Wnt signaling in adult intestinal stem cells and cancer

Signaling initiated by secreted glycoproteins of the Wnt family regulates many aspects of embryonic development and it is involved in homeostasis of adult tissues. In the gastrointestinal (GI) tract the Wnt pathway maintains the self-renewal capacity of epithelial stem cells. The stem cell attributes are conferred by mutual interactions of the stem cell with its local microenvironment, the stem cell niche. The niche ensures that the threshold of Wnt signaling in the stem cell is kept in physiological range. In addition, the Wnt pathway involves various feedback loops that balance the opposing processes of cell proliferation and differentiation. Today, we have compelling evidence that mutations causing aberrant activation of the Wnt pathway promote expansion of undifferentiated progenitors and lead to cancer.The review summarizes recent advances in characterization of adult epithelial stem cells in the gut. We mainly focus on discoveries related to molecular mechanisms regulating the output of the Wnt pathway. Moreover, we present novel experimental approaches utilized to investigate the epithelial cell signaling circuitry in vivo and in vitro. Pivotal aspects of tissue homeostasis are often deduced from studies of tumor cells; therefore, we also discuss some latest results gleaned from the deep genome sequencing studies of human carcinomas of the colon and rectum.     

Epithelial stem cells: turning over new leaves

Most epithelial tissues self-renew throughout adult life due to the presence of multipotent stem cells and/or unipotent progenitor cells. Epithelial stem cells are specified during development and are controlled by epithelial-mesenchymal interactions. Despite morphological and functional differences among epithelia, common signaling pathways appear to control epithelial stem cell maintenance, activation, lineage determination, and differentiation. Additionally, deregulation of these pathways can lead to human disorders including cancer. Understanding epithelial stem cell biology has major clinical implications for the diagnosis, prevention, and treatment of human diseases, as well as for regenerative medicine.     

Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling.

The continuously growing mouse incisor is an excellent model to analyze the mechanisms for stem cell lineage. We designed an organ culture method for the apical end of the incisor and analyzed the epithelial cell lineage by 5-bromo-2'-deoxyuridine and DiI labeling. Our results indicate that stem cells reside in the cervical loop epithelium consisting of a central core of stellate reticulum cells surrounded by a layer of basal epithelial cells, and that they give rise to transit-amplifying progeny differentiating into enamel forming ameloblasts. We identified slowly dividing cells among the Notch1-expressing stellate reticulum cells in specific locations near the basal epithelial cells expressing lunatic fringe, a secretory molecule modulating Notch signaling. It is known from tissue recombination studies that in the mouse incisor the mesenchyme regulates the continuous growth of epithelium. Expression of Fgf-3 and Fgf-10 were restricted to the mesenchyme underlying the basal epithelial cells and the transit-amplifying cells expressing their receptors Fgfr1b and Fgfr2b. When FGF-10 protein was applied with beads on the cultured cervical loop epithelium it stimulated cell proliferation as well as expression of lunatic fringe. We present a model in which FGF signaling from the mesenchyme regulates the Notch pathway in dental epithelial stem cells via stimulation of lunatic fringe expression and, thereby, has a central role in coupling the mitogenesis and fate decision of stem cells.     

Epithelial stem cells: An epigenetic and wnt‐centric perspective

Epithelial stem cells, such as those present in mammalian skin, intestine, or mammary gland, are tissue stem cells capable of both long‐term self‐renewal and multi‐lineage differentiation. Here we review studies implicating epigenetic control mechanisms in mammalian epithelial stem cell development and homeostasis. We also provide an update of recent progresses in the involvement of canonical Wnt signaling and note an interesting link between the Wnt pathway and chromatin regulation in epithelial stem cells. We anticipate that epigenetic and epigenomic studies of these cells will increase exponentially in the near future. J. Cell. Biochem. 106: 1279–1287, 2010. © 2010 Wiley‐Liss, Inc.     

