© 2018. Published by The Company of Biologists Ltd | Development (2018) 145, dev160382. doi:10.1242/dev.160382 STEM CELLS AND REGENERATION RESEARCH ARTICLE Podoplanin regulates mammary stem cell function and tumorigenesis by potentiating Wnt/β-catenin signaling Laura Bresson1,2,3, Marisa M. Faraldo1,4, Amandine Di-Cicco1, Miguel Quintanilla5, Marina A. Glukhova1,4 and Marie-Ange Deugnier1,4,* ABSTRACT K5/14), P-cadherin, smooth muscle-specific contractile proteins, Δ Stem cells (SCs) drive mammary development, giving rise postnatally and the transcription factors Np63 (an isoform of Trp63) and Slug/ to an epithelial bilayer composed of luminal and basal myoepithelial Snail2, which are essential for the maintenance of basal cell identity cells. Dysregulation of SCs is thought to be at the origin of certain breast (Yalcin-Ozuysal et al., 2010; Guo et al., 2012). The luminal cell cancers; however, the molecular identity of SCs and the factors layer is characterized by the expression of K8/18. It includes a regulating their function remain poorly defined. We identified the subset of hormone-sensing cells that express estrogen, progesterone transmembrane protein podoplanin (Pdpn) as a specific marker of the and prolactin receptors (ER, PR and PrlR, respectively) and produce basal compartment, including multipotent SCs, and found Pdpn local mediators involved in the paracrine control of basal and localized at the basal-luminal interface. Embryonic deletion of Pdpn luminal cell function (Brisken and Ataca, 2015). targeted to basal cells diminished basal and luminal SC activity and It is established that both mammary lineages, basal and luminal, affected the expression of several Wnt/β-catenin signaling components originate from a common embryonic stem cell (SC) expressing basal in basal cells. Moreover, Pdpn loss attenuated mammary tumor keratins (van Keymeulen et al., 2011; Moumen et al., 2012). In the formation in a mouse model of β-catenin-induced breast cancer, limiting postnatal mammary gland, multipotent SCs able to repopulate the tumor-initiating cell expansion and promoting molecular features entire epithelium upon transplantation have been localized to the associated with mesenchymal-to-epithelial cell transition. In line with basal compartment (Visvader and Stingl, 2014). Data from lineage- the loss-of-function data, we demonstrated that mechanistically Pdpn tracing studies have revealed the existence of basal and luminal enhances Wnt/β-catenin signaling in mammary basal cells. Overall, this lineage-restricted SCs (van Keymeulen et al., 2011, 2017; Prater study uncovers a role for Pdpn in mammary SC function and, et al., 2014; Rios et al., 2014). The precise molecular characteristics importantly, identifies Pdpn as a new regulator of Wnt/β-catenin of multipotent and lineage-restricted SCs remain unknown, and signaling, a key pathway in mammary development and tumorigenesis. their respective contributions to mammary bilayer development and homeostasis after birth are still a matter of debate (Lloyd-Lewis KEY WORDS: Mammary gland, Stem cells, Breast cancer, Wnt et al., 2017). Nevertheless, in recent years, considerable interest has signaling, Podoplanin, Mouse focused on these cell subsets, from which certain breast cancers are thought to originate, particularly cancers of the triple-negative INTRODUCTION subtype (TNBC), lacking ER, PR and amplified HER2 (or ERBB2), Although mammary development initiates during embryogenesis often associated with a poor prognosis (Visvader and Stingl, 2014; the majority occurs postnatally. During puberty, the mammary ducts Skibinski and Kuperwasser, 2015). elongate and ramify extensively, generating a ductal network in Many studies have shown that canonical Wnt/β-catenin (β-cat) sexually mature females. Pregnancy is characterized by ductal side- signaling is essential for normal mammary development (Yu et al., branching and alveoli formation. Lactational differentiation is 2016). In addition, this pathway is frequently dysregulated in followed by involution at weaning. Systemic hormonal cues and TNBCs (Pohl et al., 2017). Wnt/β-cat signaling has been shown to various local stimuli, including growth factors, cell-cell and cell- play a major role in controlling the expansion of the basal cell matrix interactions, control the morphogenesis and remodeling of population during postnatal mammary development (Teulierè et al., the postnatal mammary gland (Macias and Hinck, 2012; Glukhova 2005; Zeng and Nusse, 2010; Macias et al., 2011; van Amerongen and Streuli, 2013). et al., 2012; Cai et al., 2014; Rajaram et al., 2015). Basal cells The mammary epithelium is organized as a bilayer, with an outer display a complex Wnt receptor machinery, including Fzd7, Lrp5/6 layer of basal myoepithelial cells and an inner layer of luminal cells. and the R-spondin (Rspo) receptors Lgr4/5/6, known to modulate During lactation, the luminal cells produce milk, whereas the Wnt/β-cat signal strength (Badders et al., 2009; de Visser et al., myoepithelial cells are contractile and serve for milk expulsion. 