Tannishtha Reya

Wnt signalling in stem cells and cancer.

The canonical Wnt cascade has emerged as a critical regulator of stem cells. In many tissues, activation of Wnt signalling has also been associated with cancer. This has raised the possibility that the tightly regulated self-renewal mediated by Wnt signalling in stem and progenitor cells is subverted in cancer cells to allow malignant proliferation. Insights gained from understanding how the Wnt pathway is integrally involved in both stem cell and cancer cell maintenance and growth in the intestinal, epidermal and haematopoietic systems may serve as a paradigm for understanding the dual nature of self-renewal signals.

A role for Wnt signalling in self-renewal of haematopoietic stem cells

Haematopoietic stem cells (HSCs) have the ability to renew themselves and to give rise to all lineages of the blood; however, the signals that regulate HSC self-renewal remain unclear. Here we show that the Wnt signalling pathway has an important role in this process. Overexpression of activated β-catenin expands the pool of HSCs in long-term cultures by both phenotype and function. Furthermore, HSCs in their normal microenvironment activate a LEF-1/TCF reporter, which indicates that HCSs respond to Wnt signalling in vivo. To demonstrate the physiological significance of this pathway for HSC proliferation we show that the ectopic expression of axin or a frizzled ligand-binding domain, inhibitors of the Wnt signalling pathway, leads to inhibition of HSC growth in vitro and reduced reconstitution in vivo. Furthermore, activation of Wnt signalling in HSCs induces increased expression of HoxB4 and Notch1, genes previously implicated in self-renewal of HSCs. We conclude that the Wnt signalling pathway is critical for normal HSC homeostasis in vitro and in vivo, and provide insight into a potential molecular hierarchy of regulation of HSC development.

Wnt proteins are lipid-modified and can act as stem cell growth factors

Wnt signalling is involved in numerous events in animal development, including the proliferation of stem cells and the specification of the neural crest. Wnt proteins are potentially important reagents in expanding specific cell types, but in contrast to other developmental signalling molecules such as hedgehog proteins and the bone morphogenetic proteins, Wnt proteins have never been isolated in an active form. Although Wnt proteins are secreted from cells, secretion is usually inefficient and previous attempts to characterize Wnt proteins have been hampered by their high degree of insolubility. Here we have isolated active Wnt molecules, including the product of the mouse Wnt3a gene. By mass spectrometry, we found the proteins to be palmitoylated on a conserved cysteine. Enzymatic removal of the palmitate or site-directed and natural mutations of the modified cysteine result in loss of activity, and indicate that the lipid is important for signalling. The purified Wnt3a protein induces self-renewal of haematopoietic stem cells, signifying its potential use in tissue engineering.

Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance

A fundamental question in hematopoietic stem cell (HSC) biology is how self-renewal is controlled. Here we show that the molecular regulation of two critical elements of self-renewal, inhibition of differentiation and induction of proliferation, can be uncoupled, and we identify Notch signaling as a key factor in inhibiting differentiation. Using transgenic Notch reporter mice, we found that Notch signaling was active in HSCs in vivo and downregulated as HSCs differentiated. Inhibition of Notch signaling led to accelerated differentiation of HSCs in vitro and depletion of HSCs in vivo. Finally, intact Notch signaling was required for Wnt-mediated maintenance of undifferentiated HSCs but not for survival or entry into the cell cycle in vitro. These data suggest that Notch signaling has a dominant function in inhibiting differentiation and provide a model for how HSCs may integrate multiple signals to maintain the stem cell state.

Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia

Although the role of Hedgehog (Hh) signalling in embryonic pattern formation is well established, its functions in adult tissue renewal and maintenance remain unclear, and the relationship of these functions to cancer development has not been determined. Here we show that the loss of Smoothened (Smo), an essential component of the Hh pathway, impairs haematopoietic stem cell renewal and decreases induction of chronic myelogenous leukaemia (CML) by the BCR–ABL1 oncoprotein. Loss of Smo causes depletion of CML stem cells—the cells that propagate the leukaemia—whereas constitutively active Smo augments CML stem cell number and accelerates disease. As a possible mechanism for Smo action, we show that the cell fate determinant Numb, which depletes CML stem cells, is increased in the absence of Smo activity. Furthermore, pharmacological inhibition of Hh signalling impairs not only the propagation of CML driven by wild-type BCR–ABL1, but also the growth of imatinib-resistant mouse and human CML. These data indicate that Hh pathway activity is required for maintenance of normal and neoplastic stem cells of the haematopoietic system and raise the possibility that the drug resistance and disease recurrence associated with imatinib treatment of CML might be avoided by targeting this essential stem cell maintenance pathway.