Mammary development and breast cancer: the role of stem cells

The mammary gland is a highly regenerative organ that can undergo multiple cycles of proliferation, lactation and involution, a process controlled by stem cells. The last decade much progress has been made in the identification of signaling pathways that function in these stem cells to control self-renewal, lineage commitment and epithelial differentiation in the normal mammary gland. The same signaling pathways that control physiological mammary development and homeostasis are also often found deregulated in breast cancer. Here we provide an overview on the functional and molecular identification of mammary stem cells in the context of both normal breast development and breast cancer. We discuss the contribution of some key signaling pathways with an emphasis on Notch receptor signaling, a cell fate determination pathway often deregulated in breast cancer. A further understanding of the biological roles of the Notch pathway in mammary stem cell behavior and carcinogenesis might be relevant for the development of future therapies.     

Wingless signaling regulates the maintenance of ovarian somatic stem cells in Drosophila

Identifying the signals involved in maintaining stem cells is critical to understanding stem cell biology and to using stem cells in future regenerative medicine. In the Drosophila ovary, Hedgehog is the only known signal for maintaining somatic stem cells (SSCs). Here we report that Wingless (Wg) signaling is also essential for SSC maintenance in the Drosophila ovary. Wg is expressed in terminal filament and cap cells, a few cells away from SSCs. Downregulation of Wg signaling in SSCs through removal of positive regulators of Wg signaling, dishevelled and armadillo, results in rapid SSC loss. Constitutive Wg signaling in SSCs through the removal of its negative regulators, Axin and shaggy, also causes SSC loss. Also, constitutive wg signaling causes over-proliferation and abnormal differentiation of somatic follicle cells. This work demonstrates that wg signaling regulates SSC maintenance and that its constitutive signaling influences follicle cell proliferation and differentiation. In mammals, constitutive beta-catenin causes over-proliferation and abnormal differentiation of skin cells, resulting in skin cancer formation. Possibly, mechanisms regulating proliferation and differentiation of epithelial cells, including epithelial stem cells, is conserved from Drosophila to man.     

Epithelial stem cells and implications for wound repair

Activation of epithelial stem cells and efficient recruitment of their proliferating progeny plays a critical role in cutaneous wound healing. The reepithelialized wound epidermis has a mosaic composition consisting of progeny that can be traced back both to epidermal and several types of hair follicle stem cells. The contribution of hair follicle stem cells to wound epidermis is particularly intriguing as it involves lineage identity change from follicular to epidermal. Studies from our laboratory show that hair follicle-fated bulge stem cells commit only transient amplifying epidermal progeny that participate in the initial wound re-epithelialization, but eventually are outcompeted by other epidermal clones and largely disappear after a few months. Conversely, recently described stem cell populations residing in the isthmus portion of hair follicle contribute long-lasting progeny toward wound epidermis and, arguably, give rise to new interfollicular epidermal stem cells. The role of epithelial stem cells during wound healing is not limited to regenerating stratified epidermis. By studying regenerative response in large cutaneous wounds, our laboratory uncovered that epithelial cells in the center of the wound can acquire greater morphogenetic plasticity and, together with the underlying wound dermis, can engage in an embryonic-like process of hair follicle neogenesis. Future studies should uncover the cellular and signaling basis of this remarkable adult wound regeneration phenomenon.     

Endothelial induced EMT in breast epithelial cells with stem cell properties

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.     

Hair follicle stem cells

The increasing use of the hair follicle as a stem cell paradigm is due in part to the complex interplay between epithelial, dermal and other cell types, each with interesting differentiation potential and prospective therapeutic applications. This review focuses on research into the environmental niche, gene expression profiles and plasticity of hair follicle stem cell populations, where many recent advances have come about through novel technological and experimental approaches. We discuss major developmental pathways involved in the establishment and control of the epithelial stem cell niche, and evidence of plasticity between stem and transit amplifying cell populations.     

