The hedgehog signaling network regulates pattern formation, proliferation, cell fate and stem/progenitor cell self-renewal in many organs. Altered hedgehog signaling is implicated in 20-25% of all cancers, including breast cancer. We demonstrated previously that heterozygous disruption of the gene encoding the patched-1 (PTCH1) hedgehog receptor, a negative regulator of smoothened (Smo) in the absence of ligand, led to mammary ductal dysplasia in virgin mice. We now show that expression of activated human SMO (SmoM2) under the mouse mammary tumor virus (MMTV) promoter in transgenic mice leads to increased proliferation, altered differentiation, and ductal dysplasias distinct from those caused by Ptch1 heterozygosity. SMO activation also increased the mammosphere-forming efficiency of primary mammary epithelial cells. However, limiting-dilution transplantation showed a decrease in the frequency of regenerative stem cells in MMTV-SmoM2 epithelium relative to wild type, suggesting enhanced mammosphere-forming efficiency was due to increased survival or activity of division-competent cell types under anchorageindependent growth conditions, rather than an increase in the proportion of regenerative stem cells per se. In human clinical samples, altered hedgehog signaling occurs early in breast cancer development, with PTCH1 expression reduced in ~50% of ductal carcinoma in situ (DCIS) and invasive breast cancers (IBC). Conversely, SMO is ectopically expressed in 70% of DCIS and 30% of IBC. Surprisingly, in both human tumors and MMTV-SmoM2 mice, SMO rarely colocalized with the Ki67 proliferation marker. Our data suggest that altered hedgehog signaling may contribute to breast cancer development by stimulating proliferation, and by increasing the pool of division-competent cells capable of anchorage-independent growth.

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Recent studies have unequivocally identified multipotent stem/progenitor cells in mammary glands, offering a tractable model system to unravel genetic and epigenetic regulation of epithelial stem/progenitor cell development and homeostasis. In this study, we show that Pygo2, a member of an evolutionarily conserved family of plant homeo domain–containing proteins, is expressed in embryonic and postnatal mammary progenitor cells. Pygo2 deficiency, which is achieved by complete or epithelia-specific gene ablation in mice, results in defective mammary morphogenesis and regeneration accompanied by severely compromised expansive self-renewal of epithelial progenitor cells. Pygo2 converges with Wnt/β-catenin signaling on progenitor cell regulation and cell cycle gene expression, and loss of epithelial Pygo2 completely rescues β-catenin–induced mammary outgrowth. We further describe a novel molecular function of Pygo2 that is required for mammary progenitor cell expansion, which is to facilitate K4 trimethylation of histone H3, both globally and at Wnt/β-catenin target loci, via direct binding to K4-methyl histone H3 and recruiting histone H3 K4 methyltransferase complexes.

In the present study we developed a model of diet-induced obesity (DIO) in male C57 BL/6J mice using an 8-wk high fat diet. This model should better reflect the physiology of the majority of the human obese patients than mouse genetic models of obesity with defects in leptin or leptin signaling. At the end of the diet, DIO mice displayed an increased weight (20%) and higher leptin, insulin, glucose, and corticosterone plasma levels compared with mice fed a standard diet during the same period. Moreover, they became resistant to the central effect of peripheral administration of leptin. Oligonucleotide microarray studies were conducted in adipose tissue. They showed that a great number of genes are differentially expressed. The majority of these genes (69%) are down-regulated in DIO mice. Among those are genes encoding enzymes of the lipid metabolism or markers of adipocyte differentiation, enzymes involved in detoxification processes, as well as structural components of the cytoskeleton. Some other groups of genes displayed increased expression, such as those encoding inflammatory markers. The results of the microarray analysis were confirmed by semiquantitative RT-PCR studies run on a selected number of genes that were differentially expressed or not modified.

Altered hedgehog signaling is implicated in the development of approximately 20-25% of all cancers, especially those of soft tissues. Genetic evidence in mice as well as immunolocalization studies in human breast cancer specimens suggest that deregulated hedgehog signaling may contribute to breast cancer development. Indeed, two recent studies demonstrated that anchorage-dependent growth of some human breast cancer cell lines is impaired by cyclopamine, a potent hedgehog signaling antagonist targeting the Smoothened (SMO) protein. However, specificity of cyclopamine at the dosage required for growth inhibition (> or =10 microM) remained an open question. In this paper we demonstrate that hedgehog signaling antagonists, including cyclopamine, and a second compound, CUR0199691, can inhibit growth of estrogen receptor (ER)-positive and ER-negative tumorigenic breast cancer cells at elevated doses. However, our results indicate that, for most breast cancer cell lines, growth inhibition by these compounds can be independent of detectable Smo gene expression. Rather, our results suggest that cyclopamine and CUR0199691 have unique secondary molecular targets at the dosages required for growth inhibition that are unrelated to hedgehog signaling.

