Anti-apoptotic action of API2-MALT1 fusion protein involved in t(11;18)(q21;q21) MALT lymphoma

At least three distinct chromosomal translocations, t(11;18)(q21;q21), t(1;14)(p22;q32) and t(14;18)(q32;q21) involving the API2 (also known as c-IAP2)-MALT1 fusion protein, BCL10, and MALT1, respectively, have been implicated in the molecular pathogenesis of mucosa associated lymphoid tissue (MALT) lymphoma. Our findings showed that several variants of the API2-MALT1 fusion protein can occur in patients with t(11;18)(q21;q21), and that API2-MALT1 can potently enfance activation of nuclear factor (NF)-B signaling, which may be relevant to the pathogenesis of MALT lymphomas. We also found that MALT1 is rapidly degraded via the ubiquitin-proteasome pathway, as is the case with API2, but upon the synthesis of fusion, API2-MALT1 becomes stable against this pathway. This stability of API2-MALT1 may thus result in inappropriate nuclear factor (NF)-B activation, thereby contributing to the pathogenesis of MALT lymphoma. Recent biochemical and genetic studies have clearly shown that BCL10 and MALT1 form a physical and functional complex and are both required for NF-B activation by antigen receptor stimulation in T and B lymphocytes. It has also been shown that CARMA1, a newly discovered member of the membrane-associated guanylate kinase (MAGUK) families, is critical for antigen receptor-stimulated NF-B activation. It can be assumed that API2-MALT1 can bypass this normal BCL10/MALT1 cellular signaling pathway linked to NF-B activation, thereby inducing antigen receptor-independent proliferation of lymphocytes. Furthermore, BCL10/MALT1- and API2-MALT1-induced NF-B activation may contribute to anti-apoptotic action probably through NF-B-mediated upregulation of apoptotic inhibitor genes. We recently provided direct evidence that API2-MALT1 indeed exerts anti-apoptotic action, in part, through its direct interaction with apoptotic regulators including Smac. Taken together, these findings prompt us to hypothesize that the anti-apoptotic action of API2-MALT1 may be mediated partly by the direct interaction with apoptotic regulators as well as partly by upregulation of apoptotic inhibitor genes. Further studies can be expected to stimulate research into the development of therapeutic drugs that specifically inhibit the antigen receptor signaling-stimulated NF-B activation pathway: such molecule targeting drugs should be useful for interfering with inappropriate proliferation of lymphocytes associated with inflammatory and neoplastic disorders.     

Notch signaling regulates myogenic regenerative capacity of murine and human mesoangioblasts

Somatic stem cells hold attractive potential for the treatment of muscular dystrophies (MDs). Mesoangioblasts (MABs) constitute a myogenic subset of muscle pericytes and have been shown to efficiently regenerate dystrophic muscles in mice and dogs. In addition, HLA-matched MABs are currently being tested in a phase 1 clinical study on Duchenne MD patients (EudraCT #2011-000176-33). Many reports indicate that the Notch pathway regulates muscle regeneration and satellite cell commitment. However, little is known about Notch-mediated effects on other resident myogenic cells. To possibly potentiate MAB-driven regeneration in vivo, we asked whether Notch signaling played a pivotal role in regulating MAB myogenic capacity. Through different approaches of loss- and gain-of-function in murine and human MABs, we determined that the interplay between Delta-like ligand 1 (Dll1)-activated Notch1 and Mef2C supports MAB commitment in vitro and ameliorates engraftment and functional outcome after intra-arterial delivery in dystrophic mice. Furthermore, using a transgenic mouse model of conditional Dll1 deletion, we demonstrated that Dll1 ablation, either on the injected cells, or on the receiving muscle fibers, impairs MAB regenerative potential. Our data corroborate the perspective of advanced combinations of cell therapy and signaling tuning to enhance therapeutic efficaciousness of somatic stem cells.Notch signaling consists of a conserved pathway, triggered by physical interaction between one ligand and one receptor, both transmembrane proteins exposed by contacting cells.¹ Notch signaling has been involved in different stages of muscle formation² and regeneration.^3,4 The canonical signaling encompasses five ligands (Dll1/3/4, Jagged1/2) and four receptors (Notch1–4); however, the axis Dll1-Notch1 appears consistently involved during myogenic fate specification, for example, neural crest-driven somite maturation.⁵ Moreover, murine embryos expressing a hypomorphic allele of the Notch ligand Dll1 displayed marked impairment of skeletal muscle formation.⁶ Interestingly, the Notch pathway may exert different effects according to the cell context. Culture on DLL1-coated plastic improved ex vivo proliferation and in vivo engraftment of canine satellite cells.⁷ Expression of the active Notch1 intracellular domain (NICD) robustly committed murine and rat mesenchymal stem cells toward the myogenic fate both in vitro and in vivo.⁸ However, Notch-mediated effects on the regenerative potential of non-satellite resident myogenic cells are still unknown.Mesoangioblasts (MABs) are non-satellite resident myogenic stem cells, able to circulate and regenerate dystrophic skeletal muscles.^9,10 HLA-matched MABs are currently under phase 1 clinical study on Duchenne muscular dystrophy patients (EudraCT #2011-000176-33). In this view, understanding the cell-specific effects and mechanisms of myogenic cues will help improving clinical translation of MAB-based therapies in vivo. Recently, it has been shown that Notch synergizes with Pdgf-bb to convert fetal myoblasts into myogenic pericytes.¹¹ However, knowledge about Notch-triggered effects on the regenerative potency of somatic MABs is still scant, particularly in the contexts of cell–cell (in vitro) and fiber–cell (in vivo) contact.Therefore, we asked whether the Dll1-Notch1 axis regulates the myogenic potential of murine and human MABs and how to tune this pathway to ameliorate in vivo MAB-driven regeneration.     

