Minoti Hiremath

Parathyroid Hormone-Related Protein Activates Wnt Signaling to Specify the Embryonic Mammary Mesenchyme

Minoti Hiremath et al. — Fri, 30 Nov 2012 07:50:16 PST

Parathyroid hormone-related protein (PTHrP) regulates cell fate and specifies the mammary mesenchyme during embryonic development. Loss of PTHrP or its receptor (Pthr1) abolishes the expression of mammary mesenchyme markers and allows mammary bud cells to revert to an epidermal fate. By contrast, overexpression of PTHrP in basal keratinocytes induces inappropriate differentiation of the ventral epidermis into nipple-like skin and is accompanied by ectopic expression of Lef1, β-catenin and other markers of the mammary mesenchyme. In this study, we document that PTHrP modulates Wnt/β-catenin signaling in the mammary mesenchyme using a Wnt signaling reporter, TOPGAL-C. Reporter expression is completely abolished by loss of PTHrP signaling and ectopic reporter activity is induced by overexpression of PTHrP. We also demonstrate that loss of Lef1, a key component of the Wnt pathway, attenuates the PTHrP-induced abnormal differentiation of the ventral skin. To characterize further the contribution of canonical Wnt signaling to embryonic mammary development, we deleted β-catenin specifically in the mammary mesenchyme. Loss of mesenchymal β-catenin abolished expression of the TOPGAL-C reporter and resulted in mammary buds with reduced expression of mammary mesenchyme markers and impaired sexual dimorphism. It also prevented the ectopic, ventral expression of mammary mesenchyme markers caused by overexpression of PTHrP in basal keratinocytes. Therefore, we conclude that a mesenchymal, canonical Wnt pathway mediates the PTHrP-dependent specification of the mammary mesenchyme.

Parathyroid Hormone-Related Protein is Not Required for Normal Ductal or Alveolar Development in the Post-Natal Mammary Gland

Kata Boras-Granic et al. — Fri, 06 Jan 2012 12:42:18 PST

PTHrP is necessary for the formation of the embryonic mammary gland and, in its absence, the embryonic mammary bud fails to form the neonatal duct system. In addition, PTHrP is produced by the breast during lactation and contributes to the regulation of maternal calcium homeostasis during milk production. In this study, we examined the role of PTHrP during post-natal mammary development. Using a PTHrP-lacZ transgenic mouse, we surveyed the expression of PTHrP in the developing post-natal mouse mammary gland. We found that PTHrP expression is restricted to the basal cells of the gland during pubertal development and becomes expressed in milk secreting alveolar cells during pregnancy and lactation. Based on the previous findings that overexpression of PTHrP in cap and myoepithelial cells inhibited ductal elongation during puberty, we predicted that ablation of native PTHrP expression in the post-natal gland would result in accelerated ductal development. To address this hypothesis, we generated two conditional models of PTHrP-deficiency specifically targeted to the postnatal mammary gland. We used the MMTV-Cre transgene to ablate the floxed PTHrP gene in both luminal and myoepithelial cells and a tetracycline-regulated K14-tTA;tetO-Cre transgene to target PTHrP expression in just myoepithelial and cap cells. In both models of PTHrP ablation, we found that mammary development proceeds normally despite the absence of PTHrP. We conclude that PTHrP signaling is not required for normal ductal or alveolar development.

PTHrP-Induced Wnt Signaling Plays a Role in Specification of the Mammary Mesenchyme

M. Hiremath et al. — Wed, 27 Apr 2011 15:27:52 PDT

PTHrP regulates mammary cell fate by specifying the mammary mesenchyme during embryonic mammary development. Loss of PTHrP or its receptor allows mammary bud cells to adopt an epidermal fate, whereas, overexpression of PTHrP in the epidermis induces inappropriate differentiation of the ventral epidermis into nipple-like skin. Previous studies have suggested that the Wnt pathway is one of the downstream pathways activated by PTHrP in specifying the mammary mesenchyme. We confirmed that PTHrP modulates Wnt signaling in the mammary mesenchyme using a reporter of Wnt signaling, TOPGAL-C. Reporter expression is completely abolished by loss of PTHrP signaling and ectopic mesenchymal Wnt signaling is induced by overexpression of PTHrP. To further investigate the requirement for Wnt signaling, we examined mutant mice overexpressing PTHrP but lacking Lef1, an essential component and target of the Wnt pathway. Our observations suggest that loss of Lef1 partially reverses the nipple-like differentiation of the ventral skin. Therefore, we concluded that PTHrP-driven mammary mesenchyme specification is partially mediated by Wnt signaling. Surprisingly, ectopic TOPGAL-C expression is maintained in K14-PTHrP;Lef1-null mice. These data suggest that PTHrP induces a Wnt signal that activates Lef1 expression in the mesenchyme. To determine if Wnt signaling regulates mesenchyme specification and Lef1 expression, we deleted beta-catenin, the primary mediator of the canonical Wnt pathway, in the mammary mesenchyme. Reduced mesenchymal Wnt signaling correlates with abnormal bud morphology and reduced expression of mammary mesenchyme markers. To identify other Wnt pathway components that are regulated by PTHrP during mammary mesenchyme specification, we performed microarray analyses of RNA from ventral skins of WT and K14-PTHrP mice and from RNA extracted from mammary buds of PTHrP-null and WT mice. We identified genes that are upregulated in PTHrP-overexpressing ventral skin, compared to WT, and genes that are downregulated in PTHrP-null mammary buds, compared to WT, as candidate genes that are regulated by PTHrP. Of these, we are currently investigating RSPO1 and WNT11 as Wnt pathway regulators that potentially act downstream of PTHrP during mesenchyme specification.

