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Netrin-1, an axon navigation cue was proposed to play a crucial role during colorectal tumorigenesis by regulating apoptosis. The netrin-1 receptors DCC and UNC5H were shown to belong to the family of dependence receptors that share the ability to induce apoptosis in the absence of their ligands. Such a trait confers on these receptors a tumor suppressor activity. Expression of one of these dependence receptors at the surface of a tumor cell is indeed speculated to render this cell dependent on ligand availability for its survival, hence inhibiting uncontrolled cell proliferation or metastasis. Consequently, it is a selective advantage for a tumor cell to lose this dependence receptor activity, as previously described with losses of DCC and UNC5H expression in human cancers. However, the model predicts that a similar advantage may be obtained by gaining autocrine expression of the ligand. We describe here that, unlike human nonmetastatic breast tumors, a large fraction of metastatic breast cancers overexpress netrin-1. Moreover, we show that netrin-1-expressing mammary metastatic tumor cell lines undergo apoptosis when netrin-1 expression is experimentally decreased or when decoy soluble receptor ectodomains are added. Such treatments prevent metastasis formation both in a syngenic mouse model of lung colonization of a mammary cancer cell line and in a model of spontaneous lung metastasis of xenografted human breast tumor. Thus, netrin-1 expression observed in a large fraction of human metastatic breast tumors confers a selective advantage for tumor cell survival and potentially represents a promising target for alternative anticancer therapeutic strategies.

BACKGROUND & AIMS:Netrin-1 was recently proposed to play a crucial role during colorectal tumorigenesis by regulating apoptosis. Because netrin-1 receptors belong to the family of dependence receptors, a selective advantage for a tumor is either to lose netrin-1 receptors or to gain autocrine expression of netrin-1. We have investigated whether netrin-1 is up-regulated in colorectal cancer and have searched for a link between NF-kappaB activation and netrin-1 up-regulation.METHODS:The level of netrin-1, netrin-1 receptors, ie, DCC, UNC5H1, UNC5H2, UNC5H3, and the proinflammatory markers cyclooxygenase-2 and inhibitor of nuclear factor-kappaB (IkappaB) alpha were analyzed in a panel of 59 primary sporadic colorectal carcinomas. Netrin-1 expression was investigated in tumor cells and in mouse colonic crypts in response to NF-kappaB activation but also in a mouse model of inflammation-induced colorectal cancer. Binding of NF-kappaB to netrin-1 promoter and effect of NF-kappaB activation to the proapoptotic activity of UNC5H2 were also analyzed.RESULTS:We show that colorectal tumors with a gain of netrin-1 are tumors that display increased activation of the NF-kappaB pathway. Moreover, netrin-1 up-regulation, which is associated with tumor formation in mice, is observed in mouse colonic crypts in response to NF-kappaB activation but also in a mouse model of inflammation-induced colorectal cancer. We demonstrate that the netrin-1 gene is a direct transcriptional target of NF-kappaB. We show that NF-kappaB-induced netrin-1 expression inhibits proapoptotic activity of the netrin-1 receptors.CONCLUSIONS:We propose that NF-kappaB activation that occurs in response to inflammation confers a selective advantage for tumor development through NF-kappaB-mediated netrin-1 up-regulation.

UNC5H receptors (UNC5H1, UNC5H2, UNC5H3) are putative tumor suppressors whose expression is lost in numerous cancers. These receptors have been shown to belong to the so-called family of dependence receptors. Such receptors induce apoptosis when their ligand netrin-1 is absent, thus conferring a state of cellular dependence towards ligand presence. Along this line, these receptors may limit tumor progression because they induce the death of tumor cells that grow in settings of ligand unavailability. We show here that UNC5H receptors are localized to cholesterol-and sphingolipid-enriched membrane domains called lipid rafts. We then demonstrate that the lipid raft localization of UNC5H2 is required for the pro-apoptotic activity of unbound UNC5H2. We also propose that this lipid raft localization is probably mediated via the recruitment of adaptor protein(s) within the death domain of UNC5H2 but is not dependent on the post-translational modification by palmitoylation of UNC5H2 even though this palmitoylation is required for UNC5H2 pro-apoptotic activity. Moreover we show that the interaction of UNC5H2 with the downstream pro-apoptotic serine threonine kinase DAPk is dependent on both UNC5H2 lipid raft localization and palmitoylation. Thus, we propose that the UNC5H dependence receptors require lipid raft localization and palmitoylation to trigger apoptosis.

A novel way of seeing cellular receptors has recently emerged. While a receptor used to be considered as inactive until bound by its ligand, it has been proposed that some receptors may also be active in the absence of their ligand. These so-called dependence receptors induce a specific death signal when the ligand is absent from the cell: Therefore, the expression of one of these receptors leads to the cell becoming dependent on the presence of the ligand for its survival. We have hypothesized that such a trait is a mechanism that allows inhibition of tumor growth, by inducing apoptosis "abnormal" cells that would usually grow in settings of ligand unavailability, i.e. local growth of tumor cells or growth beyond primary tumor site. Along this line, back in the early 90s, Vogelstein and colleagues suggested that a gene called DCC (for deleted in colorectal cancer) could be a tumor suppressor gene because it was found to be deleted in more than 70% of colorectal cancers, as well as in many other cancers. During the last fifteen years, controversial data have failed to firmly establish whether DCC is indeed a tumor suppressor gene. However, our observation that DCC behaves as a dependence receptor that induces cell death unless its ligand netrin-1 is present, together with the fact that mice engineered to block DCC-induced cell death by overexpressing netrin-1 are predisposed to develop colorectal tumors, strengthen the role of dependence receptors as tumor suppressors. In this review, we will describe the implication of the netrin-1/receptor pairs as novel negative regulators of tumor development.