Differentiation of human neuroblastoma cells toward the osteogenic lineage by mTOR inhibitor

Current hypothesis suggest that tumors can originate from adult cells after a process of ''reprogramming'' driven by genetic and epigenetic alterations. These cancer cells, called cancer stem cells (CSCs), are responsible for the tumor growth and metastases. To date, the research effort has been directed to the identification, isolation and manipulation of this cell population. Independently of whether tumors were triggered by a reprogramming of gene expression or seeded by stem cells, their energetic metabolism is altered compared with a normal cell, resulting in a high aerobic glycolytic ''Warburg'' phenotype and dysregulation of mitochondrial activity. This metabolic alteration is intricately linked to cancer progression.The aim of this work has been to demonstrate the possibility of differentiating a neoplastic cell toward different germ layer lineages, by evaluating the morphological, metabolic and functional changes occurring in this process. The cellular differentiation reported in this study brings to different conclusions from those present in the current literature. We demonstrate that ''in vitro'' neuroblastoma cancer cells (chosen as experimental model) are able to differentiate directly into osteoblastic (by rapamycin, an mTOR inhibitor) and hepatic lineage without an intermediate ''stem'' cell step. This process seems owing to a synergy among few master molecules, metabolic changes and scaffold presence acting in a concerted way to control the cell fate.Cancer stem cells are currently viewed as the cells capable of generating cancer (tumor-initiating cells), owing to their intrinsic features of self-renewal and longevity.¹ However, emerging evidence suggests a surprising ability of normal committed cells to act as reserve stem cells upon reprogramming following tissue damage resulting from inflammation and wound healing. This brings to the alternative hypothesis that tumors may originate from differentiated cells that have recovered stem cell properties owing to genetic or epigenetic reprogramming.^{1, 2} Possibly, both models are correct, and consequently there is a continuum of cells capable of generating cancer, ranging from early primitive stem cells to committed progenitor or even terminally differentiated cells.The development of methods for reprogramming somatic cells to induced pluripotent stem cells (iPSCs) through ectopic expression of a few pluripotency factors holds the promise for disease modeling, drug screening studies and treatment of several diseases.³ Generating iPSCs from cancer cells might also clarify the mechanisms that underlie oncogenic transformation.^{4, 5} Thus reprogramming and oncogenic transformation are processes that have interesting common steps, while iPSCs generated from cancer cells could give clues to molecular mechanisms underlying the pathogenesis of human cancer.⁶ To date, there are only few reports demonstrating a successful reprogramming of human primary cancer cells. Only one report describes the reprogramming of human primary cancer cells⁷ while the remaining studies used established cell lines.^{8, 9, 10, 11, 12} Carrete et al.⁹ generated iPSCs from the chronic myeloid leukemia (CML) cell line KBM7 carrying the BCR-ABL fusion oncogene by expressing four ectopic reprogramming factors (OCT4, KLF4, SOX2, and c-Myc (OKSM)). Conversely, Choi et al.¹⁰ reprogrammed EBV-immortalized B lymphocytes to pluripotency using non-integrative episomal vectors. Lin et al.¹¹ reprogrammed human skin cancer cell lines to pluripotency using the microRNA miR-302. Miyoshi et al.¹² reprogrammed gastrointestinal-transformed cell lines using retroviral vectors expressing c-Myc and BCL2. Finally, Hu et al.⁸ successfully reprogrammed primary human lymphoblasts from a BCR-ABL⁺CML patient using transgene-free iPSC technology to ectopically express OKSM and LIN28. In addition, Ramos-Mejia et al.⁴ in a recent review emphasize the importance of deciphering the barriers underlying the reprogramming process of primary cancer cells to obtain information on the links between pluripotency and oncogenic transformation that would be instrumental for therapy development.Cancer cells show distinct metabolic features. In fact, neoplastic cells adapt their metabolic pathways to face the demands of abnormal proliferation. For example, cancer cells increase glucose uptake and the rate of glycolysis even under normoxic conditions; this process of aerobic glycolysis was first described by Warburg et al.^{13, 14} and thence called Warburg effect. Recent studies are increasingly highlighting the importance of metabolic manipulation in cancer cells and how bio-energetic and biosynthetic changes could be exploited to stop tumor cells progression.^{15, 16, 17} Reprogramming, pluripotency, oncogenic transformation and metabolic changes are therefore connected processes that share interesting similarities.^{18, 19} The fact that the same alterations driving tumorigenesis can influence the reprogramming of non-cancer somatic cells is a double-edged sword. It poses safety concerns for the cell therapy applications with iPSCs, while at the same time it promotes further studies aimed to analyzing the mechanisms and barriers underlying the direct reprogramming of cancer cells. This is a fundamental attempt to acquire valuable new insight on reprogramming and cell transformation.²⁰Along these concepts, here we have investigated the possibility to revert cancer progression by targeting cancer cells, seen as deprogrammed cell and therefore similar to adult stem cells. Using a human neuroblastoma cell line (SH-SY5Y) as model, our work has been designed to experimentally explore different aspects. We show how these cells can differentiate toward a germ line different from the original one, modifying their morphology and acquiring metabolic changes which are distinctive of a more normal phenotype.     