A Mouse Transgenic Approach to Induce β-Catenin Signaling in a Temporally Controlled Manner

Atish Mukherjee et al. — Wed, 27 Apr 2011 15:15:15 PDT

Although constitutive murine transgenic models have provided important insights into β-catenin signaling in tissue morphogenesis and tumorigenesis, these models are unable to express activated β-catenin in a temporally controlled manner. Therefore, to enable the induction (and subsequent de-induction) of β-catenin signaling during a predetermined time-period or developmental stage, we have generated and characterized a TETO-ΔN89β-catenin responder transgenic mouse. Crossed with the MTB transgenic effector mouse, which targets the expression of the reverse tetracycline transactivator (rtTA) to the mammary epithelium, we demonstrate that the stabilized (and activated) form of β-catenin (ΔN89β-catenin) is expressed only in the presence doxycycline-activated rtTA in the mammary epithelial compartment. Furthermore, we show that transgene-derived ΔN89β-catenin elicits significant mammary epithelial proliferation and precocious alveologenesis in the virgin doxycycline-treated MTB/TETO-ΔN89β-catenin bitransgenic. Remarkably, deinduction of TETO-ΔN89β-catenin transgene expression (through doxycycline withdrawal) results in the reversal of these morphological changes. Importantly, continued activation of the TETO-ΔN89β-catenin transgene results in palpable mammary tumors (within 7–9 months) in the doxycycline-treated virgin MTB/TETO-ΔN89β-catenin bigenic but not in the same bitransgenic without doxycycline administration. Collectively, these mammary epithelial responses to ΔN89β-catenin expression agree with previous reports using conventional transgenesis and therefore confirm that ΔN89β-catenin functions as expected in this doxycycline-responsive bigenic system. In sum, our mammary gland studies demonstrate “proof-of-principle” for using the TETO-ΔN89β-catenin transgenic responder to activate (and then de-activate) β-catenin signaling in any tissue of interest in a spatiotemporal specific fashion.

β-Catenin and Tcfs in Mammary Development and Cancer

Sarah Hatsell et al. — Wed, 27 Apr 2011 15:15:13 PDT

β-Catenin regulates cell–cell adhesion and transduces signals from many pathways to regulate the transcriptional activities of Tcf/Lef DNA binding factors. Gene ablation and transgenic expression studies strongly support the concept that β-catenin together with Lef/Tcf factors act as a switch to determine cell fate and promote cell survival and proliferation at several stages during mammary gland development. Mice expressing the negative regulator of Wnt/β-catenin signaling (K14-Dkk) fail to form mammary buds, and those lacking Lef-1 show an early arrest in this process at stage E13.5. Stabilized N89 β-catenin initiates precocious alveologenesis during pubertal development, and negative regulators of endogenous β-catenin signaling suppress normal alveologenesis during pregnancy. Stabilized β-catenin induces hyperplasia and mammary tumors in mice. Each of the β-catenin-induced phenotypes is accompanied by upregulation of the target genes cyclin D1 and c-myc. Cyclin D1, however, is dispensable for tumor formation and the initiation of alveologenesis but is essential for later alveolar expansion.

Analysis of Gene Expression in PTHrP−/− Mammary Buds Supports a Role for BMP Signaling and MMP2 in the Initiation of Ductal Morphogenesis

Julie Hens et al. — Wed, 27 Apr 2011 15:15:12 PDT

Parathyroid hormone–related protein (PTHrP) acts on the mammary mesenchyme and is required for proper embryonic mammary development. In order to understand PTHrP's effects on mesenchymal cells, we profiled gene expression in WT and PTHrP−/− mammary buds, and in WT and K14-PTHrP ventral skin at E15.5. By cross-referencing the differences in gene expression between these groups, we identified 35 genes potentially regulated by PTHrP in the mammary mesenchyme, including 6 genes known to be involved in BMP signaling. One of these genes was MMP2. We demonstrated that PTHrP and BMP4 regulate MMP2 gene expression and MMP2 activity in mesenchymal cells. Using mammary bud cultures, we demonstrated that MMP2 acts downstream of PTHrP to stimulate ductal outgrowth. Future studies on the functional role of other genes on this list should expand our knowledge of how PTHrP signaling triggers the onset of ductal outgrowth from the embryonic mammary buds.

