The secreted protease Adamts18 links hormone action to activation of the mammary stem cell niche

Estrogens and progesterone control breast development and carcinogenesis via their cognate receptors expressed in a subset of luminal cells in the mammary epithelium. How they control the extracellular matrix, important to breast physiology and tumorigenesis, remains unclear. Here we report that both hormones induce the secreted protease Adamts18 in myoepithelial cells by controlling Wnt4 expression with consequent paracrine canonical Wnt signaling activation. Adamts18 is required for stem cell activation, has multiple binding partners in the basement membrane and interacts genetically with the basal membrane-specific proteoglycan, Col18a1, pointing to the basement membrane as part of the stem cell niche. In vitro, ADAMTS18 cleaves fibronectin; in vivo, Adamts18 deletion causes increased collagen deposition during puberty, which results in impaired Hippo signaling and reduced Fgfr2 expression both of which control stem cell function. Thus, Adamts18 links luminal hormone receptor signaling to basement membrane remodeling and stem cell activation. How hormonal signaling in the mammary epithelium controls the surrounding extracellular matrix is unclear. Here, the authors show that a secreted protease, Adamts18, induced by upstream estrogen-progesterone activated Wnt4 in myoepithelial cells, remodels the basement membrane and contributes to mammary epithelial stemness.

What signals operate in the mammary niche?

Cathrin Brisken, Tamara Tanos

Adult stem cells reside in a specialized microenvironment, the niche, which controls their behavior. As mammary stem cells, and consequently their niches, are still poorly defined, we look at better-characterized adult mammalian stem cell niches in the hematopoietic system and the skin. We attempt to define the mammary stem cell niche functionally, based on the widely used mammary fat pad reconstitution assay. We note that the concept of the niche needs to be extended from the specialized microenvironment described in the hematopoietic system, to a model that takes into account the macroenviroment, as recently shown in the skin, and systemic clues as we will illustrate for the mammary gland where the reproductive hormones are major determinants of stem cell activation. In fact, in the mammary gland a special type of stem cells is determined only during pregnancy. Reproductive hormones act on hormone receptor positive cells, sensor cells, in the mammary epithelium to induce paracrine signaling that leads to activation of stem cells. Some of the downstream mediators are in common with other niches such as Wnt and possibly Notch signaling. Other signals are specific to the mammary gland such as amphiregulin and RANKL.

2008

Role of canonical Wnt signaling in mammary gland development

Renuga Devi Rajaram

The female reproductive hormones estrogens, progesterone, and prolactin control postnatal breast development and are important to breast carcinogenesis. In particular, exposure to progesterone is tightly linked to breast cancer risk. Using the mouse as a model system to study the mechanisms through which hormones elicit morphogenetic changes in the mammary gland in vivo, we demonstrated that progesterone acts by a paracrine mechanism and identified Wnt4 as a target of progesterone important in mediating side branch formation. The mechanisms underlying Wnt4 function remains poorly understood. To understand the pattern of canonical Wnt signaling activation during mammary gland development and to address which cellular compartment of the mammary gland is activated by canonical Wnt signaling, a reporter mouse model was utilized in which the lacZ gene is under the control of endogenous Axin2 promoter, classical target promoter of canonical Wnt signaling. Analysis of reporter mice at different developmental stages of mammary gland development showed the highest reporter activity during early pregnancy which correlated with the time of side branch formation. At this stage, β-galactosidase activity was found exclusively in myoepithelial cells. Overexpression of Wnt1 from MMTV promoter increases reporter activity in the myoepithelium. Gene expression analysis on FACS sorted luminal and myoepithelial cells shows that known canonical Wnt signaling pathway components such as receptors and co receptors as well as targets are specifically expressed in the myoepithelial compartment further supporting the notion that myoepithelial cells are the target cells for Wnt1 and Wnt4 secreted by luminal cells. Consistent with a model whereby progesterone induced Wnt4 secretion results in canonical Wnt signaling activation, reporter activity was induced by progesterone treatment and lacZ expression increased during diestrus when serum progesterone levels are high under physiological conditions. Analysis of contralateral mammary glands engrafted with PR-/-.Axin2 lacZ and PR+/-.Axin2 lacZ epithelium during pregnancy revealed that intact PR signaling is required to activate canonical Wnt signaling. Similar experiments with Wnt4-/-.Axin2 lacZ and Wnt4+/+.Axin2 lacZ mammary epithelium showed that reporter activation relies on Wnt4. Histological analysis on mammary glands in which β-catenin was ablated using MMTV-Cre revealed that β-catenin deleted cells were present only in the luminal epithelial compartment and showed that β-catenin deleted myoepithelial cells cannot participate in side branch formation. Similarly, ectopic expression of dominant negative β-catenin in wt MECs reduced side branching indicating that canonical Wnt signaling mediated by β-catenin is required for side branching. Furthermore, constitutive activation of β-catenin in the mammary epithelium was sufficient to induce side branch formation in virgin mice. In Wnt4 mutant epithelium, proliferation of progesterone receptor positive and negative cells were significantly reduced in response to progesterone treatment. Wnt signaling has been implicated in stem/progenitor activation in several tissues including mammary gland. Consistent with Wnt4 activating stem/progenitors, serial transplantation assays revealed that Wnt4-/- epithelium failed to reconstitute already at 2nd to 3rd generation when the wt has the ability to reconstitute up to seven generations indicating that Wnt4 is required for stem cell function while Wnt4 is dispensable for embryonic and rudimentary mammary gland formation. Histology and gene expression analysis in late pregnancy showed that Wnt4 mutant epithelia were able to differentiate and produce milk proteins, further supporting the thought that Wnt4 function is important for stem/progenitor cell activity and not required for differentiation. Taken together, I report that Wnt4 induces canonical Wnt signaling in myoepithelial cells and activation of canonical Wnt signaling is required and sufficient for side branching. Moreover, Wnt4 function is essential for mammary stem/progenitor cells.

EPFL2010

Adamts18 Function in Mammary Gland Development

Dalya Ataca

Ovarian hormones control mammary gland development and impinge on breast carcinogenesis acting via their cognate receptors in the luminal epithelium. The extracellular matrix has a key role in physiology and disease but how the two are linked is poorly understood. Here we show that deletion of the secreted protease Adamts18 delays mammary gland development. The proteaseâs role is mammary epithelial intrinsic and required for stem cell function and epithelial regeneration potential. Progesterone controls Adamts18 in the myoepithelium through upregulation of Wnt-4 expression with ensuing canonical Wnt signaling activation in basal cells. IP-Mass Spec shows that ADAMTS18 has binding partners located in the ECM and basement membrane. Consistent with Adamts18 being an important regulator of ECM function, deletion results in increased Collagen I and IV, Laminin, and Fibronectin deposition and synergizes genetically with the basal membrane proteoglycan Collagen18a1 in controlling mammary stem cell function. In vitro, Collagen18a1 and Fibronectin are cleaved by Adamts18. RNAseq analysis reveals that the stem cell regulatory hippo pathway is downstream of Adamts18 with consequent reduction of Fgfr2 expression. Our findings link hormonal signaling in luminal mammary epithelial cells to ECM remodeling via Adamts18, and demonstrate the importance of the ECM for stem cell function in the mammary gland.

EPFL2019

Role of canonical Wnt signaling in mammary gland development

Renuga Devi Rajaram

EPFL2010