Blood loses it when nerves go bad

Myeloproliferative neoplasms are diseases that arise in the stem cells of the blood. In a recent paper published in Nature, Arranz et al. demonstrated that abrogation of sympathetic nerve fibers reduced bone marrow Nestin⁺ mesenchymal cells, which in turn led to an expansion of hematopoietic stem cells and progression of myeloproliferative neoplasms.The stromal cell compartment, or non-hematopoietic cells, of the bone marrow has emerged as an important driver of cell state in hematopoietic stem cells (HSCs) and hematopoietic stem/progenitor cells (HSPCs) in a non-autonomous manner. The HSC niche is not well defined and a number of studies suggest that there are specialized niches for the unique regulation of HSCs and HSPCs¹. Several studies suggest that HSCs reside in a perivascular niche in which a heterogenic population of perivascular mesenchymal stromal cells (MSCs) with overlapping expression of Nestin, LepR, Prx1, or Mx1 each synthesize multiple factors (e.g., CXCL12 and SCF) that promote maintenance and/or localization of HSCs^1,2. Endothelial cells (Tie2⁺)³, and MSCs (NG2⁺/LepR⁻) surrounding arterial vessels⁴, are other units of the niche reported to regulate HSC numbers and quiescence, respectively. Furthermore, osteoprogenitors lining the endosteal surface have a more indirect role in the regulation of HSCs⁶. Thus, it appears that hematopoietic regulation and differentiation in the bone marrow microenvironment is governed at multiple levels⁵.Increasing evidence suggests that dysregulation of the bone marrow microenvironment may participate in blood malignancies. For instance, perturbations of the miRNA processing or ribosomal components through Dicer1 deletion in immature osteolineage cells induced myelodysplasia in mice, followed by the rare emergence of acute myeloid leukemia (AML)⁶. Others reported that β-catenin stabilization in mature osteolineage cells resulted in Notch pathway activation, myelodysplastic syndrome, and highly penetrant AML in mice⁷. In humans, ∼5% of post-transplant AML patients relapse with a leukemia of donor cell origin, suggesting that some patients may have a microenvironmental driver of leukemogenesis⁸. Together, these studies are consistent with a role of the bone marrow microenvironment in maintaining the integrity of hematopoiesis and restricting oncogenesis. When the well-orchestrated regulation of hematopoiesis is disrupted, blood malignancies might occur.The study by Arranz et al.⁹ is a continuation of prior work identifying perivascular bone marrow Nestin⁺ MSCs affected by sympathetic nerve fibers to regulate HSCs¹⁰. Previous studies that a perturbed bone marrow microenvironment modulates myeloproliferative neoplasms (MPNs)^6,7 prompted the authors to further investigate the role of Nestin⁺ MSCs in MPN, specifically MPN associated with Janus kinase 2 (JAK2) mutations^11,12.The authors first analyzed Nestin expression in bone marrow samples from MPN patients and discovered that despite elevated blood-vessel density, Nestin⁺ cell numbers and mRNA expression were reduced. Similar findings were observed in genetically engineered mice that recapitulate human MPNs (e.g., Mx1-cre; JAK2^V617F), indicating that Nestin⁺ MSCs might play a role in MPN. Arranz et al. proceeded to investigate whether a selective depletion of Nestin⁺ MSCs mimics