More than 10 years after its initial discovery, netrin-1 - the first described chimioattractive molecule controlling the guidance of the commissural axons - has recently known a unsuspected wave of interest because of its implication in the development of the nervous system but also, more recently, fot its role in angiogenesis and tumorigenesis. Because, of a series of recent publications on netrin-1 signaling, we propose here to describe the recent insight in netrin-1 signaling via its main receptor DCC (deleted in colorectal cancer), and the recent discovery that netrin controls the assymetric distribution of beta-actin in the growth cone. Thus, it seems that netrin-1, but also the neurotrophic factor BDNF, controls acute growth cone responses such as collapse and turning by the regulation of localized protein translation, such as beta-actin. This process involves both transport of beta-actin mRNA, bound to Vg1RBP, to specific locations, and mRNA translation upon stimulation by local activation of the translation initiation regulator eIF-4E-binding protein 1. Indeed, Netrin-1 induces the movement of Vg1RBP granules into filopodia, and triggers a polarized increase in beta-actin translation on the near side of the growth cone before growth cone turning. The binding of BDNF to its receptor Trk has a similar effect for growth cone attraction, althought it is differentially regulated. Thus, this asymetrically synthesized beta-actin may direct actin polymerization and consequently the migration of the growth cone toward the cue.

Background: Some receptors are active in the absence of ligand and actively trigger cell death through apoptosis. These receptors are called “dependence receptors” (DRs) as their expression at the cell surface renders cells critically dependent for their survival on ligand availability. Deleted in Colorectal Carcinoma (DCC) and UNC5H are prototypic DRs which induce apoptosis unless their ligand netrin-1 is present. It has been shown that netrin-1 is up-regulated in a large fraction of tumors and that interference with netrin-1/receptors interaction is associated with inhibition of tumor growth and metastasis in various preclinical models. We have generated the first humanized netrin-1 antibody, NP137, and characterized its antitumor activity. Preclinical results: NP137 is a humanized monoclonal IgG1 antibody, which binds netrin-1 in the nanomolar range affinity and efficiently inhibits the binding of netrin-1 to its receptor UNC5H2 (IC50: 0.5 nM). NP137 recognizes a netrin-1 specific epitope located in the second laminin-type EGF-like repeat. A crystal structure analysis shows that this region is crucial for the binding of netrin-1 to its receptor, providing a structural basis for the biological activity of NP137. In vitro, NP137 induced apoptosis in netrin-1-dependent cell lines as assessed by cell density measurement and caspase 3 activation. Ex vivo analysis performed on fresh human breast tumor slices (3D cultures) showed a specific and significant induction of apoptosis in tumor cells in approximately half of cases. In vivo, NP137 exhibited a significant anti-tumor effect associated with good pharmacokinetic properties in models of both solid tumors and hematologic malignancies. A dose-effect relationship was observed with an optimal pharmacologically active dose established at 10 mg/kg twice a week. We also showed that NP137 potentiates cancer cell death induced by cytotoxic drugs like doxorubicin or platinum derivatives as well as demethylating agents, which induce upregulation of netrin-1 and its receptors. In this context, a strong synergy with doxorubicin was shown in a rat syngeneic model of osteosarcoma, reversing its chemoresistant phenotype. Toxicological studies are ongoing with first results suggesting a high therapeutic margin. Conclusion: We are developing the first therapeutic antibody targeting netrin-1 and inducing dependence receptors-mediated tumor cell death. Preclinical results suggest a therapeutic potential both in solid tumors and hematological malignancies. A high therapeutic margin is expected, which may accelerate combination strategies. Based on these encouraging results, a first-in-human trial is planned at the end of 2015, with the support of EORTC. Note: This abstract was not presented at the meeting. Citation Format: Benjamen Ducarouge, Jean-Guy Delcros, Riad Abès, David Goldschneider, Benjamin Gibert, John Blachier, David Neves, Patrick Mehlen, Agnès Bernet, Stéphane Depil. Preclinical characteristics of NP137, a first-in-class monoclonal antibody directed against netrin-1 and inducing dependence receptors-mediated cell death. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2921. doi:10.1158/1538-7445.AM2015-2921

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase, initially discovered as part of the NPM-ALK fusion protein, resulting from the t(2;5) translocation that is frequently associated with anaplastic large-cell lymphomas. The native ALK protein is normally expressed in the developing and, at a weaker level, adult nervous system. We recently demonstrated that the oncogenic, constitutively kinase-activated NPM-ALK protein was antiapoptotic when expressed in Jurkat lymphoblastic cells treated with cytotoxic drugs. In contrast, we now show that Jurkat cells overexpressing the wild-type ALK receptor are more sensitive to doxorubicin-induced apoptosis than parental cells. Moreover, the ALK protein is cleaved during apoptosis in a caspase-dependent manner. Mutation of aspartic residues to asparagine allowed us to map the caspase cleavage site in the juxtamembrane region of ALK. In order to assess the role of ALK in neural cell-derived tissue, we transiently expressed ALK in the 13.S.1.24 rat neuroblast immortalized cell line. ALK expression led to apoptotic cell death of the neuroblasts. ALK ligation by specific activating antibodies decreased ALK-facilitated apoptosis in both lymphoid and neuronal cell lines. Moreover, ALK transfection reduced the survival of primary cultures of cortical neurons. Thus, ALK has a proapoptotic activity in the absence of ligand, whereas it is antiapoptotic in the presence of its ligand and when the kinase is intrinsically activated. These properties place ALK in the growing family of dependence receptors.

