Adriana P. Visbal

Altered differentiation and paracrine stimulation of mammary epithelial cell proliferation by conditionally activated Smoothened

The Hedgehog (Hh) signaling network is critical for patterning and organogenesis in mammals, and has been implicated in a variety of cancers. Smoothened (Smo), the gene encoding the principal signal transducer, is overexpressed frequently in breast cancer, and constitutive activation in MMTV-SmoM2 transgenic mice caused alterations in mammary gland morphology, increased proliferation, and changes in stem/progenitor cell number. Both in transgenic mice and in clinical specimens, proliferative cells did not usually express detectable Smo, suggesting the hypothesis that Smo functioned in a non-cell autonomous manner to stimulate proliferation. Here, we employed a genetically tagged mouse model carrying a Cre-recombinase-dependent conditional allele of constitutively active Smo (SmoM2) to test this hypothesis. MMTV-Cre- or adenoviral-Cre-mediated SmoM2 expression in the luminal epithelium, but not in the myoepithelium, was required for the hyper-proliferative phenotypes. High levels of proliferation were observed in cells adjacent or in close-proximity to Smo expressing cells demonstrating that SmoM2 expressing cells were stimulating proliferation via a paracrine or juxtacrine mechanism. In contrast, Smo expression altered luminal cell differentiation in a cell-autonomous manner. SmoM2 expressing cells, purified by fluorescence activated cell sorting (FACS) via the genetic fluorescent tag, expressed high levels of Ptch2, Gli1, Gli2, Jag2 and Dll-1, and lower levels of Notch4 and Hes6, in comparison to wildtype cells. These studies provide insight into the mechanism of Smo activation in the mammary gland and its possible roles in breast tumorigenesis. In addition, these results also have potential implications for the interpretation of proliferative phenotypes commonly observed in other organs as a consequence of hedgehog signaling activation.

CCAAT/Enhancer Binding Protein Beta Regulates Stem Cell Activity and Specifies Luminal Cell Fate in the Mammary Gland

The bZIP transcription factor C/EBPβ is important for mammary gland development and its expression is deregulated in human breast cancer. To determine whether C/EBPβ regulates mammary stem cells (MaSCs), we employed two different knockout strategies. Using both a germline and a conditional knockout strategy, we demonstrate that mammosphere formation was significantly decreased in C/EBPβ‐deficient mammary epithelial cells (MECs). Functional limiting dilution transplantation assays indicated that the repopulating ability of C/EBPβ‐deleted MECs was severely impaired. Serial transplantation experiments demonstrated that C/EBPβ deletion resulted in decreased outgrowth potential and premature MaSC senescence. In accord, fluorescence‐activated cell sorting analysis demonstrated that C/EBPβ‐null MECs contained fewer MaSCs, the loss of luminal progenitors and an increase in differentiated luminal cells as compared with wild‐type. Gene profiling of C/EBPβ‐null stem cells revealed an alteration in cell fate specification, exemplified by the expression of basal markers in the luminal compartment. Thus, C/EBPβ is a critical regulator of both MaSC repopulation activity and luminal cell lineage commitment. These findings have critical implications for understanding both stem cell biology and the etiology of different breast cancer subtypes. STEM CELLS 2010;28:535–544

Hedgehog signaling in the normal and neoplastic mammary gland.

The hedgehog signal transduction network is a critical regulator of metazoan development. Inappropriate activation of this network is implicated in several different cancers, including breast. Genetic evidence in mice as well as molecular biological studies in human cells clearly indicate that activated signaling can lead to mammary hyperplasia and, in some cases, tumor formation. However, the exact role(s) activated hedgehog signaling plays in the development or progression of breast cancer also remain unclear. In this review, we have discussed recent data regarding the mechanism(s) by which the hedgehog network may signal in the mammary gland, as well as the data implicating activated signaling as a contributing factor to breast cancer development. Finally, we provide a brief update on the available hedgehog signaling inhibitors with respect to ongoing clinical trials, some of which will include locally advanced or metastatic breast cancers. Given the growing intensity with which the hedgehog signaling network is being studied in the normal and neoplastic mammary gland, a more complete understanding of this network should allow more effective targeting of its activities in breast cancer treatment or prevention.