the MPN mouse model. Mice depleted of Nestin⁺ MSCs showed an expansion of HSCs, due to a drop in CXCL12 expression, accompanied by increased hematopoietic progenitors in bone marrow, peripheral blood and spleen, indicative of MPN. Extensive genome-wide RNA-sequencing studies revealed enrichment of Schwann cell- and neural-related genes in Nestin⁺ MSCs derived from MPN mice. This result prompted the authors to explore the role of sympathetic nerve fibers and nonmyelinating Schwann cells in MPN patients and the MPN mouse model. Strikingly, both MPN patients and MPN mice had reduced sympathetic nerve fibers and nonmyelinating Schwann cells adjacent to Nestin⁺ cells in the bone marrow. Multiplex ELISA experiments identified that mutant HSCs secrete IL-1β, which induced apoptosis in bone marrow Schwann cells by Caspase-1 activation followed by neuronal damage. Neural-glial damage in turn compromised Nestin⁺ MSCs survival and led to MPN. Finally, the authors rescued the MPN phenotype partially in MPN mice by treating the mutant mice with IL-1R antagonist, a neuroglial protection agent (4-methylcatechol), or a β3-adrenergic agonist (BRL37344) which compensated for deficient sympathetic stimulation. This treatment was selective against mutant hematopoietic progenitors and preserved normal HSCs, and this effect could only be observed in vivo. Therefore, the authors concluded that the effect was niche-dependent (Figure 1).Open in a separate windowFigure 1Bone marrow neuropathy leads to mutant HSC expansion in MPN. (1) IL-1β is released by mutant HSCs, which induces Caspase-1-dependent apoptosis in sympathetic nerve fibers, ensheathed by nonmyelinating Schwann cells. (2) Neural-glial damage leads to a reduced noradrenergic sympathetic stimulation of Nestin⁺ MSCs and loss of MSCs. (3) Aberrant neural regulation sensitizes Nestin⁺ MSCs to IL-1β-induced apoptosis with a subsequent drop in CXCL12 expression. (4) HSC and progenitor cell proliferation is increased, followed by MPN pathogenesis. (5) Nestin⁺ MSCs survival and function can be restored by the neuroglial protective agent 4-methylcatechol, the β3-adrenergic agonist BRL37344, or by blocking IL-1R. MPN, myeloproliferative neoplasm; HSC, hematopoietic stem cell; MSC, mesenchymal stem cell; NA, noradrenaline; AR, adrenergic receptor; IL, Interleukin; IL-1R, Interleukin-1 receptor.While the prevailing understanding of cancer as a disease in which changes in the cell of origin drive oncogenic transformation, these studies point to the potential for the microenvironment as a critical cooperator in the malignant process for at least some neoplasms. This study emphasizes that there may be a two-way perturbation process required for MPN. HSCs acquire a mutation (e.g., JAK2-V617F mutation) that leads to cell expansion and the mutant HSC perturbs the bone marrow niche, which further drive HSCs into neoplasia. Based on these findings, the authors postulated that neural-glial protective agents and β3-adrenergic agonists may subvert the process and be therapeutically useful. Therefore, this model provides insight into how the neural compartment of the bone marrow microenvironment can serve as a modulator of malignancy and offers a novel, testable approach for treating MPNs — by not only targeting the malignant cell, but also by selectively targeting the unhealthy niche.     