Recent studies have led a different view about membrane receptors. While a receptor used to be considered as inactive until bound by its ligand, it has been proposed that some receptors may also be active in the absence of their ligand. These so-called dependence receptors induce a specific death signal when the ligand is absent from the cell. Therefore, the expression of one of these receptors drives the cell to become dependent on the presence of the ligand for its survival. We have hypothesized that this mechanism allows inhibition of tumor growth, by inducing apoptosis of "abnormal" cells that would usually grow when ligand are unavailable--i.e., during local growth of tumor cells or growth beyond primary tumor site -. Along this line, back in the early 90s, Vogelstein and colleagues suggested that a gene called DCC (for "deleted in colorectal cancer") could be a tumor suppressor gene because it was found to be deleted in more than 70 % of colorectal cancers, as well as in many other cancers. During the last fifteen years, controversial data have failed to firmly establish whether DCC is indeed a tumor suppressor gene. However, our observation that DCC behaves as a dependence receptor that induces cell death unless its ligand netrin-1 is present, together with the fact that mice engineered to block DCC-induced cell death by overexpressing netrin-1 are predisposed to develop colorectal tumors, strengthen the role of dependence receptors as tumor suppressors. In this review, we will describe the implication of the netrin-1/receptor pairs as novel negative regulators of tumor development.

Netrin-1 has been shown to play a crucial role in neuronal navigation during nervous system development mainly through its interaction with its receptors DCC and UNC5H. However, initially the DCC (deleted in colorectal cancer) gene was proposed as a putative tumor suppressor gene. It was then difficult to reconcile the two activities of DCC until the observation that DCC belongs to an emerging family of receptors named dependence receptors. Such receptors share the property of inducing apoptosis in the absence of ligand, hence creating a cellular state of dependence on the ligand. Thus, netrin-1 may not only be a chemotropic factor for neurons but also a survival factor. We will review here the identification of netrin-1 and its receptors, the signaling pathways initiated in the presence or absence of netrin-1. We will suggest some possible roles of netrin-1 in nervous system development, neovascularisation, adhesion and tumorigenesis.

The notion of "morphogens" is an important one in developmental biology. By definition, a morphogen is a molecule that emanates from a specific set of cells that is present in a concentration gradient and that specifies the fate of each cell along this gradient. The strongest candidate morphogens are members of the transforming growth factor-beta (TGF-beta), Hedgehog (Hh), and Wnt families. While these morphogens have been extensively described as differentiation inducers, some reports also suggest their possible involvement in cell death and cell survival. It is frequently speculated that the cell death induction that is found associated with experimental removal of morphogens is the manifestation of abnormal differentiation signals. However, several recent reports have raised controversy about this death by default, suggesting that cell death regulation is an active process for shaping tissues and organs. In this review, we will present morphogens, with a specific emphasis on Sonic Hedgehog, a mammalian member of the Hh family, not as a positive regulators of cell differentiation but as key regulators of cell survival.

Cells depend for their survival on stimulation by trophic factors and other prosurvival signals, the withdrawal of which induces apoptosis, both via the loss of antiapoptotic signaling and the activation of proapoptotic signaling via specific receptors. These receptors, dubbed dependence receptors, activate apoptotic pathways following the withdrawal of trophic factors and other supportive stimuli. Such receptors may feature in developmental cell death, carcinogenesis (including metastasis), neurodegeneration, and possibly subapoptotic events such as neurite retraction and somal atrophy. Mechanistic studies of dependence receptors suggest that these receptors form ligand-dependent complexes that include specific caspases. Complex formation in the absence of ligand leads to caspase activation by a mechanism that is typically dependent on caspase cleavage of the receptor itself, releasing proapoptotic peptides. Cellular dependence receptors, considered in the aggregate, may thus form a system of molecular integration, analogous to the electrical integration system provided by dendritic arbors in the nervous system.

Although cancer is a multifaceted disease, all cancer types share identical molecular and cellular mechanisms. These mechanisms involve a collection of alterations critical to the normal physiological functioning of cells, such as alterations of growth factor signalling pathways, angiogenesis, cell adhesion signals, DNA replication and apoptotic cell death. Many genes involved in the processes enumerated above are functionally inactive in tumour cells, designating them as putative 'tumour suppressor genes'. Back in the early 1990s, Vogelstein and colleagues suggested that a gene called DCC (for Deleted in Colorectal Cancer) could be a tumour suppressor gene because it was found to be deleted in more than 70% of colorectal cancers, as well as in many other cancers. During the last 15 years, controversial data have failed to firmly establish whether DCC is indeed a tumour suppressor gene. However, the recent observations that DCC triggers cell death and is a receptor for netrin-1, a molecule recently implicated in colorectal tumorigenesis, have prompted a renewal of interest in the role of DCC in tumorigenesis and suggest that the netrin-1/receptor pairs act as novel negative regulators of tumour development.

During development, axons migrate long distances in responses to attractive or repulsive signals that are detected by their growth cones. One of these signals is mediated by netrin-1, a diffusible laminin-related molecule that both attracts and repels growth cones via interaction with its receptor DCC (deleted in colorectal cancer). Here we show that DCC in both commissural neurons and immortalized cells, is partially associated with cholesterol- and sphingolipid-enriched membrane domains named lipid rafts. This localization of DCC in lipid rafts is mediated by the palmitoylation within its transmembrane region. Moreover, this raft localization of DCC is required for netrin-1-induced DCC-dependent ERK activation, and netrin-1-mediated axon outgrowth requires lipid raft integrity. Thus, the presence of axon guidance-related receptors in lipid rafts appears to be a crucial pre-requisite for growth cone response to chemo-attractive or repulsive cues.

Programmed cell death (PCD) is physiologically involved in the regulation of cell division and differentiation. It encompasses caspase-dependent mitochondrial and nonmitochondrial pathways. Additional caspase-independent pathways have been characterized in mitochondrial PCDs but remain hypothetical in nonmitochondrial PCDs. Epidermal growth factor (EGF) has been shown to inhibit division of pituitary somato-lactotrope cells occurring in parallel with EGF-mediated differentiation of these precursors into lactotrope cells. We show here that in somato-lactotrope pituitary cell line GH4C1, EGF triggers a PCD characterized by an apoptosis-like DNA fragmentation, insensitivity to broad-range caspase inhibitors, and absence of either cytochrome c or apoptosis-inducing factor release from mitochondria. Dying cells display loose chromatin clustering and numerous cytoplasmic vacuoles, a fraction of which are autophagic, thus conferring a heterogeneous phenotype to this PCD. Moreover, overexpression of cell death inhibitor Bcl-2 prevented not only the EGF-induced PCD but also its prodifferentiation effects, thus pointing to a mechanistic relationship existing between these two phenomena. Overall, the characterized differentiation-linked cell death represents an original form of caspase-independent PCD. The mechanisms underlying this PCD involve combinatorial engagement of discrete death effectors leading to a heterogeneous death phenotype that might be evolutionary related to PCD seen during the differentiation of some unicellular organisms.

