Diverse regulation of mammary epithelial growth and branching morphogenesis through noncanonical Wnt signaling Kai Kessenbrocka,1, Prestina Smithb,1, Sander Christiaan Steenbeekc, Nicholas Pervolarakisa, Raj Kumarc, Yasuhiro Minamid, Andrei Gogae, Lindsay Hinckb,2, and Zena Werbc,2 aDepartment of Biological Chemistry, University of California, Irvine, CA 92697; bDepartment of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, CA 95064; cDepartment of Anatomy and Biomedical Sciences Program, University of California, San Francisco CA 94143-0452; dDivision of Cell Physiology, Department of Physiology and Cell Biology, Graduate School of Medicine, Kobe University, Kobe, Hyogo, 650-0017, Japan; and eDepartment of Cell and Tissue Biology, University of California, San Francisco, CA 94143-0452 Contributed by Zena Werb, February 1, 2017 (sent for review June 15, 2016; reviewed by Caroline M. Alexander and Rosa Serra) The mammary gland consists of an adipose tissue that, in a process The role of the noncanonical WNT signaling pathway in the reg- called branching morphogenesis, is invaded by a ductal epithelial ulation of mammary gland development and breast cancer function is network comprising basal and luminal epithelial cells. Stem and obscure. WNT5A and WNT5B represent two noncanonical WNT progenitor cells drive mammary growth, and their proliferation is ligands expressed in the mammary gland (11–14). Within the mam- regulated by multiple extracellular cues. One of the key regulatory mary epithelium, expression of both Wnt5a and Wnt5b is restricted to pathways for these cells is the β-catenin–dependent, canonical the more differentiated luminal epithelial cell lineage (14). Several wingless-type MMTV integration site family (WNT) signaling path- receptors have been implicated in mediating the function of WNT5A way; however, the role of noncanonical WNT signaling within the and WNT5B. These include the noncanonical receptors, receptor mammary stem/progenitor system remains elusive. Here, we fo- tyrosine kinase-like orphan receptor 1 (ROR1) and ROR2, which are cused on the noncanonical WNT receptors receptor tyrosine expressed in both the basal and luminal compartments (14, 15), kinase-like orphan receptor 2 (ROR2) and receptor-like tyrosine whereas the expression of the receptorRYKremainslesswellde- CELL BIOLOGY kinase (RYK) and their activation by WNT5A, one of the hallmark fined. Although several studies have reported inhibitory roles for noncanonical WNT ligands, during mammary epithelial growth and branching morphogenesis. We found that WNT5A inhibits noncanonical WNT ligands, WNT5A during branching morphogen- mammary branching morphogenesis in vitro and in vivo through esis (16) and WNT5B in mammary stem and progenitor outgrowth the receptor tyrosine kinase ROR2. Unexpectedly, WNT5A was (16, 17), the receptors mediating these inhibitory functions remain able to enhance mammary epithelial growth, which is in contrast poorly characterized. to its next closest relative WNT5B, which potently inhibits mam- Even though the actions of WNT5A and WNT5B have been mary stem/progenitor proliferation. We found that RYK, but not associated with events occurring during breast cancer initiation ROR2, is necessary for WNT5A-mediated promotion of mammary and progression, the role of noncanonical WNT signaling in growth. These findings provide important insight into the biology breast cancer remains elusive. Although there is evidence sug- of noncanonical WNT signaling in adult stem/progenitor cell reg- gesting that secretion of WNT5A by stromal cells may inhibit the ulation and development. Future research will determine how these interactions go awry in diseases such as breast cancer. Significance mammary stem cells | noncanonical Wnt signaling | receptor tyrosine The extracellular signals that regulate cell growth and tissue kinase | epithelial morphogenesis rearrangements during organogenesis are still poorly un- derstood. One large family of secreted signaling molecules, he mammary gland is composed of a highly dynamic epithelial called wingless-type MMTV integration site family member Tstructure that undergoes multiple rounds of remodeling during (WNTs), governs many of these processes by controlling cell– puberty, pregnancy, lactation, and involution (1). At puberty, the cell interactions. Here, we show that two closely related mammary gland forms a branching ductal network, which con- members of the WNT superfamily influence the development nects the nipple to the milk-producing lobuloalveolar structures of the mammary gland by signaling through different tyrosine that arise during pregnancy (2). Development and growth of the kinase receptors. WNT5A enhances the growth of mammary mammary