched basal cells, reduces expression of E‐cadherin and β‐catenin, and leads to a closed ductal lumen. RNA‐sequencing analysis reveals reduced expression of oxidative phosphorylation (OXPHOS)‐associated proteins and desmosomal polarity proteins. Functional assays show reduced numbers of mitochondria in the mammary epithelial cells of ΔNp63 cKO compared to wild‐type, supporting the reduced OXPHOS phenotype. These findings identify a novel role for ∆Np63 in cellular metabolism and mammary epithelial cell polarity.

p63 and Its Target Follistatin Maintain Salivary Gland Stem/Progenitor Cell Function through TGF-β/Activin Signaling

“…Our findings are in line with genetic lineage tracing experiments performed in salivary glands of adult mice showing that the transition of actively proliferating K14 + basal cells to both the ductal intercalated and excretory luminal cells of the gland occurs fairly quickly-within 14 days (Kwak et al, 2016;Kwak and Ghazizadeh, 2015). Moreover, the wide-spread defects observed upon the loss of DNp63 in the SG are also in good agreement with similar studies reported in the mammary gland (Forster et al, 2014;Kumar et al, 2019). Interestingly, conditional deletion of DNp63 in the pubertal mammary gland results in aberrant ductal formation presumably owing to altered stem cell differentiation (Kumar et al, 2019).…”

“…Moreover, the wide-spread defects observed upon the loss of ΔNp63 in the SG are also in good agreement with similar studies reported in the mammary gland ( Forster et al., 2014 ; Kumar et al., 2019 ). Interestingly, conditional deletion of ΔNp63 in the pubertal mammary gland results in aberrant ductal formation presumably owing to altered stem cell differentiation ( Kumar et al., 2019 ). Moreover, ΔNp63 has also been shown to be important during pregnancy as ΔNp63KO mice showed defects in luminal progenitor cell proliferation and differentiation ( Forster et al., 2014 ).…”

“…To examine the role of ΔNp63 in adult SG maintenance, we crossed ΔNp63 fl/fl mice to a transgenic strain that ubiquitously expresses Cre-recombinase fused to the estrogen-ligand binding domain ERT2 ( UBC CreERT2 ). This inducible conditional mouse model system allowed for overcoming the perinatal lethality associated with loss of p63 and also for the excision of ΔNp63 in both the basal and myoepithelial cell populations as shown in other organs such as the skin and mammary glands ( Kumar et al., 2019 ; Chakravarti et al., 2014 ). Tamoxifen (TAM) was administered to adult ΔNp63 fl/fl (control) and UBC CreERT2 ;ΔNp63 fl/fl (ΔNp63KO) mice, and SGs were harvested 8–10 days post TAM administration and analyzed.…”

“…However, our current understanding of the basic physiological mechanisms and the transcriptional networks regulating stem/progenitor (SP) cell self-renewal, proliferation, and differentiation programs in the SG remains elusive. p63, specifically the DNp63 isoform, is a lineage-specific master transcription factor that is highly expressed in epithelial-rich tissues and organs where it is plays important roles in stem cell self-renewal, cell fate decisions and lineage commitment, morphogenesis, and directing differentiation programs (Romano et al, 2012;Fan et al, 2018;Chakrabarti et al, 2014;Senoo et al, 2007;Packard et al, 2011;Blanpain and Fuchs, 2007;Kumar et al, 2019). Interestingly, these studies have unearthed important p63-driven signaling pathways that direct stem cell fate choices in various stratified epithelial tissues and glandular secretory organs.…”

Characterization of Gene Expression Signatures for the Identification of Cellular Heterogeneity in the Developing Mammary Gland

“…In line with previous studies, myoepithelial cells were defined by the expression of basal compartment-biased genes Krt5, Cytokeratin 14 (Krt14), Cytokeratin 17 (Krt17), Acta2, Secreted Protein Acidic and Cysteine Rich (Sparc), Myosin light chain kinase (Mylk), Podoplanin (Pdpn) and C-X-C Motif Chemokine Ligand (Cxcl14) [15,22,26,[34][35][36]. Myoepithelial progenitor/stem cells were marked by the expression of genes previously shown to contribute to tissue reconstruction in fat pad transplantation assays and overall mammary developmental processes, such as Tumor protein p63 (Tp63) [37], Bromodomain PHD Finger Transcription Factor (Bptf) [17], and classical markers of mammary stem-like state, such as Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (Lgr5) [38], Neuregulin 1 (Nrg1) [39] and Inhibitor of DNA Binding 4, HLH Protein (Id4) [40] (Fig. 2B and Supplementary Fig.…”