The recently described family of dependence receptors is a new family of functionally related receptors. These proteins have little sequence similarity but display the common feature of inducing two completely opposite intracellular signals depending on ligand availability: in the presence of ligand, these receptors transduce a positive signal leading to survival, differentiation or migration, while in the absence of ligand, the receptors initiate or amplify a negative signal for apoptosis. Thus, cells that express these proteins manifest a state of dependence on their respective ligands. The mechanisms that trigger cell death induction in the absence of ligand are in large part unknown, but typically require cleavage by specific caspases. In this review we will present the proposed mechanisms for cell death induction by these receptors and their potential function in nervous system development and regulation of tumorigenesis.

A new family of functionally-related receptors has recently been proposed, dubbed dependence receptors. These proteins, only some of which share sequence similarities, display the common property that they transduce two different intracellular signals: in the presence of ligand, these receptors transduce a positive signal leading to survival, differentiation or migration; conversely, in the absence of ligand, the receptors initiate or amplify a signal for programmed cell death. Thus cells that express these proteins at sufficient concentrations manifest a state of dependence on their respective ligands. The signaling that mediates cell death induction upon ligand withdrawal is in large part uncharacterized, but typically includes a required interaction with, and cleavage by, specific caspases. Here, we review the current knowledge concerning dependence receptors, including the shared mechanisms for cell death induction and their potential relevance in nervous system development and regulation of tumorigenesis.

The expression of the protein DCC (deleted in colorectal cancer) is lost or markedly reduced in numerous cancers and in the majority of colorectal cancers due to loss of heterozygosity in chromosome 18q, and has therefore been proposed to be a tumour suppressor. However, the rarity of mutations found in DCC, the lack of cancer predisposition of DCC mutant mice, and the presence of other tumour suppressor genes in 18q have raised doubts about the function of DCC as a tumour suppressor. Unlike classical tumour suppressors, DCC has been shown to induce apoptosis conditionally: by functioning as a dependence receptor, DCC induces apoptosis unless DCC is engaged by its ligand, netrin-1 (ref. 3). Here we show that inhibition of cell death by enforced expression of netrin-1 in mouse gastrointestinal tract leads to the spontaneous formation of hyperplastic and neoplastic lesions. Moreover, in the adenomatous polyposis coli mutant background associated with adenoma formation, enforced expression of netrin-1 engenders aggressive adenocarcinomatous malignancies. These data demonstrate that netrin-1 can promote intestinal tumour development, probably by regulating cell survival. Thus, a netrin-1 receptor or receptors function as conditional tumour suppressors.The expression of the protein DCC (deleted in colorectal cancer) is lost or markedly reduced in numerous cancers and in the majority of colorectal cancers due to loss of heterozygosity in chromosome 18q, and has therefore been proposed to be a tumour suppressor. However, the rarity of mutations found in DCC, the lack of cancer predisposition of DCC mutant mice, and the presence of other tumour suppressor genes in 18q have raised doubts about the function of DCC as a tumour suppressor. Unlike classical tumour suppressors, DCC has been shown to induce apoptosis conditionally: by functioning as a dependence receptor, DCC induces apoptosis unless DCC is engaged by its ligand, netrin-1 (ref. 3). Here we show that inhibition of cell death by enforced expression of netrin-1 in mouse gastrointestinal tract leads to the spontaneous formation of hyperplastic and neoplastic lesions. Moreover, in the adenomatous polyposis coli mutant background associated with adenoma formation, enforced expression of netrin-1 engenders aggressive adenocarcinomatous malignancies. These data demonstrate that netrin-1 can promote intestinal tumour development, probably by regulating cell survival. Thus, a netrin-1 receptor or receptors function as conditional tumour suppressors.

Programmed cell death occurs in response to both the presence of various extracellular factors and the lack of specific factors. Receptors that can mediate cell death in the absence of ligand binding are called dependence receptors, and they were the topic of the meeting held during the summer of 2003 in Fondation des Treilles, France. Not only is progress being made in the identification of new dependence receptors, but the partners that carry out this "negative" signal are also coming to light. With several of the receptors implicated in various human developmental disorders or disease states, gaining an understanding of the molecular mechanisms controlling dependence receptor-mediated cell death has clear clinical relevance.

During early development in vertebrates, Sonic hedgehog (Shh) is produced by the notochord and the floor plate. A ventrodorsal gradient of Shh directs ventrodorsal patterning of the neural tube. However, Shh is also required for the survival of neuroepithelial cells. We show that Patched (Ptc) induces apoptotic cell death unless its ligand Shh is present to block the signal. Moreover, the blockade of Ptc-induced cell death partly rescues the chick spinal cord defect provoked by Shh deprivation. Thus, the proapoptotic activity of unbound Ptc and the positive effect of Shh-bound Ptc on cell differentiation probably cooperate to achieve the appropriate spinal cord development.

Neuronal growth cones are guided to their targets by attractive and repulsive guidance cues. In mammals, netrin-1 is a bifunctional cue, attracting some axons and repelling others. Deleted in colorectal cancer (Dcc) is a receptor for netrin-1 that mediates its chemoattractive effect on commissural axons, but the signalling mechanisms that transduce this effect are poorly understood. Here we show that Dcc activates mitogen-activated protein kinase (MAPK) signalling, by means of extracellular signal-regulated kinase (ERK)-1 and -2, on netrin-1 binding in both transfected cells and commissural neurons. This activation is associated with recruitment of ERK-1/2 to a Dcc receptor complex. Inhibition of ERK-1/2 antagonizes netrin-dependent axon outgrowth and orientation. Thus, activation of MAPK signalling through Dcc contributes to netrin signalling in axon growth and guidance.

