Faculty Bibliography

Desmosomes are adhesive junctions that provide mechanical coupling between cells. Plakoglobin (PG) is a major component of the intracellular plaque that serves to connect transmembrane elements to the cytoskeleton. We have used electron tomography and immunolabeling to investigate the consequences of PG knockout on the molecular architecture of the intracellular plaque in cultured keratinocytes. Although knockout keratinocytes form substantial numbers of desmosome-like junctions and have a relatively normal intercellular distribution of desmosomal cadherins, their cytoplasmic plaques are sparse and anchoring of intermediate filaments is defective. In the knockout, beta-catenin appears to substitute for PG in the clustering of cadherins, but is unable to recruit normal levels of plakophilin-1 and desmoplakin to the plaque. By comparing tomograms of wild type and knockout desmosomes, we have assigned particular densities to desmoplakin and described their interaction with intermediate filaments. Desmoplakin molecules are more extended in wild type than knockout desmosomes, as if intermediate filament connections produced tension within the plaque. On the basis of our observations, we propose a particular assembly sequence, beginning with cadherin clustering within the plasma membrane, followed by recruitment of plakophilin and desmoplakin to the plaque, and ending with anchoring of intermediate filaments, which represents the key to adhesive strength

Experiments involving beta-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 beta-catenin signaling has not been investigated. In this study, we demonstrate that activated beta-catenin rescues alveologenesis in progesterone receptor (PR; Pgr)-null mice during pregnancy. Two distinct subsets of mammary cells respond to expression of DeltaN89beta-catenin. Cells at ductal tips are inherently beta-catenin-responsive and form alveoli in the absence of PR. However, PR activity confers beta-catenin responsiveness to progenitor cells along the lateral ductal borders in the virgin gland. Once activated by beta-catenin, responding cells switch on an alveolar differentiation program that is indistinguishable from that observed in pregnancy and is curtailed by PR signaling

The Hedgehog pathway is vital for the development of many epidermal appendages, but its role in mammary development has been unclear. Here, we show that although Gli2 and Gli3 are expressed during embryonic mammary development, transcriptional reporters of positive Hedgehog signaling are absent. Nevertheless, Gli3(xt/xt) embryos show aberrant early mammary marker expression and lack two pairs of mammary buds, demonstrating that Gli3 is essential for mammary bud formation and preceding patterning events. Misactivation of the Hedgehog pathway by targeted expression of the constitutive activator Gli1, from the Gli2 promoter in Gli3(xt/+) mice, also induces mammary bud loss. Moreover, loss of Gli3 expression induces Gli1 misexpression in mammary mesenchyme. These results establish that the essential function of Gli3 during embryonic mammary development is to repress Hedgehog/Gli1-inducible targets. During postnatal mammary development, Gli2 and Gli3 are expressed in stromal and myoepithelial cells, and Gli3 is also found within the lumenal epithelium. Again, transcriptional reporters of positive Hedgehog signaling are absent from these cell types, yet are expressed robustly within mammary lymphatics. Thus, positive Hedgehog signaling is absent throughout mammary development, distinguishing the mammary gland from other epidermal appendages, such as hair follicles, which require Hedgehog pathway activity

Recent studies show that cadherins and catenins are hormonally regulated and carry out physiological roles during mammary development but have pathological effects when deregulated. E-cadherin expression is irreversibly lost in invasive lobular breast cancer (ILC). Animal models of ILC provide mechanistic insight, confirming that E-cadherin serves as both a tumor suppressor and an invasion suppressor in ILC. Ductal breast cancer involves complex, reversible, epigenetic modulation of multiple cadherins. Transcriptional regulators of E-cadherin have been identified that induce epithelial-to-mesenchymal transitions. Catenins are lost or mislocalized in tumors lacking cadherins. However, beta-catenin signaling is upregulated by numerous pathways in >50% of breast tumors and animal models suggest its oncogenic function in breast relates to its role in mammary progenitor cell expansion

The majority (75%) of human breast cancers express estrogen receptor (ER). Although ER-positive tumors usually respond to antiestrogen therapies, 30% of them do not. It is not known what controls the ER status of breast cancers or their responsiveness to antihormone interventions. In this report, we document that transgenic (TG) expression of Wnt-1 in mice induces ER-positive tumors. Loss of Pten or gain of Ras mutations during the evolution of tumors in Wnt-1 TG mice has no effect on the expression of ER, but overexpression of Neu or loss of p53 leads to ER-negative tumors. Thus, our results provide compelling evidence that expression of ER in breast cancer may be influenced by specific genetic changes that promote cancer progression. These findings constitute a first step to explore the molecular mechanisms leading to ER-positive or ER-negative mammary tumors. In addition, we find that ER-positive tumors arising in Wnt-1 TG mice are refractory to both ovariectomy and the ER antagonist tamoxifen, but lose ER expression with tamoxifen, suggesting that antiestrogen selects for ER-negative tumor cells and that the ER-positive cell fraction is dispensable for growth of these tumors. This is a first report of a mouse model of antiestrogen-resistant ER-positive breast cancers, and could provide a powerful tool to study the molecular mechanisms that control antiestrogen resistance.

Hair follicle morphogenesis and cycling were examined in transgenic mice that overexpress the bone morphogenetic protein (BMP) inhibitor Noggin under the control of the neuron-specific enolase promoter. The Noggin transgene was misexpressed in the proximal portion of the hair follicle, primarily the matrix cells, apart from the usual expression in neurons. Transgene expression appeared only after induction of both the primary (tylotrich) and secondary (nontylotrich) pelage hair follicles had already occurred, thus allowing examination of the role of BMP signaling in follicles that had been induced normally in the presence of BMPs. The overexpression of Noggin in these animals resulted in a dramatic loss of hair postnatally. There was an apparently normal, but shortened period of postnatal hair follicle morphogenesis, followed by premature initiation of hair follicle cycling via entry into the first catagen transformation. This resulted in a complete loss of hair shafts from the nontylotrich hair follicles in these mice while the tylotrich hair follicles were normal. The onset of anagen of the first postnatal hair follicle cycle was also accelerated in the transgenic mice. Our results show that BMP signaling is specifically required for proper proliferation and differentiation during late morphogenesis of nontylotrich hair follicles and that inhibition of this signaling pathway may be one of the triggers for the onset of catagen when the follicles are in anagen and the onset of anagen when the follicles are in telogen. Ectopic sebocyte differentiation was another hallmark of the phenotype of these transgenic mice suggesting that BMP signaling may be an important determinant of lineage selection by common progenitor cells in the skin. BMPs likely promote a hair follicle-type differentiation pathway of keratinocytes while suppressing the sebaceous differentiation pathway of skin epithelium

Beta-catenin and cyclin D1 have attracted considerable attention due to their proto-oncogenic roles in human cancer. The finding of cyclin D1 as a direct target gene of beta-catenin in colon cancer cells led to the assumption that cyclin D1 upregulation is pivotal to beta-catenin's oncogenicity. Our recent paper shows that this is not the case; cyclin D1 dampens the oncogenicity of activated beta-catenin (MMTV-DN89beta-catenin). The relationships and dependencies of beta-catenin and cyclin D1 point to distinct, essential and sequential roles during alveologenesis. These results support the concept that both beta-catenin's and cyclin D1's actions are more sophisticated than simple acceleration of the cell cycle clock. These proteins are employed at critical junctures involving cell fate decisions that we speculate require specific types of cell cycle to traverse