Capturing and profiling adult hair follicle stem cells

The hair follicle bulge possesses putative epithelial stem cells. Characterization of these cells has been hampered by the inability to target bulge cells genetically. Here, we use a Keratin1-15 (Krt1-15, also known as K15) promoter to target mouse bulge cells with an inducible Cre recombinase construct or with the gene encoding enhanced green fluorescent protein (EGFP), which allow for lineage analysis and for isolation of the cells. We show that bulge cells in adult mice generate all epithelial cell types within the intact follicle and hair during normal hair follicle cycling. After isolation, adult Krt1-15-EGFP-positive cells reconstituted all components of the cutaneous epithelium and had a higher proliferative potential than Krt1-15-EGFP-negative cells. Genetic profiling of hair follicle stem cells revealed several known and unknown receptors and signaling pathways important for maintaining the stem cell phenotype. Ultimately, these findings provide potential targets for the treatment of hair loss and other disorders of skin and hair.     

Lung stem cells

The lung is a relatively quiescent tissue comprised of infrequently proliferating epithelial, endothelial, and interstitial cell populations. No classical stem cell hierarchy has yet been described for the maintenance of this essential tissue; however, after injury, a number of lung cell types are able to proliferate and reconstitute the lung epithelium. Differentiated mature epithelial cells and newly recognized local epithelial progenitors residing in specialized niches may participate in this repair process. This review summarizes recent discoveries and controversies, in the field of stem cell biology, that are not only challenging, but also advancing an understanding of lung injury and repair. Evidence supporting a role for the numerous cell types believed to contribute to lung epithelial homeostasis is reviewed, and initial studies employing cell-based therapies for lung disease are presented. As a detailed understanding of stem cell biology, lung development, lineage commitment, and epithelial differentiation emerges, an ability to modulate lung injury and repair is likely to follow.     

Wnt signaling controls the stem cell-like asymmetric division of the epithelial seam cells during C. elegans larval development

Metazoan stem cells repopulate tissues during adult life by dividing asymmetrically to generate another stem cell and a cell that terminally differentiates. Wnt signaling regulates the division pattern of stem cells in flies and vertebrates. While the short-lived nematode C. elegans has no adult somatic stem cells, the lateral epithelial seam cells divide in a stem cell-like manner in each larval stage, usually generating a posterior daughter that retains the seam cell fate and an anterior daughter that terminally differentiates. We show that while wild-type adult animals have 16 seam cells per side, animals with reduced function of the TCF homolog POP-1 have as many as 67 seam cells, and animals with reduced function of the β-catenins SYS-1 and WRM-1 have as few as three. Analysis of seam cell division patterns showed alterations in their stem cell-like divisions in the L2-L4 stages: reduced Wnt signaling caused both daughters to adopt non-seam fates, while activated Wnt signaling caused both daughters to adopt the seam fate. Therefore, our results indicate that Wnt signaling globally regulates the asymmetric, stem cell-like division of most or all somatic seam cells during C. elegans larval development, and that Wnt pathway regulation of stem cell-like behavior is conserved in nematodes.     

Wnt signaling in stem and cancer stem cells

Canonical Wnt signaling supports the formation and maintenance of stem and cancer stem cells. Recent studies have elucidated epigenetic mechanisms that control pluripotency and stemness, and allow a first assessment how embryonic and tissue stem cells are generated and maintained, and how Wnt signaling might be involved. The core of this review highlights the roles of Wnt signaling in stem and cancer stem cells of tissues such as skin, intestine and mammary gland. Lastly, we refer to the characterization of novel and powerful inhibitors of canonical Wnt signaling and describe attempts to bring these compounds into preclinical and clinical studies.     

