SUMMARY The mechanistic underpinnings of metastatic dormancy and reactivation are poorly understood. A gain-of-function cDNA screen reveals that Coco, a secreted antagonist of TGF-β ligands, induces dormant breast cancer cells to undergo reactivation in the lung. Mechanistic studies indicate that Coco exerts this effect by blocking lung-derived BMP ligands. Whereas Coco enhances the manifestation of traits associated with cancer stem cells, BMP signaling suppresses it. Coco induces a discrete gene expression signature, which is strongly associated with metastatic relapse to the lung but not to the bone or brain in patients. Experiments in mouse models suggest that these latter organs contain niches devoid of bioactive BMP. These findings reveal that metastasis-initiating cells need to overcome organ-specific anti-metastatic signals in order to undergo reactivation.

Hereditary hypotrichosis simplex (HHS) is a rare autosomal dominant form of hair loss characterized by hair follicle (HF) miniaturization1 , 2. Using genetic linkage analysis, we mapped a novel locus for HHS to chromosome 18p11.22, and identified a mutation (L9R) in the APCDD1 gene in three families. We show that APCDD1 is a membrane-bound glycoprotein that is abundantly expressed in human HFs, and can interact in vitro with WNT3A and LRP5, two essential components of Wnt signaling. Functional studies revealed that APCDD1 inhibits Wnt signaling in a cell-autonomous manner and functions upstream of β-catenin. Moreover, APCDD1 represses activation of Wnt reporters and target genes, and inhibits the biological effects of Wnt signaling during both the generation of neurons from progenitors in the developing chick nervous system, and axis specification in Xenopus embryos. The mutation L9R is located in the signal peptide of APCDD1, and perturbs its translational processing from ER to the plasma membrane. L9R-APCDD1 likely functions in a dominant-negative manner to inhibit the stability and membrane localization of the wild-type protein. These findings describe a novel inhibitor of the Wnt signaling pathway with an essential role in human hair growth. Since APCDD1 is expressed in a broad repertoire of cell types 3 , our findings suggest that APCDD1 may regulate a diversity of biological processes controlled by Wnt signaling.

b-catenin is the major effector of the canonical Wnt signaling pathway. Mutations in components of the pathway that stabilize b-catenin result in augmented gene transcription and play a major role in many human cancers. We employed microarrays to identify transcriptional targets of deregulated b-catenin in a human epithelial cell line (293) engineered to produce mutant b-catenin and in ovarian endometrioid adenocarcinomas characterized with respect to mutations affecting the Wnt/b-catenin pathway. Two genes strongly induced in both systems-FGF20 and DKK1-were studied in detail. Elevated levels of FGF20 RNA were also observed in adenomas from mice carrying the Apc Min allele. Both XFGF20 and Xdkk-1 are expressed early in Xenopus embryogenesis under the control of the Wnt signaling pathway. Furthermore, FGF20 and DKK1 appear to be direct targets for b-catenin/TCF transcriptional regulation via LEF/TCF-binding sites. Finally, by using small inhibitory RNAs specific for FGF20, we show that continued expression of FGF20 is necessary for maintenance of the anchorage-independent growth state in RK3E cells transformed by b-catenin, implying that FGF-20 may be a critical element in oncogenesis induced by the Wnt signaling pathway.

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The TGF-β pathway plays a vital role in development and disease and regulates transcription through a complex composed of receptor-regulated Smads (R-Smads) and Smad4. Extensive biochemical and genetic studies argue that the pathway is activated through R-Smad phosphorylation; however, the dynamics of signaling remain largely unexplored. We monitored signaling and transcriptional dynamics and found that although R-Smads stably translocate to the nucleus under continuous pathway stimulation, transcription of direct targets is transient. Surprisingly, Smad4 nuclear localization is confined to short pulses that coincide with transcriptional activity. Upon perturbation, the dynamics of transcription correlate with Smad4 nuclear localization rather than with R-Smad activity. In Xenopus embryos, Smad4 shows stereotyped, uncorrelated bursts of nuclear localization, but activated RSmads are uniform. Thus, R-Smads relay graded information about ligand levels that is integrated with intrinsic temporal control reflected in Smad4 into the active signaling complex.A small number of signaling pathways are used repeatedly throughout metazoan development, and their effects depend upon timing and context (1). Extensive biochemical characterization of these developmental signaling pathways has elucidated the sequence of events leading from ligand binding at the cell surface to regulation of transcription. Proper temporal control of pathway activity is crucial for normal development; however, the dynamic aspects of signaling are difficult to infer from population data and have lagged behind dissection of pathway components (2, 3). The few cases that have been examined have revealed rich dynamics that could not have been predicted from knowledge of the molecular interactions or from bulk measurements of protein modifications or mRNA levels (2-4).The TGF-β pathway is essential for developmental processes including mesoderm specification and dorsal-ventral axis formation and is dysregulated in a variety of cancers. It also is an important model for pathway crosstalk and dynamics, because it has two branches that share several components including receptors and transcription factors (5, 6). Binding of ligands specific to each branch to receptor complexes leads to the phosphorylation of branch-specific transcription factors: TGF-β/activin/nodal ligands induce the phosphorylation of Smad2/3, whereas bone morphogenic proteins (BMPs) activate Smad1/5/8. Phosphorylation of the receptor-activated Smads (R-Smads) from either branch of the pathway results in complex formation with Smad4, nuclear accumulation, and transcriptional activation.The prevailing model is that R-Smads carry pathway information with Smad4 mirroring their activity (7,8). R-Smad phosphorylation is necessary for the nuclear accumulation and transcriptional activity of both the R-Smads and Smad4. Termination of signaling often is presumed to be caused by either degradation or dephosphorylation of activated R-Smads (9, 10), and it further is assumed that the continuous pres...