To be or not to be a stem cell: dissection of cellular and molecular components of haematopoietic stem cell niches

Nature 481 7382, 457–462 (2012); published online January252012Recent studies have identified multiple cell types that regulate haematopoietic stem cells (HSCs); however, proof that a specific cell type produces a specific factor important for HSC function and maintenance is largely lacking. Ding et al (2012) reported recently that conditional deletion of stem cell factor (SCF) in Leptin receptor (Lepr) expressing perivascular cells or endothelial and haematopoietic cells resulted in significant reductions in number but less profound reduction in function of HSCs. Although the long-term fate of HSCs in these models is largely unexplored and an underlying mechanism for reduction in HSCs not yet reported, these findings further implicate the vascular niche in the functional maintenance of HSCs in vivo and also raise intriguing questions for future studies in this field.The haematopoietic stem cell (HSC) niche has traditionally been considered a discrete site within the bone marrow; however, recent studies have shown that numerous cell types are critically important for HSC regulation and maintenance (Wang and Wagers, 2011). Imaging studies have shown that phenotypic HSCs can be found adjacent to osteoblasts or osteoprogenitor cells on the inner surface of trabecular bone, and genetic studies have further shown that expansion of trabecular bone, leads to expansion of HSCs (Calvi et al, 2003; Zhang et al, 2003; Lo Celso et al, 2009; Xie et al, 2009). Other studies have found that phenotypic HSCs are frequently localized to the central marrow, specifically near endothelial or perivascular cells (Kiel et al, 2005). Recently, endothelial cells have been shown to support the ex vivo expansion of HSCs (Butler et al, 2010); however, it was so far not known whether endothelial or perivascular cells functionally maintain in vivo HSCs.Ding and colleagues used knockin reporter mice for Scf expression and found that stem cell factor (SCF) was produced predominantly by endothelial and perivascular cells but was not concentrated near the bone surface. To investigate which cellular sources of SCF are important for HSC maintenance, they conditionally deleted Scf specifically in haematopoietic cells, osteoblasts and Nestin-Cre expressing cells but found no significant effects on HSC maintenance. In contrast, conditional deletion in both haematopoietic and endothelial cells or in Leptin receptor (Lepr) expressing perivascular stromal cells significantly reduced phenotypic and, to a lesser extent, functional HSC frequency—thus further demonstrating that the vascular niche plays a role in functionally supporting HSCs (Figure 1). These findings underscore the complexity of the HSC niche and raise crucial future questions.Open in a separate windowFigure 1(A) HSCs reside in both osteoblastic and vascular niches. The vascular niche is juxtaposed with the osteoblastic niche and includes endothelial cells, CAR cells, Nestin⁺ cells, Lepr⁺ perivascular cells and other cell types. Ding et al show that SCF is predominantly provided by endothelial and perivascular cells. (B) Cell-specific deletion of Scf in endothelial and Lepr⁺ cells results in reduced HSCs; however, other HSCs are maintained, possibly from a quiescent reserved population that is less dependent on SCF, providing significant levels of haematopoiesis.The nature and specific identity of Lepr expressing cells is uncertain. This population appears to partially overlap with Nestin-Cre expressing cells, and it is not clear to what extent Lepr expressing cells might identify with Cxcl12-abundant reticular (CAR) cells, both of which have been previously identified as HSC niche components (Sugiyama et al, 2006; Mendez-Ferrer et al, 2010). Although phenotypic HSC frequency (determined by the cell-surface markers lineage⁻, Sca-1⁺, Kit⁺, CD150⁺, CD48⁻) is dramatically reduced, functional HSC frequency is only mildly compromised following conditional deletion of Scf either ubiquitously or in endothelial/perivascular cells. This indicates that other factors or sources of SCF maintain substantial numbers of HSCs independent of SCF produced by the vascular niche or elsewhere. Indeed, these results may be consistent with the coexistence of quiescent and active HSC populations—with the quiescent, reserved population serving as a backup HSC source to support life-long haematopoiesis, especially following the loss of active HSCs in response to stress (Li and Clevers, 2010). Considering the role of SCF in promoting proliferation (Broudy, 1997), it would be interesting to know the long-term effects of cell-specific deletion of Scf on HSC maintenance. Cell-specific deletion in Nestin-Cre expressing cells apparently did not affect HSC frequency long-term (5 months); however, such long-term data were not presented for osteoblast-specific knockout of Scf. It would also be interesting to know the mechanism for HSC loss following endothelial/perivascular-specific deletion of Scf. Interesting topics to address in the future are whether HSC quiescence is compromised, or whether apoptosis or differentiation is increased.As the authors note, multiple cell types is involved in HSC maintenance. Given the juxtaposition of endothelial and perivascular cells with the bone surface, the osteoblastic and vascular niche represent not always mutually exclusive entities (Lo Celso et al, 2009). We have recently proposed that stem cells may reside in special zones, where active stem cells may provide for the daily replenishment of tissues while quiescent, reserved stem cells serve as a backup sub-population to ensure life-long tissue maintenance and replenishment of the stem cell pool following stress (Li and Clevers, 2010). It remains for future studies to continue to determine which specific niche cells produce which particular factors for maintaining long-term quiescence versus those for supporting proliferation and survival of stem cells. The results published by Ding et al present a significant step towards this goal.     