The olfactory epithelium of adult mouse, where primary sensory neurons are massively committed to apoptosis by removal of their synaptic target, was used as a model to determine in vivo mechanisms for neuronal cell death induction. A macro-array assay revealed that the death of olfactory neurons is accompanied with over-expression of the serine protease inhibitor Spi2. This over-expression is associated with decreased serine protease activity in the olfactory mucosa. Moreover, in vitro or in vivo inhibition of serine proteases induced apoptotic death of olfactory neuronal cells. Interestingly, Spi2 over-expression is not occurring in olfactory neurons but in cells of the lamina propria, suggesting that Spi2 may act extracellularly as a cell death inducer. In that sense, we present evidence that in vitro Spi2 overexpression generates a secreted signal for olfactory neuron death. Hence, taken together these results document a possible novel mechanism for apoptosis induction that might occur in response to neurodegenerative insults.

Programmed cell death (apoptosis) is a complex phenomenon that is mediated in mammals mainly via the selective cleavage of intracellular proteins by the large family of cysteine aspartate protease caspases. Apoptosis is tightly regulated by the competitive effect of numerous proteins displaying either pro-apoptotic or anti-apoptotic activity. The ETS-family transcription factor FLI-1, frequently associated with malignant transformation, has been shown to display anti-apoptotic activity in several cell types including avian erythroblasts, mouse fibroblasts or lymphoid cells. We show here that apoptosis of murine preB leukemic cells is accompanied with the specific cleavage of FLI-1 by a caspase-like activity. We also demonstrate that the two isoforms of FLI-1 are indeed cleaved at three conserved sites by caspase 3 in vitro. The conservation of these cleavage sites among species suggests that the caspase cleavage of the anti-apoptotic transcription factor FLI-1 may represent a critical step to ensure irreversible cell death.Programmed cell death (apoptosis) is a complex phenomenon that is mediated in mammals mainly via the selective cleavage of intracellular proteins by the large family of cysteine aspartate protease caspases. Apoptosis is tightly regulated by the competitive effect of numerous proteins displaying either pro-apoptotic or anti-apoptotic activity. The ETS-family transcription factor FLI-1, frequently associated with malignant transformation, has been shown to display anti-apoptotic activity in several cell types including avian erythroblasts, mouse fibroblasts or lymphoid cells. We show here that apoptosis of murine preB leukemic cells is accompanied with the specific cleavage of FLI-1 by a caspase-like activity. We also demonstrate that the two isoforms of FLI-1 are indeed cleaved at three conserved sites by caspase 3 in vitro. The conservation of these cleavage sites among species suggests that the caspase cleavage of the anti-apoptotic transcription factor FLI-1 may represent a critical step to ensure irreversible cell death.

The membrane receptors DCC and UNC5H have been shown to be crucial for axon guidance and neuronal migration by acting as receptors for netrin-1. DCC has also been proposed as a dependence receptor inducing apoptosis in cells that are beyond netrin-1 availability. Here we show that the netrin-1 receptors UNC5H (UNC5H1, UNC5H2, UNC5H3) also act as dependence receptors. UNC5H receptors induce apoptosis, but this effect is blocked in the presence of netrin-1. Moreover, we demonstrate that UNC5H receptors are cleaved in vitro by caspase in their intracellular domains. This cleavage may lead to the exposure of a fragment encompassing a death domain required for cell death induction in vivo. Finally, we present evidence that during development of the nervous system, the presence of netrin-1 is crucial to maintain survival of UNC5H- and DCC-expressing neurons, especially in the ventricular zone of the brainstem. Altogether, these results argue for a role of netrin-1 during the development of the nervous system, not only as a guidance cue but as a survival factor via its receptors DCC and UNC5H.

The common neurotrophin receptor, p75(NTR), has been shown to signal in the absence of Trk tyrosine kinase receptors, including induction of neural apoptosis and activation of NF-kappaB. However, the mechanisms by which p75(NTR) initiates these intracellular signal transduction pathways are unknown. Here we report interactions between p75(NTR) and the six members of TRAF (tumor necrosis factor receptor-associated factors) family proteins. The binding of different TRAF proteins to p75(NTR) was mapped to distinct regions in p75(NTR). Furthermore, TRAF4 interacted with dimeric p75(NTR), whereas TRAF2 interacted preferentially with monomeric p75(NTR). TRAF2-p75(NTR), TRAF4-p75(NTR), and TRAF6-p75(NTR) interactions modulated p75(NTR)-induced cell death and NF-kappaB activation with contrasting effects. Coexpression of TRAF2 with p75(NTR) enhanced cell death, whereas coexpression of TRAF6 was cytoprotective. Furthermore, overexpression of TRAF4 abrogated the ability of dimerization to prevent the induction of apoptosis normally mediated by monomeric p75(NTR). TRAF4 also inhibited the NF-kappaB response, whereas TRAF2 and TRAF6 enhanced p75(NTR)-induced NF-kappaB activation. These results demonstrate that TRAF family proteins interact with p75(NTR) and differentially modulate its NF-kappaB activation and cell death induction.

The development of colonic carcinoma is associated with the mutation of a specific set of genes. One of these, DCC (deleted in colorectal cancer), is a candidate tumour-suppressor gene, and encodes a receptor for netrin-1, a molecule involved in axon guidance. Loss of DCC expression in tumours is not restricted to colon carcinoma, and, although there is no increase in the frequency of tumour formation in DCC hemizygous mice, reestablishment of DCC expression suppresses tumorigenicity. However, the mechanism of action of DCC is unknown. Here we show that DCC induces apoptosis in the absence of ligand binding, but blocks apoptosis when engaged by netrin-1. Furthermore, DCC is a caspase substrate, and mutation of the site at which caspase-3 cleaves DCC suppresses the pro-apoptotic effect of DCC completely. These results indicate that DCC may function as a tumour-suppressor protein by inducing apoptosis in settings in which ligand is unavailable (for example, during metastasis or tumour growth beyond local blood supply) through functional caspase cascades by a mechanism that requires cleavage of DCC at Asp 1,290.

