Terminal end bud maintenance in mammary gland is dependent upon FGFR2b signaling

We previously demonstrated that Fibroblast Growth Factor 10 (FGF10) and its receptor FGFR2b play a key role in controlling the very early stages of mammary gland development during embryogenesis [Mailleux, A.A., Spencer-Dene, B., Dillon, C., Ndiaye, D., Savona-Baron, C., Itoh, N., Kato, S., Dickson, C., Thiery, J.P., and Bellusci, S. (2002). Role of FGF10/FGFR2b signaling during mammary gland development in the mouse embryo. Development 129, 53-60. Veltmaat, J. M., Relaix, F., Le, L.T., Kratochwil, K., Sala, F.G., van Veelen, W., Rice, R., Spencer-Dene, B., Mailleux, A.A., Rice, D.P., Thiery, J.P., and Bellusci, S. (2006). Gli3-mediated somitic Fgf10 expression gradients are required for the induction and patterning of mammary epithelium along the embryonic axes. Development 133, 2325-35.]. However, the role of FGFR2b signaling in postnatal mammary gland development is still elusive. We show that FGF10 is expressed at high level throughout the adipose tissue in the mammary gland of young virgin female mice whereas its main receptor FGFR2 is found mostly in the epithelium. Using a rtTA transactivator/tetracycline promoter approach allowing inducible and reversible attenuation of the FGFR2b signaling throughout the adult mouse, we are now reporting that FGFR2b signaling is also critical during postnatal mammary gland development. Ubiquitous attenuation of FGFR2b signaling in the postnatal mouse for 6 weeks starting immediately after birth is not lethal and leads to minor defects in the animal. Upon dissection of the mammary glands, a 40% reduction in size compared to the WT control is observed. Further examination shows a rudimentary mammary epithelial tree with completely absent terminal end buds (TEBs), compared to a well-branched structure observed in wild type. Transplantation of mammary gland explants into cleared fat pad of wild type mouse recipients indicates that the observed abnormal branching results from defective FGFR2b signaling in the epithelium. We also demonstrate that this rudimentary tree reforms TEBs and resumes branching upon removal of doxycycline suggesting that the regenerative capacities of the mammary epithelial progenitor cells were still functional despite long-term inactivation of the FGFR2b pathway. At the cellular level, upon FGFR2b attenuation, we show an increase in apoptosis associated with a decrease in the proliferation of the mammary luminal epithelium. We conclude that during puberty, there is a differential requirement for FGFR2b signaling in ductal vs. TEBs epithelium. FGFR2b signaling is crucial for the survival and proliferation of the mammary luminal epithelial cells, but does not affect the regenerative potential of the mammary epithelial progenitor cells.     

FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis

Branching morphogenesis of mouse submandibular glands is regulated by multiple growth factors. Here, we report that ex vivo branching of intact submandibular glands decreases when either FGFR2 expression is downregulated or soluble recombinant FGFR2b competes out the endogenous growth factors. However, a combination of neutralizing antibodies to FGF1, FGF7 and FGF10 is required to inhibit branching in the intact gland, suggesting that multiple FGF isoforms are required for branching. Exogenous FGFs added to submandibular epithelial rudiments cultured without mesenchyme induce distinct morphologies. FGF7 induces epithelial budding, whereas FGF10 induces duct elongation, and both are inhibited by FGFR or ERK1/2 signaling inhibitors. However, a PI3-kinase inhibitor also decreases FGF7-mediated epithelial budding, suggesting that multiple signaling pathways exist. We immunolocalized FGF receptors and analyzed changes in FGFR, FGF and MMP gene expression to identify the mechanisms of FGF-mediated morphogenesis. FGFR1b and FGFR2b are present throughout the epithelium, although FGFR1b is more highly expressed around the periphery of the buds and the duct tips. FGF7 signaling increases FGFR1b and FGF1 expression, and MMP2 activity, when compared with FGF10, resulting in increased cell proliferation and expansion of the epithelial bud, whereas FGF10 stimulates localized proliferation at the tip of the duct. FGF7- and FGF10-mediated morphogenesis is inhibited by an MMP inhibitor and a neutralizing antibody to FGF1, suggesting that both FGF1 and MMPs are essential downstream mediators of epithelial morphogenesis. Taken together, our data suggests that FGFR2b signaling involves a regulatory network of FGFR1b/FGF1/MMP2 expression that mediates budding and duct elongation during branching morphogenesis.     

Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and Mammary Epithelial Cells

Expression of the heparin-binding growth factor, pleiotrophin (PTN) in the mammary gland has been reported but its function during mammary gland development is not known. We examined the expression of PTN and its receptor ALK (Anaplastic Lymphoma Kinase) at various stages of mouse mammary gland development and found that their expression in epithelial cells is regulated in parallel during pregnancy. A 30-fold downregulation of PTN mRNA expression was observed during mid-pregnancy when the mammary gland undergoes lobular-alveolar differentiation. After weaning of pups, PTN expression was restored although baseline expression of PTN was reduced significantly in mammary glands of mice that had undergone multiple pregnancies. We found PTN expressed in epithelial cells of the mammary gland and thus used a monoclonal anti-PTN blocking antibody to elucidate its function in cultured mammary epithelial cells (MECs) as well as during gland development. Real-time impedance monitoring of MECs growth, migration and invasion during anti-PTN blocking antibody treatment showed that MECs motility and invasion but not proliferation depend on the activity of endogenous PTN. Increased number of mammospheres with laminin deposition after anti-PTN blocking antibody treatment of MECs in 3D culture and expression of progenitor markers suggest that the endogenously expressed PTN inhibits the expansion and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In vivo, PTN activity was found to inhibit ductal outgrowth and branching via the inhibition of phospho ERK1/2 signaling in the mammary epithelial cells. We conclude that PTN delays the maturation of the mammary gland by maintaining mammary epithelial cells in a progenitor phenotype and by inhibiting their differentiation during mammary gland development.     

Coordination of epithelial branching and salivary gland lumen formation by Wnt and FGF signals

Branching morphogenesis is a molecularly conserved mechanism that is adopted by several organs, such as the lung, kidney, mammary gland and salivary gland, to maximize the surface area of a tissue within a small volume. Branching occurs through repetitive clefting and elongation of spherical epithelial structures, called endbuds, which invade the surrounding mesenchyme. In the salivary gland, lumen formation takes place alongside branching morphogenesis, but in a controlled manner, so that branching is active at the distal ends of epithelial branches while lumen formation initiates at the proximal ends, and spreads distally. We present here data showing that interaction between FGF signaling and the canonical (β-catenin dependent) and non-canonical branches of Wnt signaling coordinates these two processes. Using the Axin2^lacZ reporter mice, we find Wnt/β-catenin signaling activity first in the mesenchyme and later, at the time of lumen formation, in the ductal epithelium. Gain and loss of function experiments reveal that this pathway exerts an inhibitory effect on salivary gland branching morphogenesis. We have found that endbuds remain devoid of Wnt/β-catenin signaling activity, a hallmark of ductal structures, through FGF-mediated inhibition of this pathway. Our data also show that FGF signaling has a major role in the control of lumen formation by preventing premature hollowing of epithelial endbuds and slowing down the canalization of presumptive ducts. Concomitantly, FGF signaling strongly represses the ductal marker Cp2l1, most likely via repression of Wnt5b and non-canonical Wnt signaling. Inhibition of canonical and non-canonical Wnt signaling in endbuds by FGF signaling occurs at least in part through sFRP1, a secreted inhibitor of Wnt signaling and downstream target of FGF signaling. Altogether, these findings point to a key function of FGF signaling in the maintenance of an undifferentiated state in endbud cells by inhibition of a ductal fate.     

Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation

Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43^−/− salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43^−/− samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43^−/− phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.     

The Wnt Co-Receptor Lrp6 Is Required for Normal Mouse Mammary Gland Development

Canonical Wnt signals are transduced through a Frizzled receptor and either the LRP5 or LRP6 co-receptor; such signals play central roles during development and in disease. We have previously shown that Lrp5 is required for ductal stem cell activity and that loss of Lrp5 delays normal mammary development and Wnt1-induced tumorigenesis. Here we show that canonical Wnt signals through the Lrp6 co-receptor are also required for normal mouse mammary gland development. Loss of Lrp6 compromises Wnt/β-catenin signaling and interferes with mammary placode, fat pad, and branching development during embryogenesis. Heterozygosity for an inactivating mutation in Lrp6 is associated with a reduced number of terminal end buds and branches during postnatal development. While Lrp6 is expressed in both the basal and luminal mammary epithelium during embryogenesis, Lrp6 expression later becomes restricted to cells residing in the basal epithelial layer. Interestingly, these cells also express mammary stem cell markers. In humans, increased Lrp6 expression is associated with basal-like breast cancer. Taken together, our results suggest both overlapping and specific functions for Lrp5 and Lrp6 in the mammary gland.     