Methods for Preparing Fluorescent and Neutral Red-Stained Whole Mounts of Mouse Mammary Glands

Whole mount preparations of mouse mammary glands are useful for evaluating overall changes in growth and morphology, and are essential for detecting and evaluating focal or regionally-localized phenotypes that would be difficult to detect or analyze using other techniques. We present three newly developed methods for preparing whole mounts of mammary glands from genetically-engineered mice expressing fluorescent proteins, as well as using either neutral red or a variety of fluorescent dyes. Unlike traditional hematoxylin- or carmine-stained preparations, neutral red-stained and some fluorescent preparations can be used for several common downstream analyses.

P190A RhoGAP is required for mammary gland development

P190A and p190B Rho GTPase activating proteins (GAPs) are essential genes that have distinct, but overlapping roles in the developing nervous system. Previous studies from our laboratory demonstrated that p190B is required for mammary gland morphogenesis, and we hypothesized that p190A might have a distinct role in the developing mammary gland. To test this hypothesis, we examined mammary gland development in p190A-deficient mice. P190A expression was detected by in situ hybridization in the developing E14.5day embryonic mammary bud and within the ducts, terminal end buds (TEBs), and surrounding stroma of the developing virgin mammary gland. In contrast to previous results with p190B, examination of p190A heterozygous mammary glands demonstrated that p190A deficiency disrupted TEB morphology, but did not significantly delay ductal outgrowth indicating haploinsufficiency for TEB development. To examine the effects of homozygous deletion of p190A, embryonic mammary buds were rescued by transplantation into the cleared fat pads of SCID/Beige mice. Complete loss of p190A function inhibited ductal outgrowth in comparison to wildtype transplants (51% vs. 94% fat pad filled). In addition, the transplantation take rate of p190A deficient whole gland transplants from E18.5 embryos was significantly reduced compared to wildtype transplants (31% vs. 90%, respectively). These results suggest that p190A function in both the epithelium and stroma is required for mammary gland development. Immunostaining for p63 demonstrated that the myoepithelial cell layer is disrupted in the p190A deficient glands, which may result from the defective cell adhesion between the cap and body cell layers detected in the TEBs. The number of estrogen- and progesterone receptor-positive cells, as well as the expression levels of these receptors was increased in p190A deficient outgrowths. These data suggest that p190A is required in both the epithelial and stromal compartments for ductal outgrowth and that it may play a role in mammary epithelial cell differentiation.

An essential role for Gα(i2) in Smoothened-stimulated epithelial cell proliferation in the mammary gland.

Gαi2 mediates proliferation stimulated by Smoothened in the mammary gland. Smoothened signals through G proteins The Hedgehog signaling pathway promotes the expression of genes that are critical during development through regulation of the Gli family of transcription factors. Because this pathway is subverted by cancer cells, and because the gene encoding the Hedgehog effector Smoothened (SMO) is aberrantly expressed in some types of breast cancers, various chemotherapeutics have been developed to target SMO. Villanueva et al. (see also the Focus by Ogden) showed that SMO promoted cell proliferation in the mammary glands of mice through the G protein Gαi2, rather than through Gli-mediated changes in gene expression. These results confirm that SMO can act as a G protein–coupled receptor in mammals and suggest that SMO-targeting drugs should also be screened for their ability to inhibit Gαi2 activation. Hedgehog (Hh) signaling is critical for organogenesis, tissue homeostasis, and stem cell maintenance. The gene encoding Smoothened (SMO), the primary effector of Hh signaling, is expressed aberrantly in human breast cancer, as well as in other cancers. In mice that express a constitutively active form of SMO that does not require Hh stimulation in mammary glands, the cells near the transgenic cells proliferate and participate in hyperplasia formation. Although SMO is a seven-transmembrane receptor like G protein–coupled receptors (GPCRs), SMO-mediated activation of the Gli family of transcription factors is not known to involve G proteins. However, data from Drosophila and mammalian cell lines indicate that SMO functions as a GPCR that couples to heterotrimeric G proteins of the pertussis toxin (PTX)–sensitive Gαi class. Using genetically modified mice, we demonstrated that SMO signaling through G proteins occurred in the mammary gland in vivo. SMO-induced stimulation of proliferation was PTX-sensitive and required Gαi2, but not Gαi1, Gαi3, or activation of Gli1 or Gli2. Our findings show that activated SMO functions as a GPCR to stimulate proliferation in vivo, a finding that may have clinical importance because most SMO-targeted agents have been selected based largely on their ability to block Gli-mediated transcription.