The mammalian small stress protein hsp27 is an oligomeric phosphoprotein which interferes with the cell death induced by several stimuli. In that sense, we and others have recently shown that human hsp27 expression induced cellular protection against tumor necrosis factor (TNFalpha), a protection which depends on the ability of hsp27 to decrease the level of reactive oxygen species and increase that of glutathione. Here, we have analyzed unphosphorylatable mutants of human hsp27 in which serines 15, 78, and 82 were replaced by alanines, glycines, or aspartic acids. Depending on the amino acid which was used to substitute the serine sites, a different pattern of hsp27 structural organization was observed. Alanine substitution generated large hsp27 aggregates while glycine and aspartic acid did the reverse. Hence, these phosphorylatable serine residues can be considered as key elements affecting hsp27 structural organization. Only the large aggregates of hsp27 were able to modulate reactive oxygen species and glutathione and generated cellular protection against TNFalpha. Moreover, using drugs that modulate the intracellular level of glutathione, we show that an increase in glutathione by itself was sufficient to generate large hsp27 structures while the reverse was observed in the case of glutathione deprivation.

Small stress proteins are developmentally regulated and linked to cell growth and differentiation. The early phase of murine embryonic stem (ES) cell differentiation, characterized by a gradual growth arrest, is accompanied with hsp27 transient accumulation. This differentiation process also correlated with changes in hsp27 phosphorylation and oligomerization. The role of hsp27 was investigated in ES clones stably transfected with murine or human hsp27 genes, placed in sense or antisense orientation. Several clones were obtained that either underexpressed endogenous murine hsp27 or overexpressed murine or human hsp27. Maintained undifferentiated, these clones showed similar growth rates. We report here that hsp27 constitutive overexpression enhanced the differentiation-mediated decreased rate of ES cell proliferation but did not alter morphological changes. In contrast, hsp27 underexpression, which attenuated cell growth arrest, induced differentiation abortion because of an overall cell death by apoptosis. Recently, we showed that hsp27 interfered with cell death probably because of its ability to modulate intracellular glutathione. hsp27 accumulation during ES cell differentiation was also correlated with an increase in glutathione, which was attenuated by hsp27 down-expression. Hence, hsp27 transient expression seems essential for preventing differentiating ES cells from undergoing apoptosis, a switch that may be redox regulated.

> Il est classiquement admis qu’un recepteur est uniquement actif lorsqu’il interagit avec son ligand. La famille des recepteurs dits « a dependance » est l’exception qui confirme la regle. En effet, en presence de ligand, ces recepteurs induisent des signaux de proliferation, differenciation ou migration, mais ils sont egalement actifs en absence de leur ligand puisqu’ils induisent la mort de la cellule par apoptose. Ces recepteurs sont donc dits « a dependance » car une cellule qui les exprime devient dependante de la presence du ligand pour survivre. Cette famille emergente compte aujourd’hui une dizaine de membres : P75NTR, DCC, les recepteurs UNC5H1, UNC5H2 et UNC5H3, RET, le recepteur aux androgenes, les integrines αVβ3 et α5β1, Patched et Neogenine [1]. Le recepteur DCC (deleted in colorectal cancer) est un recepteur transmembranaire initialement decrit comme un potentiel suppresseur de tumeurs car son expression est perdue dans 70 % des cancers colorectaux ainsi que dans de nombreux autres cancers. Si l’activite suppresseur de tumeurs de DCC n’est pas encore formellement demontree, il semblerait que DCC previenne l’echappement tumoral en eliminant par apoptose les cellules cancereuses. En effet, les cellules tumorales proliferent intensement dans un environnement ou la concentration en netrine-1 reste constante, les cellules seraient donc en deficit de netrine-1 et DCC declencherait leur apoptose. De la meme maniere, une cellule tumorale metastatique qui quitterait le site primaire de tumorigenese pour envahir d’autres tissus perdrait la netrine-1 et entrerait alors en apoptose. Ainsi, une perte d’expression de DCC, suite a une perte d’heterozygotie ou a la methylation du promoteur, serait un avantage selectif qui rendrait les cellules resistantes a l’absence de netrine-1. De meme, une surexpression de netrine-1 par les cellules environnantes ou par les cellules tumorales elles-memes permettrait aux cellules cancereuses de proliferer ou d’envahir d’autres tissus. Ainsi, des souris transgeniques, dont la mort induite par DCC est inhibee par une surexpression de netrine-1, sont predisposees au developpement de tumeurs colorectales [2]. Le recepteur DCC n’est pas seulement implique dans les processus tumoraux puisqu’au cours Figure 1. Le recepteur DCC. Il s’agit d’un suppresseur de tumeurs presomptif qui controlerait l’echappement tumoral en induisant l’apoptose des cellules qui se retrouvent deficitaires en netrine-1 car elles ont prolifere ou migre vers d’autres tissus depourvus de netrine-1. La perte d’expression du recepteur et la surexpression ou l’expression autocrine de netrine-1 seraient des avantages selectifs pour le developpement tumoral. Une alteration de la localisation de DCC dans les radeaux lipidiques, a la suite d’une modification des lipides membranaires ou de la palmitoylation du recepteur, pourrait conduire a une perte de l’activite pro-apoptotique de DCC. Perte de l'activite apoptotique de DCC Perte d'expression de DCC Surexpression de netrine-1 Alteration de la localisation membranaire de DCC

Guidance molecules ensure that nerve cells grow in the correct direction during brain development. However, very little is known about the signals transmitted by these molecules. It now seems that netrin-1 — the first guidance molecule discovered — achieves this, in part, through activation of PITPα, a protein involved in lipid transfer.