Expression and Functional Role of Sprouty-2 in Breast Morphogenesis

Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2) in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting SPRY2. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D) culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD) resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an important regulator of branching morphogenesis and epithelial to mesenchymal transition in the mammary gland.     

Role of FGF10/FGFR2b signaling during mammary gland development in the mouse embryo.

The mouse develops five pairs of mammary glands that arise during mid-gestation from five pairs of placodes of ectodermal origin. We have investigated the molecular mechanisms of mammary placode development using Lef1 as a marker for the epithelial component of the placode, and mice deficient for Fgf10 or Fgfr2b, both of which fail to develop normal mammary glands. Mammary placode induction involves two different signaling pathways, a FGF10/FGFR2b-dependent pathway for placodes 1, 2, 3 and 5 and a FGF10/FGFR2b-independent pathway for placode 4. Our results also suggest that FGF signaling is involved in the maintenance of mammary bud 4, and that Fgf10 deficient epithelium can undergo branching morphogenesis into the mammary fat pad precursor.     

Genetic mosaic analysis reveals FGF receptor 2 function in terminal end buds during mammary gland branching morphogenesis

FGF signaling is associated with breast cancer and is required for mammary placode formation in the mouse. In this study, we employed a genetic mosaic analysis based on Cre-mediated recombination to investigate FGF receptor 2 (Fgfr2) function in the postnatal mammary gland. Mosaic inactivation of Fgfr2 by the MMTV-Cre transgene enabled us to compare the behavior of Fgfr2 null and Fgfr2 heterozygous cells in the same gland. Fgfr2 null cells were at a competitive disadvantage to their Fgfr2 heterozygous neighbors in the highly proliferative terminal end buds (TEBs) at the invasion front, owing to a negative effect of loss of Fgfr2 function on cell proliferation. However, Fgfr2 null cells were tolerated in mature ducts. In these genetic mosaic mammary glands, the epithelial network is apparently built by TEBs that over time are composed of a progressively larger proportion of Fgfr2-positive cells. However, subsequently, most cells lose Fgfr2 function, presumably due to additional rounds of Cre-mediated recombination. Using an independent strategy to create mosaic mammary glands, which employed an adenovirus-Cre that acts only once, we confirmed that Fgfr2 null cells were out-competed by neighboring Fgfr2 heterozygous cells. Together, our data demonstrate that Fgfr2 functions in the proliferating and invading TEBs, but it is not required in the mature ducts of the pubertal mammary gland.     

FGF10: A multifunctional mesenchymal–epithelial signaling growth factor in development,health, and disease

The FGF family comprises 22 members with diverse functions in development and health. FGF10 specifically activates FGFR2b in a paracrine manner with heparan sulfate as a co-factor. FGF10and FGFR2b are preferentially expressed in the mesenchyme and epithelium, respectively. FGF10 is a mesenchymal signaling molecule in the epithelium. FGF10 knockout mice die shortly after birth due to the complete absence of lungs as well as fore- and hindlimbs. FGF10 is also essential for the development of multiple organs. The phenotypes of Fgf10 knockout mice are very similar to those of FGFR2b knockout mice, indicating that FGF10 acts as a ligand that is specific to FGFR2b in mouse multi-organ development. FGF10 also plays roles in epithelial–mesenchymal transition, the repair of tissue injury, and embryonic stem cell differentiation. In humans, FGF10 loss-of-function mutations result in inherited diseases including aplasia of lacrimal and salivary gland, lacrimo-auriculo-dento-digital syndrome, and chronic obstructive pulmonary disease. FGF10 is also involved in the oncogenicity of pancreatic and breast cancers. Single nucleotide polymorphisms in FGF10 are also potential risk factors for limb deficiencies, cleft lip and palate, and extreme myopia. These findings indicate that FGF10 is a crucial paracrine signal from the mesenchyme to epithelium for development, health, and disease.     