Epithelial and non-epithelial Patched-1 (Ptch1) play opposing roles to regulate proliferation and morphogenesis of the mouse mammary gland

Patched 1 (Ptch1) has epithelial, stromal and systemic roles in murine mammary gland organogenesis, yet specific functions remain undefined. Cre-recombinase-mediated Ptch1 ablation in mammary epithelium increased proliferation and branching, but did not phenocopy transgenic expression of activated smoothened (SmoM2). The epithelium showed no evidence of canonical hedgehog signaling, and hyperproliferation was not blocked by smoothened (SMO) inhibition, suggesting a non-canonical function of PTCH1. Consistent with this possibility, nuclear localization of cyclin B1 was increased. In non-epithelial cells, heterozygous Fsp-Cre-mediated Ptch1 ablation increased proliferation and branching, with dysplastic terminal end buds (TEB) and ducts. By contrast, homozygous Ptch1 ablation decreased proliferation and branching, producing stunted ducts filled with luminal cells showing altered ovarian hormone receptor expression. Whole-gland transplantation into wild-type hosts or estrogen/progesterone treatment rescued outgrowth and hormone receptor expression, but not the histological changes. Bone marrow transplantation failed to rescue outgrowth. Ducts of Fsp-Cre;Ptch1fl/fl mice were similar to Fsp-Cre;SmoM2 ducts, but Fsp-Cre;SmoM2 outgrowths were not stunted, suggesting that the histology might be mediated by Smo in the local stroma, with systemic Ptch1 required for ductal outgrowth and proper hormone receptor expression in the mammary epithelium. Summary: Systemic and tissue-specific depletion of patched 1 in epithelial and stromal compartments of the mammary gland defines functions in ductal patterning, proliferation and gene expression.

Hedgehog Signaling in Mammary Gland Development and Breast Cancer

The Hedgehog signal transduction network is an essential regulator of metazoan embryonic development and functions in tissue homeostasis in the adult. In the mammary gland, this network mediates epithelial–stromal interactions regulating branching morphogenesis and histoarchitecture of mammary ducts, and may control alveolar differentiation. Inappropriate activation of the Hedgehog network is associated with a wide variety of cancers, including breast. Genetic evidence in mice, as well as molecular biological studies in human cells, demonstrated that activated signaling can lead to mammary hyperplasia and, in some cases, tumor formation. However, the mechanism(s) by which Hedgehog signaling regulates normal gland development, and exactly how activated Hedgehog signaling contributes to development or progression of breast cancer remain unclear. Because evidence of inappropriate activation of Hedgehog signaling is observed in some breast cancers, and network activity is associated with breast cancer cell self-renewal, the Hedgehog network has become a leading candidate for the development of targeted therapeutics.