MMTV-Wnt1 and -DN89b-Catenin Induce Canonical Signaling in Distinct Progenitors and Differentially Activate Hedgehog Signaling within Mammary Tumors

Brigitte Teissedre et al. — Wed, 27 Apr 2011 15:15:10 PDT

Canonical Wnt/b-catenin signaling regulates stem/progenitor cells and, when perturbed, induces many human cancers. A significant proportion of human breast cancer is associated with loss of secreted Wnt antagonists and mice expressing MMTVWnt1 and MMTV-DN89b-catenin develop mammary adenocarcinomas. Many studies have assumed these mouse models of breast cancer to be equivalent. Here we show that MMTV-Wnt1 and MMTV-DN89b-catenin transgenes induce tumors with different phenotypes. Using axin2/conductin reporter genes we show that MMTV-Wnt1 and MMTV-DN89b-catenin activate canonical Wnt signaling within distinct cell-types. DN89b-catenin activated signaling within a luminal subpopulation scattered along ducts that exhibited a K18+ER2PR2CD24highCD49flow profile and progenitor properties. In contrast, MMTV-Wnt1 induced canonical signaling in K14+ basal cells with CD24/CD49f profiles characteristic of two distinct stem/progenitor cell-types.MMTVWnt1 produced additional profound effects on multiple cell-types that correlated with focal activation of the Hedgehog pathway. We document that largemelanocytic nevi are a hitherto unreported hallmark of early hyperplasticWnt1 glands. These nevi formed along the primary mammary ducts and were associated with Hedgehog pathway activity within a subset of melanocytes and surrounding stroma. Hh pathway activity also occurred within tumor-associated stromal and K14+/p63+ subpopulations in a manner correlated with Wnt1 tumor onset. These data show MMTV-Wnt1 and MMTV-DN89b-catenin induce canonical signaling in distinct progenitors and that Hedgehog pathway activation is linked to melanocytic nevi and mammary tumor onset arising from excess Wnt1 ligand. They further suggest that Hedgehog pathway activation maybe a critical component and useful indicator of breast tumors arising from unopposed Wnt1 ligand.

Molecular, Cellular, and Developmental Biology of Breast Cancer

S. J. Hatsell et al. — Wed, 27 Apr 2011 15:15:09 PDT

Cadherins in Metastasis

Minoti Hiremath et al. — Wed, 27 Apr 2011 15:15:07 PDT

Cadherins participate in calcium-dependent cell-cell adhesion. Although numerous models have been proposed for cadherin-cadherin interactions, the exact mechanism remains elusive. Structural and mutational analyses have emphasized the importance of the N-terminus and the HAV domain, and have determined that specificity of cadherin interaction is determined by amino acids in the 1st cadherin repeat. E-cadherin expression is irreversibly lost in invasive lobular cancer (ILC) and epigenetic alterations in cadherins frequently accompany ductal breast carcinomas. Animal models of lobular cancer provide mechanistic insight into the role of E-cadherin as a tumor and invasion suppressor in ILC. Transcriptional regulators of E-cadherin that act as master regulators of epithelial-mesenchymal transition in development have been identified. Mis-expression of cadherins promotes a migratory phenotype in breast cancer. E-cadherin binding partners, such as β-catenin and p120^ctn, that participate in cell signaling, are lost or mislocalized in tumors lacking cadherins. In conclusion, cadherins act as suppressors of invasion and metastasis through multiple routes.

The Pattern Of β-Catenin-Responsiveness within the Mammary Gland is Regulated by Progesterone Receptor

Minoti Hiremath et al. — Wed, 27 Apr 2011 15:15:05 PDT

Experiments involving β-catenin loss- and gain-of-function in the mammary gland have decisively demonstrated the role of this protein in normal alveologenesis. However, the relationship between hormonal and β-catenin signaling has not been investigated. In this study, we demonstrate that activated β-catenin rescues alveologenesis in progesterone receptor (PR; Pgr)-null mice during pregnancy. Two distinct subsets of mammary cells respond to expression of ΔN89β-catenin. Cells at ductal tips are inherently β-catenin-responsive and form alveoli in the absence of PR. However, PR activity confers β-catenin responsiveness to progenitor cells along the lateral ductal borders in the virgin gland. Once activated by β-catenin, responding cells switch on an alveolar differentiation program that is indistinguishable from that observed in pregnancy and is curtailed by PR signaling.