Tumor Suppressor Function of Syk in Human MCF10A In Vitro and Normal Mouse Mammary Epithelium In Vivo

The normal function of Syk in epithelium of the developing or adult breast is not known, however, Syk suppresses tumor growth, invasion, and metastasis in breast cancer cells. Here, we demonstrate that in the mouse mammary gland, loss of one Syk allele profoundly increases proliferation and ductal branching and invasion of epithelial cells through the mammary fat pad during puberty. Mammary carcinomas develop by one year. Syk also suppresses proliferation and invasion in vitro. siRNA or shRNA knockdown of Syk in MCF10A breast epithelial cells dramatically increased proliferation, anchorage independent growth, cellular motility, and invasion, with formation of functional, extracellular matrix-degrading invadopodia. Morphological and gene microarray analysis following Syk knockdown revealed a loss of luminal and differentiated epithelial features with epithelial to mesenchymal transition and a gain in invadopodial cell surface markers CD44, CD49F, and MMP14. These results support the role of Syk in limiting proliferation and invasion of epithelial cells during normal morphogenesis, and emphasize the critical role of Syk as a tumor suppressor for breast cancer. The question of breast cancer risk following systemic anti-Syk therapy is raised since only partial loss of Syk was sufficient to induce mammary carcinomas.     

mTOR Directs Breast Morphogenesis through the PKC-alpha-Rac1 Signaling Axis

Akt phosphorylation is a major driver of cell survival, motility, and proliferation in development and disease, causing increased interest in upstream regulators of Akt like mTOR complex 2 (mTORC2). We used genetic disruption of Rictor to impair mTORC2 activity in mouse mammary epithelia, which decreased Akt phosphorylation, ductal length, secondary branching, cell motility, and cell survival. These effects were recapitulated with a pharmacological dual inhibitor of mTORC1/mTORC2, but not upon genetic disruption of mTORC1 function via Raptor deletion. Surprisingly, Akt re-activation was not sufficient to rescue cell survival or invasion, and modestly increased branching of mTORC2-impaired mammary epithelial cells (MECs) in culture and in vivo. However, another mTORC2 substrate, protein kinase C (PKC)-alpha, fully rescued mTORC2-impaired MEC branching, invasion, and survival, as well as branching morphogenesis in vivo. PKC-alpha-mediated signaling through the small GTPase Rac1 was necessary for mTORC2-dependent mammary epithelial development during puberty, revealing a novel role for Rictor/mTORC2 in MEC survival and motility during branching morphogenesis through a PKC-alpha/Rac1-dependent mechanism.     

IRE1 Phosphatase PP2Ce Regulates Adaptive ER Stress Response in the Postpartum Mammary Gland

We recently reported that the PPM1l gene encodes an endoplasmic reticulum (ER) membrane targeted protein phosphatase (named PP2Ce) with highly specific activity towards Inositol-requiring protein-1 (IRE1) and regulates the functional outcome of ER stress. In the present report, we found that the PP2Ce protein is highly expressed in lactating epithelium of the mammary gland. Loss of PP2Ce in vivo impairs physiological unfolded protein response (UPR) and induces stress kinase activation, resulting in loss of milk production and induction of epithelial apoptosis in the lactating mammary gland. This study provides the first in vivo evidence that PP2Ce is an essential regulator of normal lactation, possibly involving IRE1 signaling and ER stress regulation in mammary epithelium.     

Site-specific inductive and inhibitory activities of MMP-2 and MMP-3 orchestrate mammary gland branching morphogenesis

During puberty, mouse mammary epithelial ducts invade the stromal mammary fat pad in a wave of branching morphogenesis to form a complex ductal tree. Using pharmacologic and genetic approaches, we find that mammary gland branching morphogenesis requires transient matrix metalloproteinase (MMP) activity for invasion and branch point selection. MMP-2, but not MMP-9, facilitates terminal end bud invasion by inhibiting epithelial cell apoptosis at the start of puberty. Unexpectedly, MMP-2 also represses precocious lateral branching during mid-puberty. In contrast, MMP-3 induces secondary and tertiary lateral branching of ducts during mid-puberty and early pregnancy. Nevertheless, the mammary gland is able to develop lactational competence in MMP mutant mice. Thus, specific MMPs refine the mammary branching pattern by distinct mechanisms during mammary gland branching morphogenesis.     