gland depends on the function of adult mammary stem stem/progenitor cells while restricting the process of branching cells (MaSCs) (1). These MaSCs are capable of reconstituting a morphogenesis. In contrast, WNT5B represses mammary stem/ complete mammary epithelial ductal structure when implanted as progenitor cell proliferation through a different receptor. To- a single cell into a cleared fat pad in vivo (3–5). The morphoge- gether, the distinct actions of these highly homologous extra- netic changes of the breast epithelium are closely coordinated cellular cues regulate the developmental processes that supply within the context of its microenvironment, which consists of a cells and shape tissues during periods of rapid growth. variety of stromal cells such as adipocytes, macrophages, and fi- broblasts (6, 7). MaSCs respond to extracellular signals such as Author contributions: K.K., P.S., L.H., and Z.W. designed research; P.S., S.C.S., and R.K. per- formed research; Y.M. and A.G. contributed new reagents/analytic tools; K.K., P.S., S.C.S., N.P., wingless-type MMTV integration site family (WNT) ligands pro- R.K., and L.H. analyzed data; and K.K., P.S., L.H., and Z.W. wrote the paper. vided by stromal cells of the microenvironment. For example, Reviewers: C.M.A., University of Wisconsin–Madison; and R.S., University of Alabama, hyperactivation of the canonical WNT/β-catenin signaling pathway Birmingham. in the mammary gland expands the MaSC population by sixfold The authors declare no conflict of interest. (4), and WNT ligands are necessary for self-renewal properties of 1K.K. and P.S. contributed equally to this work. MaSCs (8). Constitutive overexpression of the gene encoding the 2To whom correspondence may be addressed. Email: [email protected] or lhinck@ucsc. canonical Wnt1 ligand in this organ ultimately gives rise to tumors, edu. suggesting a direct link between MaSC accumulation and tumor This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. susceptibility (9, 10). 1073/pnas.1701464114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1701464114 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 actions of tumor-initiating cells in breast cancer (18), other A BC studies show that WNT5A/B may promote epithelial to mesen- Control +WNT5A +WNT5B chymal transition and metastatic progression in breast and other cancers through a noncanonical Frizzled2 pathway (19). Overall, the role of noncanonical WNT signaling in breast cancer appears to be particularly context-dependent, and the diverse effects of noncanonical WNT ligands on cell and developmental pathways remain largely unexplored. Here, we focused on the role of WNT5A and WNT5B as two of the main mediators of noncanonical WNT signaling in the mammary gland. In particular, we sought to understand how DE200 100 * )m noncanonical WNT signaling is involved in the regulation of 80 * MaSC function and branching morphogenesis. Our results show 150 ( that despite their high degree of similarity, WNT5A and WNT5B eziSe 60 100 * may work in a distinct manner involving different receptor mole- r 40 e cules. These findings further shed light on our understanding of hp 50 * the intricacies of the WNT signaling pathway and provide crucial S 20 Number of Spheres insights to better understand their role in diseases such as 0 0 breast cancer. Results Control WNT5A WNT5B Control WNT5A WNT5B F 1.6 WNT5A and WNT5B Differentially Regulate Mammary Growth and Control Progenitor Cell Proliferation. We previously observed that WNT5B nois 1.4 +WNT5A +WNT5B is capable of inhibiting mammary epithelial stem and progenitor serpxEdloF 1.2 cell growth capacity in vitro and in vivo, using lentiviral-mediated 1.0 overexpression in transplanted MaSCs (17). WNT5A and WNT5B 0.8 show a high degree of amino acid sequence similarity, at 83% (Fig. S1A), which initially suggested that these two WNT li- 0.6 * e 0.4 v gands may function in a redundant manner. To test whether these itale two noncanonical WNT ligands are indeed functionally re- 0.2 * dundant in the context of MaSC/progenitor regulation, we first R 0 K8 Gata3 K5 K14 Mcm2 Ki67 used the clonal mammosphere assay as a proliferative analysis tool Luminal Basal Proliferation in which single mammary epithelial cells (MECs) are embedded into a 3-dimensional Matrigel-based culture. Using this assay, we Fig. 1. Differential effects of WNT5A and WNT5B on mammosphere out- then compared the effects of exogenous recombinant WNT5A and growth. Primary WT MECs (2,000 cells per well) were used as single cells in the mammosphere assay and treated with vehicle control or medium con- WNT5B to vehicle-treated mammosphere formation. Interestingly, taining either WNT5A (0.5 μg/mL) or WNT5B (0.5 μg/mL) recombinant