Wnt Signaling Regulates B Lymphocyte Proliferation through a LEF-1 Dependent Mechanism

Lymphocyte enhancer factor-1 (LEF-1) is a member of the LEF-1/TCF family of transcription factors, which have been implicated in Wnt signaling and tumorigenesis. LEF-1 was originally identified in pre-B and T cells, but its function in B lymphocyte development remains unknown. Here we report that LEF-1-deficient mice exhibit defects in pro-B cell proliferation and survival in vitro and in vivo. We further show that Lef1-/- pro-B cells display elevated levels of fas and c-myc transcription, providing a potential mechanism for their increased sensitivity to apoptosis. Finally, we establish a link between Wnt signaling and normal B cell development by demonstrating that Wnt proteins are mitogenic for pro-B cells and that this effect is mediated by LEF-1.

Itraconazole, a Commonly Used Antifungal that Inhibits Hedgehog Pathway Activity and Cancer Growth

In a screen of drugs previously tested in humans we identified itraconazole, a systemic antifungal, as a potent antagonist of the Hedgehog (Hh) signaling pathway that acts by a mechanism distinct from its inhibitory effect on fungal sterol biosynthesis. Systemically administered itraconazole, like other Hh pathway antagonists, can suppress Hh pathway activity and the growth of medulloblastoma in a mouse allograft model and does so at serum levels comparable to those in patients undergoing antifungal therapy. Mechanistically, itraconazole appears to act on the essential Hh pathway component Smoothened (SMO) by a mechanism distinct from that of cyclopamine and other known SMO antagonists, and prevents the ciliary accumulation of SMO normally caused by Hh stimulation.

Regulation of myeloid leukaemia by the cell-fate determinant Musashi

Chronic myelogenous leukaemia (CML) can progress from a slow growing chronic phase to an aggressive blast crisis phase, but the molecular basis of this transition remains poorly understood. Here we have used mouse models of CML to show that disease progression is regulated by the Musashi–Numb signalling axis. Specifically, we find that the chronic phase is marked by high levels of Numb expression whereas the blast crisis phase has low levels of Numb expression, and that ectopic expression of Numb promotes differentiation and impairs advanced-phase disease in vivo. As a possible explanation for the decreased levels of Numb in the blast crisis phase, we show that NUP98–HOXA9, an oncogene associated with blast crisis CML, can trigger expression of the RNA-binding protein Musashi2 (Msi2), which in turn represses Numb. Notably, loss of Msi2 restores Numb expression and significantly impairs the development and propagation of blast crisis CML in vitro and in vivo. Finally we show that Msi2 expression is not only highly upregulated during human CML progression but is also an early indicator of poorer prognosis. These data show that the Musashi–Numb pathway can control the differentiation of CML cells, and raise the possibility that targeting this pathway may provide a new strategy for the therapy of aggressive leukaemias.

Imaging Hematopoietic Precursor Division in Real Time

Stem cells are thought to balance self-renewal and differentiation through asymmetric and symmetric divisions, but whether such divisions occur during hematopoietic development remains unknown. Using a Notch reporter mouse, in which GFP acts as a sensor for differentiation, we image hematopoietic precursors and show that they undergo both symmetric and asymmetric divisions. In addition we show that the balance between these divisions is not hardwired but responsive to extrinsic and intrinsic cues. Precursors in a prodifferentiation environment preferentially divide asymmetrically, whereas those in a prorenewal environment primarily divide symmetrically. Oncoproteins can also influence division pattern: although BCR-ABL predominantly alters the rate of division and death, NUP98-HOXA9 promotes symmetric division, suggesting that distinct oncogenes subvert different aspects of cellular function. These studies establish a system for tracking division of hematopoietic precursors and show that the balance of symmetric and asymmetric division can be influenced by the microenvironment and subverted by oncogenes.

Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells

Hematopoietic stem cell (HSC) self-renewal is regulated by both intrinsic and extrinsic signals. Although some of the pathways that regulate HSC self-renewal have been uncovered, it remains largely unknown whether these pathways can be triggered by deliverable growth factors to induce HSC growth or regeneration. Here we show that pleiotrophin, a neurite outgrowth factor with no known function in hematopoiesis, efficiently promotes HSC expansion in vitro and HSC regeneration in vivo. Treatment of mouse bone marrow HSCs with pleiotrophin caused a marked increase in long-term repopulating HSC numbers in culture, as measured in competitive repopulating assays. Treatment of human cord blood CD34+CDCD38−Lin− cells with pleiotrophin also substantially increased severe combined immunodeficient (SCID)-repopulating cell counts in culture, compared to input and cytokine-treated cultures. Systemic administration of pleiotrophin to irradiated mice caused a pronounced expansion of bone marrow stem and progenitor cells in vivo, indicating that pleiotrophin is a regenerative growth factor for HSCs. Mechanistically, pleiotrophin activated phosphoinositide 3-kinase (PI3K) signaling in HSCs; antagonism of PI3K or Notch signaling inhibited pleiotrophin-mediated expansion of HSCs in culture. We identify the secreted growth factor pleiotrophin as a new regulator of both HSC expansion and regeneration.