Cancer progression involves the gradual loss of a differentiated phenotype and acquisition of progenitor and stem-cell-like features. Here, we provide novel stemness indices for assessing the degree of oncogenic dedifferentiation. We used an innovative one-class logistic regression (OCLR) machine-learning algorithm to extract transcriptomic and epigenetic feature sets derived from non-transformed pluripotent stem cells and their differentiated progeny. Using OCLR, we were able to identify previously undiscovered biological mechanisms associated with the dedifferentiated oncogenic state. Analyses of the tumor microenvironment revealed unanticipated correlation of cancer stemness with immune checkpoint expression and infiltrating immune cells. We found that the dedifferentiated oncogenic phenotype was generally most prominent in metastatic tumors. Application of our stemness indices to single-cell data revealed patterns of intra-tumor molecular heterogeneity. Finally, the indices allowed for the identification of novel targets and possible targeted therapies aimed at tumor differentiation.

beta-Catenin is an essential molecule in Wnt/wingless signaling, which controls decisive steps in embryogenesis. To study the role of beta-catenin in skin development, we introduced a conditional mutation of the gene in the epidermis and hair follicles using Cre/loxP technology. When beta-catenin is mutated during embryogenesis, formation of placodes that generate hair follicles is blocked. We show that beta-catenin is required genetically downstream of tabby/downless and upstream of bmp and shh in placode formation. If beta-catenin is deleted after hair follicles have formed, hair is completely lost after the first hair cycle. Further analysis demonstrates that beta-catenin is essential for fate decisions of skin stem cells: in the absence of beta-catenin, stem cells fail to differentiate into follicular keratinocytes, but instead adopt an epidermal fate.

Metastatic growth in distant organs is the major cause of cancer mortality. The development of metastasis is a multistage process with several rate-limiting steps 1 . Although dissemination of tumour cells seems to be an early and frequent event 2 , the successful initiation of metastatic growth, a process termed 'metastatic colonization', is inefficient for many cancer types and is accomplished only by a minority of cancer cells that reach distant sites 3,4 . Prevalent target sites are characteristic of many tumour entities 5 , suggesting that inadequate support by distant tissues contributes to the inefficiency of the metastatic process. Here we show that a small population of cancer stem cells is critical for metastatic colonization, that is, the initial expansion of cancer cells at the secondary site, and that stromal niche signals are crucial to this expansion process. We find that periostin (POSTN), a component of the extracellular matrix, is expressed by fibroblasts in the normal tissue and in the stroma of the primary tumour. Infiltrating tumour cells need to induce stromal POSTN expression in the secondary target organ (in this case lung) to initiate colonization. POSTN is required to allow cancer stem cell maintenance, and blocking its function prevents metastasis. POSTN recruits Wnt ligands and thereby increases Wnt signalling in cancer stem cells. We suggest that the education of stromal cells by infiltrating tumour cells is an important step in metastatic colonization and that preventing de novo niche formation may be a novel strategy for the treatment of metastatic disease.We aimed to explore limiting factors that determine metastatic success using the MMTV-PyMT mouse breast cancer model, which spontaneously metastasizes to the lungs 6 . We reasoned that the recently identified cancer stem cells (CSCs, also called tumour-initiating cells), a subset of cancer cells that allow long-term tumour growth and are thought to be responsible for remissions 7,8 , might also be relevant to the development of metastatic disease ( Supplementary Fig. 1). We measured the relative size of the population of CSCs from primary MMTV-PyMT tumours and their pulmonary metastases using the previously established markers CD90 and CD24, which label a subset of the CD24 1 CD29hi or CD241 CD49f hi population used earlier to isolate CSCs and normal mammary gland stem cells 9-13 (Supplementary Fig. 2). This CSC subset accounts for 3 6 2.1% (s.d.) of all tumour cells from both primary tumours and metastases (Fig. 1a). When CD90 1 CD241 CSCs or CD90 1 CD24 1 -depleted non-CSCs are separately isolated from GFP 1 tumours and directly introduced into mice through tail vein injection (GFP, green fluorescent protein), only the CSC population is able to produce lung metastases (Fig. 1b). Moreover, CD90 1 CD241 cells isolated subsequently from pulmonary metastases are again the only tumour cell population that efficiently initiates secondary metastases (Fig. 1c). This is not due to differences in the extravasation capabilities of CSCs an...

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The anterior-posterior axis of the mouse embryo is defined before formation of the primitive streak, and axis specification and subsequent anterior development involves signaling from both embryonic ectoderm and visceral endoderm. Τhe Wnt signaling pathway is essential for various developmental processes, but a role in anterior-posterior axis formation in the mouse has not been previously established. β-Catenin is a central player in the Wnt pathway and in cadherin-mediated cell adhesion. We generated β-catenin–deficient mouse embryos and observed a defect in anterior-posterior axis formation at embryonic day 5.5, as visualized by the absence of Hex and Hesx1 and the mislocation of cerberus-like and Lim1 expression. Subsequently, no mesoderm and head structures are generated. Intercellular adhesion is maintained since plakoglobin substitutes for β-catenin. Our data demonstrate that β-catenin function is essential in anterior-posterior axis formation in the mouse, and experiments with chimeric embryos show that this function is required in the embryonic ectoderm.

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SUMMARY Wnt/β-catenin and NF-κB signaling mechanisms provide central controls in development and disease, but how these pathways intersect is unclear. Using hair follicle induction as a model system, we show that patterning of dermal Wnt/β-catenin signaling requires epithelial β-catenin activity. We find that Wnt/β-catenin signaling is absolutely required for NF-κB activation, and that Edar is a direct Wnt target gene. Wnt/β-catenin signaling is initially activated independently of Eda/Edar/NF-κB activity in primary hair follicle primordia. However, Eda/Edar/NF-κB signaling is required to refine the pattern of Wnt/β-catenin activity, and to maintain this activity at later stages of placode development. We show that maintenance of localized expression of Wnt10b and Wnt10a requires NF-κB signaling, providing a molecular explanation for the latter observation, and identify Wnt10b as a direct NF-κB target. These data reveal a complex interplay and inter-dependence of Wnt/β-catenin and Eda/Edar/NF-κB signaling pathways in initiation and maintenance of primary hair follicle placodes.

Gain of Wnt signaling through beta-catenin has been ascribed a critical function in the stimulation of hematopoietic stem cell self-renewal, whereas loss of beta-catenin is reportedly dispensable for hematopoiesis. Here we have used conditional mouse genetics and transplantation assays to demonstrate that constitutive activation of beta-catenin blocked multilineage differentiation, leading to the death of mice. Blood cell depletion was accompanied by failure of hematopoietic stem cells to repopulate irradiated hosts and to differentiate into mature cells. Activation of beta-catenin enforced cell cycle entry of hematopoietic stem cells, thus leading to exhaustion of the long-term stem cell pool. Our data suggest that fine-tuned Wnt stimulation is essential for hematopoiesis and is thus critical for therapeutic hematopoietic stem cell population expansion.