Backgrounds & Aims: The TrkC neurotrophin receptor belongs to the functional dependence receptors family, members of which share the ability to induce apoptosis in the absence of their ligands. Such a trait has been hypothesized to confer tumor-suppressor activity. Indeed, cells that express these receptors are thought to be dependent on ligand availability for their survival, a mechanism that inhibits uncontrolled tumor cell proliferation and migration. TrkC is yet a relatively classic tyrosine kinase receptor and therefore generally rather considered as a proto-oncogene. We investigated here whether, because of its dependence receptor activity, TrkC is a putative tumor suppressor in colorectal malignancies. Methods: The level of TrkC was analyzed in a panel of 45 primary sporadic colorectal carcinomas. Loss of heterozygosity and epigenetic alterations in the TrkC promoter were also analyzed. The level of TrkC was also analyzed in a panel of colorectal cancer cell lines. The TrkC tumor suppressive activity was investigated by expressing TrkC and related TrkC variants in colorectal cancer cell lines and by measuring apoptosis, anchorage-independent growth, migration and tumor growth in a chicken model. Results: We show here that TrkC expression is down-regulated in a large fraction of human colorectal cancers, mainly through promoter methylation. Moreover, we show that TrkC silencing by promoter methylation is a selective advantage for colorectal cell lines to limit tumor cell death. Consequently, we show that reestablished TrkC expression in colorectal cancer cell lines is associated with tumor cell death and inhibition of in vitro characteristics of cell transformation and in vivo tumor growth. Conclusions: The loss of TrkC expression observed in human colorectal cancer is a selective advantage for tumor cell survival. Thus, TrkC appears to be a putative new colon cancer tumor suppressor. Citation Format: Anne-Laure Genevois, Gabriel Ichim, Marie-May Coissieux, Marie-Pierre Lambert, Florian Lepinasse, Zdenko Herceg, Jean-Yves Scoazec, Servane Tauszig-Delamasure, Patrick Mehlen. The dependence receptor TrkC is a putative colon cancer tumor suppressor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3036. doi:10.1158/1538-7445.AM2013-3036

Guidance molecules called semaphorins ensure that nerve cells grow in the correct direction during development. It now appears that one semaphorin interacts with integrin proteins to produce another effect.

The Netrin-1 receptors UNC5A, B and C belong to the functional family of dependence receptors that share the ability to induce apoptosis in the absence of their ligands. Such a trait has been hypothesized to confer a tumor suppressor activity to these receptors. Expression of one of these dependence receptors at the surface of a tumor cell is thus speculated to render this cell dependent on ligand for its survival, hence inhibiting cell proliferation or migration outside the ligand availability zone. Here we show that the expression of the UNC5C gene is down-regulated in colorectal cancers, mainly because of tumor-associated specific promoter methylation. In addition, inactivation of UNC5C in mice is associated with increased intestinal tumor progression, demonstrating its tumor suppressor role. Moreover, mutations in UNC5C that turn down the pro-apoptotic activity of the receptor are found in patients with familial form of colorectal cancer. This germline coding mutation in UNC5C has been detected in five different families from France and USA and co-segregates with the disease. We further, determine that this anti-apoptotic mutation arose from a common founder. In summary, our study strongly indicates that the genetic inactivation of the tumor suppressor UNC5C may be a major promoter of familial predisposition to colorectal tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 28. doi:1538-7445.AM2012-28

The proteasome is a multicatalytic proteinase complex composed of several non-identical protein subunits with molecular weights ranging from 20 to 35 kDa. To approach the mechanisms modulating the activity of this protease, we have investigated the possible interaction of this particle with specific polypeptides as well as the phosphorylation status of its subunits. A specific antiserum was used to immunoprecipitate this particle under native conditions. Three major polypeptides, characterized by molecular masses of 53, 59 and 77 kDa co-immunoprecipitated specifically with the proteasome. Labelling experiments indicated that these proteins are leucine-rich and contain very few methionine residues. None of them were phosphorylated in vivo in normal cell growth conditions, in contrast to one of the proteasome subunit (30 kDa). These results indicate that, in vivo, the proteasome is probably associated with leucine-rich polypeptides and that this protease is a kinase substrate.

Abstract #3075 Netrin-1, an axon navigation cue was proposed to play a crucial role during colorectal tumorigenesis by regulating apoptosis. The netrin-1 receptors DCC and UNC5H belong to the family of dependence receptors that share the ability to induce apoptosis in the absence of their ligand. Cells expressing one of these dependence receptors are thus dependent on ligand availability to survive. Such a trait confers on these receptors a tumor suppressor activity by preventing uncontrolled cell proliferation. We have previously shown in breast cancer that gaining autocrine expression of the ligand netrin-1 would represent for cancer cells a selective advantage. Human and murine netrin-1-expressing mammary metastatic cell lines undergo apoptosis when netrin-1 expression is experimentally decreased or when decoy soluble receptor ectodomains are used. Such treatments prevent metastasis formation in a syngenic mouse model of lung colonization by a mammary cancer cell line (Fitamant et al., PNAS 2008, vol 105). In order to assess the clinical importance of netrin-1 expression in human breast cancer, we quantified by Q-RT-PCR the mRNA levels of netrin-1 in primary breast cancer biopsies and studied their correlation to pathological and clinical data. We used frozen tumor samples from 120 breast cancer patients treated in our institution between 1993 and 2000. At diagnosis, median age was 50, median tumor size was 40 mm. 11% of the patients had SBR1 tumor, 46% had SBR2 tumor and 43% had SBR3 tumor. Estrogen receptor was positive for 62% of the patients. 17% of the patients were pN0, M0; 71% were pN+, M0 and 12% already had distant metastasis (M+) at time of diagnosis. Although there is no correlation between netrin-1 expression and tumor size, histological SBR grade or hormone receptor status, there is a strong correlation between high netrin-1 expression and the presence of nodal involvement (p=0,007) or distant metastasis at the time of diagnosis (p Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3075.