The Emerging Roles of Steroid Hormone Receptors in Ductal Carcinoma in Situ (DCIS) of the Breast

Ductal carcinoma in situ (DCIS) is a non-obligate precursor to most types of invasive breast cancer (IBC). Although it is estimated only one third of untreated patients with DCIS will progress to IBC, standard of care for treatment is surgery and radiation. This therapeutic approach combined with a lack of reliable biomarker panels to predict DCIS progression is a major clinical problem. DCIS shares the same molecular subtypes as IBC including estrogen receptor (ER) and progesterone receptor (PR) positive luminal subtypes, which encompass the majority (60–70%) of DCIS. Compared to the established roles of ER and PR in luminal IBC, much less is known about the roles and mechanism of action of estrogen (E2) and progesterone (P4) and their cognate receptors in the development and progression of DCIS. This is an underexplored area of research due in part to a paucity of suitable experimental models of ER+/PR + DCIS. This review summarizes information from clinical and observational studies on steroid hormones as breast cancer risk factors and ER and PR as biomarkers in DCIS. Lastly, we discuss emerging experimental models of ER+/PR+ DCIS.

P4-04-02: Smoothened Function as a G-Protein Coupled Receptor in Mammary Epithelial Cells.

Background Hedgehog signaling orchestrates many key developmental processes, including cell fate determination and tissue patterning in the embryo, as well as tissue homeostasis and stem cell maintenance in the adult. Aberrant hedgehog signaling has been implicated in several cancers including skin, brain, prostate and breast. Previous work in our laboratory demonstrated that overexpression of activated Smoothened (SMO), a key effector in hedgehog signaling, leads to mammary hyperplasia, and is overexpressed in DCIS and IBCs. Consistent with recent data, we now provide evidence that SMO signals via Gαi proteins in the mouse mammary gland. Hypothesis: Smoothened promotes mammary gland proliferation by functioning as a G-protein coupled receptor(GPCR) and couples to one or more members of the PTX-sensitive Gαi family of heterotrimeric G-protein subunits. Experimental Design and Methods: Expression of Gαi subunit(s) was tested by qRT-PCR & immunofluorescence microscopy of mammary epithelial cells (MECs) and whole mammary glands from SMO and WT mice. We analyzed mammary glands from single Gαi subunit null mice for developmental abnormalities. Genetic studies are underway in which SMO mice are crossed to Gαil2&3 null mice. To elucidate downstream signaling events triggered by SMO activation, we are treating SMO derived MECs cultured in a 3D matrix and whole gland organ cultures with inhibitors of Gαi effector molecules and assessing proliferation status. We will also employ GTPγS binding assays using primary MECs from SMO overexpressing mice to test whether SMO can activate Gαi proteins in mammary gland epithelium. Results and Discussion: Treatment of MMTV-Cre dependent SMO hyperplasias with pertussis toxin (PTX), a potent inhibitor of Gαi family of G-protein signaling, significantly attenuates mammary gland hyperproliferation. This result supports the hypothesis that Smoothened functions as a GPCR interacting with one or more members of the Gαi family. We have now found that Gαi2 and Gαi3 null mice display an increased number of terminal end buds and a more completely filled fat pad relative to WT control littermates. Our data suggest that SMO-expressing cells induce proliferation of neighboring WT cells and that low-level proliferation in SMO-expressing cells is not affected by PTX. qPCR data indicates that Gαi1 is the most abundant transcript in sorted MECs which overexpress SMO and that the Notch signaling pathway may be activated. While our mechanistic data thus far mostly comprises transcriptional analyses coupled with pharmacological inhibition, they offer insight as to which pathways may be regulated by SMO activation. Genetic crosses between the SMO and Gαi null mice should provide evidence that these two proteins interact in the developing mammary gland. When associated with biochemical data, these studies ought to provide functional insight into the mechanism behind SMO-driven mammary hyperplasias. This work offers potential clinical implications towards breast cancer treatment as there exist therapeutic agents targeting the hedgehog signaling pathway in clinical trials that were developed by solely testing their ability to inhibit GLI1/2-mediated transcription. Our data identify targets for intervention other than the GLI transcription factors. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-04-02.