In vitro studies have demonstrated leptin-positive effects on the osteoblast lineage and negative effects on osteoclastogenesis. Therefore, we tested the hypothesis that leptin may prevent tail-suspension-induced bone loss characterized by an uncoupling pattern of bone remodeling, through both mechanisms. Female rats were randomly tail-suspended or not and treated either with ip administration of leptin or vehicle for 3, 7, and 14 d. As measured by dual energy x-ray absorptiometry, tail-suspension induced a progressive decrease in tibia-metaphysis bone mineral density, which was prevented by leptin. Histomorphometry showed that this was related to the prevention of the transient increase in osteoclast number observed with suspension at d 7. These effects could be mediated by the receptor activator of nuclear factor kappaB-ligand (RANKL)/osteoprotegerin (OPG) pathway since we observed using direct RT-PCR, a suspension-induced increase in RANKL gene expression in proximal tibia at d 3, which was counterbalanced by leptin administration with a similar 3-fold increase in OPG expression and a RANKL to OPG ratio close to nonsuspended conditions. In addition, leptin prevented the decrease in bone formation rate induced by tail-suspension at d 14. The latter could be related to the role of leptin in mediating the reciprocal differentiation between adipocytes and osteoblasts, because leptin concurrently blunted the disuse-induced increase in bone marrow adipogenesis. In summary, these data suggest that peripheral administration of leptin could prevent disuse-induced bone loss through, first, a major inhibitory effect on bone resorption and, second, a delayed effect preventing the decrease in bone formation.

Hedgehog (Hh) signalling is mediated through the Patched-1 (Ptch1) receptor. Hh-binding to Ptch1 blocks the inhibitory effects of Ptch1 on the activity of the transmembrane protein, Smoothened (Smo), resulting induction of target genes by the Gli-family of transcription factors. We demonstrate here that Hh-binding to Ptch1 stimulates activation of Erk1/2. This activation is insensitive to the small molecule Smo antagonists and occurs in a cell line that does not express Smo. Specifically, the C-terminus of Ptch1 harbors motifs encoding Class I and II SH3-binding sites. SH3-domain binding activity was verified using GST-c-src SH3 , -Grb2 SH3 and -p85β SH3 fusion-proteins. Ectopically-expressed Grb2 or p85β could also be co-immunoprecipitated with the Ptch1 C-terminus. Addition of Shh to serum-starved human mammary epithelial cells and Shh Light II fibroblasts stimulated phosphorylation of Erk1/2. Erk1/2 activation was observed in cells where Smo activity had been inhibited using cyclopamine and in the breast epithelial cell line, MCF10A, that does not express Smo. These data reveal novel binding activities for the C-terminal region of Ptch1 and define a signalling pathway stimulated by the Hh-ligands operating independently of pathways requiring Smo.

E-Cadherin is a cell:cell adhesion molecule critical for appropriate embryonic and mammary development. In cancer, E-Cadherin has been primarily viewed as being lost during the process of epithelial-mesenchymal transition (EMT), which occurs with a switch from E-Cadherin expression to a gain of N-Cadherin and other mesenchymal markers. EMT has been shown to play a role in the metastatic process while the reverse process, mesenchymal-epithelial transition (MET), is important for metastatic colonization. Here we report an unexpected role of E-Cadherin in regulating tumorigenicity and hypoxia responses of breast tumors in vivo. Reduced expression of E-Cadherin led to a dramatic reduction of the in vivo growth capability of SUM149, Mary-X and 4T1 tumor cells. Furthermore, over-expression of ZEB1, a known transcriptional repressor of E-Cadherin, led to reduced in vivo growth of SUM149 tumors. Gene set enrichment analysis identified the loss of hypoxia response genes as a major mechanism in mediating the lack of in vivo growth of SUM149 cells that lacked E-Cadherin or over-expressed ZEB1. The in vivo growth defect of SUM149 E-Cadherin knockdown tumors was rescued by the hypoxia-inducible 1α transcription factor (HIF-1α). Given the importance of HIF-1α in cellular metabolism, we observed reduced glycolytic capacity in SUM149 and 4T1 cells that had E-Cadherin knocked down. Our observations shed light on the complex functions of E-Cadherin in retention of an epithelial phenotype and as a mediator of survival of aggressive breast cancer under hypoxic conditions in vivo. Furthermore, we find that patients with basal subtype breast cancer and high E-Cadherin expression in their tumors had a poor clinical outcome. Our data suggests a novel function for E-Cadherin as a bona fide signaling molecule required for the in vivo growth of aggressive breast cancer tumor cells, that retain E-Cadherin expression, in mediating their metabolic function.