Inducible dimerization of FGFR1: development of a mouse model to analyze progressive transformation of the mammary gland

To develop an inducible and progressive model of mammary gland tumorigenesis, transgenic mice were generated with a mouse mammary tumor virus-long terminal repeat-driven, conditional, fibroblast growth factor (FGF)-independent FGF receptor (FGFR)1 (iFGFR1) that can be induced to dimerize with the drug AP20187. Treatment of transgenic mice with AP20187 resulted in iFGFR1 tyrosine phosphorylation, increased proliferation, activation of mitogen-activated protein kinase and Akt, and lateral budding. Lateral buds appeared as early as 3 d after AP20187 treatment and initially consisted of bilayered epithelial cells and displayed apical and basolateral polarity appeared after 13 d of AP20187 treatment. Invasive lesions characterized by multicell-layered lateral buds, decreased myoepithelium, increased vascular branching, and loss of cell polarity were observed after 2-4 wk of treatment. These data indicate that acute iFGFR1 signaling results in increased lateral budding of the mammary ductal epithelium, and that sustained activation induces alveolar hyperplasia and invasive lesions.     

Fibroblast Growth Factor 9 Regulation by MicroRNAs Controls Lung Development and Links DICER1 Loss to the Pathogenesis of Pleuropulmonary Blastoma

Pleuropulmonary Blastoma (PPB) is the primary neoplastic manifestation of a pediatric cancer predisposition syndrome that is associated with several diseases including cystic nephroma, Wilms tumor, neuroblastoma, rhabdomyosarcoma, medulloblastoma, and ovarian Sertoli-Leydig cell tumor. The primary pathology of PPB, epithelial cysts with stromal hyperplasia and risk for progression to a complex primitive sarcoma, is associated with familial heterozygosity and lesion-associated epithelial loss-of-heterozygosity of DICER1. It has been hypothesized that loss of heterozygosity of DICER1 in lung epithelium is a non-cell autonomous etiology of PPB and a critical pathway that regulates lung development; however, there are no known direct targets of epithelial microRNAs (miRNAs) in the lung. Fibroblast Growth Factor 9 (FGF9) is expressed in the mesothelium and epithelium during lung development and primarily functions to regulate lung mesenchyme; however, there are no known mechanisms that regulate FGF9 expression during lung development. Using mouse genetics and molecular phenotyping of human PPB tissue, we show that FGF9 is overexpressed in lung epithelium in the initial multicystic stage of Type I PPB and that in mice lacking epithelial Dicer1, or induced to overexpress epithelial Fgf9, increased Fgf9 expression results in pulmonary mesenchymal hyperplasia and a multicystic architecture that is histologically and molecularly indistinguishable from Type I PPB. We further show that miR-140 is expressed in lung epithelium, regulates epithelial Fgf9 expression, and regulates pseudoglandular stages of lung development. These studies identify an essential miRNA-FGF9 pathway for lung development and a non-cell autonomous signaling mechanism that contributes to the mesenchymal hyperplasia that is characteristic of Type I PPB.     

Persistence of responsiveness of adult mouse mammary gland to induction by embryonic mesenchyme

Persistence of the capacity for embryogenic morphogenesis in adult mammary epithelium was demonstrated by allowing it to interact with grafted embryonic mesenchyme in vivo. When 14-day embryonic mammary or salivary mesenchyme was transplanted in the mammary gland of syngeneic young adult virgin mice, organogenetic development of the mammary epithelial cells occurred responding to closely attached mesenchyme. An early change, within 2–4 days, that was observed equally in both types of the mesenchymes was proliferation of mammary epithelial cells in multiple layers resembling rudimental architecture. Subsequently, ductal branching occurred from the rudimental architecture by mesenchyme-dependent branching pattern, of mammary gland type with mammary mesenchyme and of salivary gland-like type with salivary mesenchyme. This developmental response did not require hormones secreted from ovaries since it was observed similarly in ovariectomized mice. The mammary epithelium at the lactating stage did not show such a potential to the transplanted salivary mesenchyme.