2012; Wang et al., 2013; Chakrabarti et al., 2014; Blaas et al., 2016; Basal myoepithelial cells express basal-specific keratins (including Driehuis and Clevers, 2017). Luminal cells have been identified as a major source of Wnt-associated ligands. In particular, they produce β 1Institut Curie, PSL Research University, CNRS, UMR144, Paris, F-75248, France. Wnt4 and Rspo1, two major regulators of paracrine Wnt/ -cat 2UniversitéParis Sud, UniversitéParis-Saclay, F-91405, Orsay, France. 3Sorbonne activation in basal cells (Cai et al., 2014; Rajaram et al., 2015). Universités, UPMC Univ Paris 06, F-75005, Paris, France. 4INSERM, Paris, F-75013, Comparative transcriptome analyses of basal and luminal cells France. 5Instituto de Investigaciones Biomedicas Alberto Sols, CSIC-UAM, Madrid, Spain. isolated from adult mouse and human mammary glands indicated that podoplanin (Pdpn) was among the top-ranking genes *Author for correspondence ([email protected]) characterizing the basal cell signature (Lim et al., 2010). However, M.-A.D., 0000-0002-2199-1099 its functional importance remains unknown. Pdpn is a small mucin- type transmembrane protein composed of a glycosylated extracellular Received 9 October 2017; Accepted 15 January 2018 domain, a transmembrane region and a short cytoplasmic tail devoid DEVELOPMENT 1 STEM CELLS AND REGENERATION Development (2018) 145, dev160382. doi:10.1242/dev.160382 of enzymatic activity (Renart et al., 2015). Widely used as a marker of cytoskeleton linkers ezrin and moesin (members of the ERM lymphatic endothelial cells, Pdpn is also displayed by various other family). Moreover, Pdpn has been reported to modulate Rho cell types, including certain epithelial cells, and is overexpressed in GTPase activity in fibroblasts and epithelial cells (Martin-Villar human carcinomas of various tissue origin (Schacht et al., 2005; et al., 2006; Wicki et al., 2006; Cueni et al., 2010; Acton et al., 2014; Wicki and Christofori, 2007; Ugorski et al., 2016; Suzuki-Inoue Astarita et al., 2015; Asai et al., 2016). et al., 2017). We used a conditional gene deletion approach to investigate the Pdpn null mice die before or shortly after birth, exhibiting defects role of Pdpn in mammary development and tumorigenesis. Our data in lung organogenesis, cardiac function and blood-lymph separation reveal that Pdpn participates in the control of basal SC function (Ramirez et al., 2003; Schacht et al., 2003; Mahtab et al., 2009). through positive regulation of the Wnt/β-cat signaling pathway. Pdpn is therefore crucial for the early development of several Moreover, using a mouse model of β-cat-induced TNBC, we found tissues. Most of the data concerning the physiological function of that Pdpn loss limited tumor-initiating cell expansion, attenuating Pdpn come from studies on the immune system, focusing on the mammary tumorigenesis. ability of the extracellular domain of Pdpn to bind the C-type lectin Clec2 (or Clec1b) (Suzuki-Inoue et al., 2007, 2017). Heterotypic RESULTS signaling from Pdpn- to Clec2-expressing immune cells is crucial In the postnatal mammary gland, Pdpn is expressed for platelet activation, blood-lymph separation and dendritic cell exclusively in the basal compartment including migration (Astarita et al., 2012; Suzuki-Inoue et al., 2017). multipotent SCs Pdpn function in epithelial cells has been mostly investigated in Pdpn was present throughout the mammary bud on embryonic day culture. The ectopic expression of Pdpn in various epithelial cell (E) 15, and was strongly expressed at cell-cell contacts (Fig. 1A). lines promotes cell motility, modifying actin cytoskeleton After birth, Pdpn expression was restricted to the basal cell layer. organization (Martin-Villar et al., 2006; Wicki et al., 2006; Cueni Ductal myoepithelial cells and the basal cap cells of the terminal et al., 2010; Asai et al., 2016). At the molecular level, Pdpn has been end buds (TEBs), specialized structures driving ductal growth shown to interact, via its intracellular domain, with the membrane and branching during puberty, expressed Pdpn, as did ductal and Fig. 1. Pdpn specifically marks the mammary basal cell population, including multipotent SCs. (A-D) Immunofluorescence labeling of sections through (A) an embryonic mammary bud at E15; (B) a TEB (upper panel) and epithelial ducts (middle and lower panels) from 6-week-old mice; (C) alveoli and a small duct from 15-day pregnant (G-15d) mouse; and (D) 4-day lactating (L4)