Cancer stem cells

Cancer stem cells (CSCs) are a small subpopulation of cells within tumors with capabilities of self-renewal, differentiation, and tumorigenicity when transplanted into an animal host. A number of cell surface markers such as CD44, CD24, and CD133 are often used to identify and enrich CSCs. A regulatory network consisting of microRNAs and Wnt/β-catenin, Notch, and Hedgehog signaling pathways controls CSC properties. The clinical relevance of CSCs has been strengthened by emerging evidence, demonstrating that CSCs are resistant to conventional chemotherapy and radiation treatment and that CSCs are very likely to be the origin of cancer metastasis. CSCs are believed to be an important target for novel anti-cancer drug discovery. Herein we summarize the current understanding of CSCs, with a focus on the role of miRNA and epithelial–mesenchymal transition (EMT), and discuss the clinical application of targeting CSCs for cancer treatment.     

Therapeutic use of limbal stem cells

Stem cells are defined as relatively undifferentiated cells that have the capacity to generate more differentiated daughter cells. Limbal stem cells are responsible for epithelial tissue repair and regeneration throughout the life. Limbal stem cells have been localized to the Palisades of Vogt in the limbal region. Limbal stem cells have a higher proliferative potential compared to the cells of peripheral and central cornea. Limbal stem cells have the capacity to maintain normal corneal homeostasis. However, in some pathological states, such as chemical and thermal burns, Stevens-Johnson syndrome, and ocular pemphigoid limbal stem cells fail to maintain the corneal epithelial integrity. In such situations, limbal stem cell transplantation has been required as a therapeutic option. In unilateral disorders, the usual source of stem cells is the contralateral eyes, but if the disease is bilateral stem cell allografts have to be dissected from family members or cadaver eyes. The advent of ex vivo expansion of limbal stem cells from a small biopsy specimen has reduced the risk of limbal deficiency in the donor eye. Concomitant immunosuppressive therapy promotes donor-derived epithelial cell viability, but some evidences suggest that donor-derived epithelial stem cell viability is not sustained indefinitely. Thus, long-term follow-up studies are required to ascertain whether donor limbal stem cell survival or promotion of recolonization by resident recipient stem cells occurs in restored recipient epithelium. However, this is not an easy task since a definitive limbal stem cell marker has not been identified yet. This review will discuss the therapeutic usage of limbal stem cells in the corneal epithelial disorders.     

Notch-dependent differentiation of adult airway basal stem cells

The epithelium lining the airways of the adult human lung is composed of ciliated and secretory cells together with undifferentiated basal cells (BCs). The composition and organization of this epithelium is severely disrupted in many respiratory diseases. However, little is known about the mechanisms controlling airway homeostasis and repair after epithelial damage. Here, we exploit the mouse tracheobronchial epithelium, in which BCs function as resident stem cells, as a genetically tractable model of human small airways. Using a reporter allele we show that the low level of Notch signaling at steady state is greatly enhanced during repair and the generation of luminal progenitors. Loss-of-function experiments show that Notch signaling is required for the differentiation, but not self-renewal, of BCs. Moreover, sustained Notch activation in BCs promotes their luminal differentiation, primarily toward secretory lineages. We also provide evidence that this function of Notch signaling is conserved in BCs from human airways.     

WNTing embryonic stem cells

Embryonic stem cells (ESCs) - undifferentiated cells originating from preimplantation stage embryos - have prolonged self-renewal capacity and are pluripotent. Activation of the canonical Wnt pathway is implicated in maintenance of and exit from the pluripotent state. Recent findings demonstrate that the essential mediator of canonical Wnt signaling, β-catenin, is dispensable for ESC maintenance; however, its activation inhibits differentiation through derepression of T cell factor 3 (Tcf3)-bound genes. Wnt agonists are useful in deriving ESCs from recalcitrant mouse strains and the rat and in nuclear reprogramming of somatic stem cells. We discuss recent advances in our understanding of the role of canonical Wnt signaling in the regulation of ESC self-renewal and how its manipulation can improve pluripotent ESC derivation and maintenance.