Environmental stimuli and intestinal stem cell behavior

Comment on: Wong VWY, et al. Nat Cell Biol 2012; 14:401-8.The intestine carries out important functions related to digestion and absorption. It is composed of three distinct layers, an outer muscle layer, a mesenchymal layer and the epithelial layer. The epithelial layer forms the protective barrier that faces the luminal content of the intestine. In order to maintain barrier function the epithelial layer needs constant replenishment. This is ensured by continuous cellular replication in proliferative crypt compartments. Following exit from the crypt, cells adopt fates along either secretory or absorptive lineage and will, after three to four days, be exfoliated into the lumen of the intestine from the tips of the villi. Intestinal stem cells located at the bottom of the proliferative crypt compartment ensure lifelong maintenance of the organ (Fig. 1A).Open in a separate windowFigure 1. Diagram of the intestinal stem cell niche. (A) Lgr5-expressing columnar-based crypt cells (CBCs) intercalated between Paneth cells are indicated in green. Stem cells located in position +4 are yellow. Lrig1 is expressed in a gradient along the niche axis with highest expression in the CBCs indicated with the thickness of the red line. Proliferation in the stem cell niche ensures continuous replenishment of the transit-amplifying (TA) compartment. (B) Within the stem cell niche, Lgr5-expressing CBCs are actively dividing and will give rise to both HopX-expressing +4 cells and TA cells. HopX-expressing cells, which are less mitotically active, will give rise to fewer TA cells and occasionally an Lgr5-expressing stem cell. Lrig1 expression in the stem cell niche reduces the amplitude of ErbB activation and is essential for controlling stem cell proliferation.Adult stem cell niches are far more heterogeneous than previously anticipated.¹ The intestinal stem cell niche can be subdivided by the relative position within the crypt. Stem cells located in position +4, just above secretory Paneth cells, express HopX, Bmi1 and Tert. These cells are generally less mitotically active than Lgr5-expressing stem cells located at the bottom of the proliferative crypts intercalated between Paneth cells (Fig. 1A).^2,3 It has been argued that both populations represent the most primitive stem cell; however, recent studies suggest that stem cells can interconvert between the two states (Fig. 1B).³ Fate mapping from cells in position 4 and at the bottom of the crypt supports this.^2,4 The positional cues responsible for cellular sorting into different functional stem cell compartments are poorly characterized. The only known effector of cellular positioning is Wnt (wingless-related MMTV integration site) signaling.⁵ Wnt is highly expressed by Paneth cells along with other mitotic factors, such as ErbB and Notch ligands.⁶ This could functionally account for the differences observed in proliferative potential along the stem cell axis. The discrete expression patterns of Lgr5 and HopX also support the existence of distinct microenvironments that supports cellular identities. A thorough characterization of the factors responsible for stem cell identity will help delineate and define the functional relationship between the distinct stem cell populations.Tissue homeostasis is governed by balanced loss and gain of cells. The stem cell niche supports constant proliferation via pro-mitotic stimuli. In order to control the amplitude of signaling strength, many pathways have developed negative feedback loops. Lrig1 (Leucine-rich repeats and immunoglobulin-like domains 1) is a negative feedback regulator of ErbB-mediated growth factor signaling.⁷ Lrig1 marks stem cells in various epithelial tissues including the intestinal epithelium, where it is expressed within the entire stem cell niche including the +4 and Lgr5-expressing cells (Fig. 1).^8,9 The functional relevance of Lrig1 and negative feedback regulation is clear from the pronounced expansion of the intestinal stem cell compartment observed in the Lrig1-KO mouse model.⁹ This is mediated via increased ErbB signaling and demonstrates the importance of balanced signaling strength within the stem cell niche.⁹ Moreover, an independent study reveals that Lrig1-KO animals have a higher incidence of colorectal cancer, suggesting that unbalanced stem cell proliferation increases tumor susceptibility.¹⁰ Future studies will address whether additional feedback regulators control signaling strength within the intestinal stem cell niche and how homeostasis within the stem cell compartment affects tumor susceptibility.