> Il est classiquement admis qu’un recepteur ne devient actif qu’apres son interaction avec son ligand. Au cours des dernieres annees, cependant, un nouveau concept a peu a peu emerge suggerant que certains recepteurs dits « a dependance » peuvent delivrer deux messages: en presence de ligand, ces recepteurs transduisent un signal positif classique, stimulant par exemple une voie de differenciation; en l’absence de leur ligand en revanche, ils induisent la mort de la cellule par apoptose [1] (➜). Un article recemment paru dans la revue Science, emanant d’un travail collaboratif entre notre groupe et celui de N. Le Douarin, revele que le recepteur de Sonic Hedgehog (Shh), Patched (Ptc), fonctionne selon ce schema et que les deux actions de Ptc participent a la mise en place de la moelle epiniere [2]. L’activite pro-apoptotique de ces recepteurs en l’absence de leur ligand laisse supposer: (1) que ces recepteurs pourraient posseder une activite suppresseur de tumeur en eliminant les cellules tumorales qui se developperaient en dehors des zones d’expression normale du ligand ; (2) qu’un exces de mort cellulaire devrait survenir chez des animaux dont on aurait elimine experimentalement le ligand physiologique de ces recepteurs. Or, le recepteur Ptc repond a ces deux caracteristiques, ce qui explique notre interet pour cette proteine. Ptc est une proteine a douze domaines transmembranaires qui est le recepteur specifique de Shh, une molecule centrale lors du developpement, en particulier parce qu’elle induit le programme de determination-differenciation des neurones presents dans le tube neural, un tube s’etendant selon l’axe antero-posterieur des vertebres a l’origine de la moelle epiniere et du cerveau. Pourtant Ptc pourrait aussi etre un suppresseur de tumeur implique dans differents carcinomes. De plus, N. Le Douarin et al. avaient montre que, chez le poulet, le retrait experimental de Shh conduisait a une mort massive des neurones formant le tube neural [3]. Ce dernier resultat avait alors ete interprete par une communaute scientifique tres dogmatique, comme la consequence de l’absence d’un signal adequat de differenciation conduisant a la mort de ces cellules. Cependant, notre etude montre que le recepteur Ptc induit spontanement la mort des cellules tant que son ligand Shh n’est pas present. Cette induction d’apoptose semble passer par un clivage du dernier domaine intracellulaire de Ptc par les proteases centrales de l’apoptose que sont les caspases. Par ailleurs, grâce a l’utilisation d’une forme mutante de Ptc qui agit comme un dominant negatif de l’activite pro-apoptotique de Ptc, nous avons pu montrer que lors du developpement du poulet, on peut bloquer la mort des cellules du tube neural induite par le retrait experimental de Shh. De plus, l’action de ce dominant negatif ne se limite pas a bloquer la mort, mais conduit egalement a un developpement partiel du tube neural. Cette observation est conceptuellement alors tres interessante car elle suggere que la mort induite « experimentalement » par l’absence de Shh n’est pas la simple consequence d’une absence de differenciation mais revele en fait un mecanisme actif de modelage du tube neural par l’apoptose. On peut alors postuler que le developpement du tube neural controle par Shh passe a la fois par un mecanisme positif de determination-differenciation des neurones, mais aussi par l’inhibition d’un mecanisme negatif qui conduirait a la mort des neurones se developpant dans un contexte inapproprie. ◊ Patched is a dependence receptor NOUVELLE

Une nouvelle famille de recepteurs cellulaires a recemment ete identifiee : les dependence receptors. Ces proteines, qui ne possedent pas necessairement d'homologie de structure, ont toutes en commun la caracteristique de transduire deux signalisations totalement differentes : en presence de leur ligand dans le systeme nerveux et dans certaines tumeurs, ces recepteurs transduisent un signal classique de proliferation, de differenciation ou de migration ; a l'inverse, en l'absence de ligand, ils ne sont pas inactifs mais provoquent la mise en route d'un programme de mort cellulaire. Les cellules synthetisant ces proteines se trouvent donc dans un etat de dependance vis-a-vis du ligand. La signalisation permettant a ces recepteurs d'induire l'apoptose reste encore largement inconnue meme si elle semble passer par leur clivage proteolytique et par leur interaction avec les caspases, les proteases centrales de l'apoptose. La recherche des mecanismes moleculaires permettant cette double signalisation est alors d'autant plus importante que ces recepteurs sont a la fois impliques dans le developpement du systeme nerveux et dans la tumorigenese.

The DCC (Deleted in Colorectal Cancer) gene has been found to be deleted in more than 50% of primary colorectal cancers and in numerous other carcinomas, although the role of DCC as a tumor suppressor remains controversial. DCC is also a receptor for netrin-1, a lamininrelated molecule involved in axon guidance. Recently, we reported that DCC is actually a dependence receptor; such receptors create cellular states of dependence on their respective ligands by inducing apoptosis when unoccupied by ligand, but inhibiting apoptosis in the presence of ligand. Here, we show that DCC drives apoptosis through a mechanism requiring caspase-9 activation. Unexpectedly, if DCC kills through caspase-9, it does not go through the classic ‘mitochondria pathway’ since Bcl-2 and an inhibitor of cytochrome c release, cyclosporin A, are unable to block CCC-induced ceil death. Of interest, DCC intera& with both caspase3 and caspase-9 suggesting that DCC could play the role of an initiator complex for caspase activation. Moreover, in vitro translated DCC drives the activation of caspase-3 through caspase-9 without a requirement for cytochrome c. Hence, DCC defines the first pathway for cytochrome c-independent-and thus mitochondria-independentactivation of caspase-9.

Expression of the mammalian small stress protein Hsp27 has been increasingly linked to cell growth regulation and differentiation. Hsp27 is a phosphoprotein which forms oligomers with native sizes ranging between 100 and 800 kDa. We have examined the fate of Hsp27 transiently expressed during the retinoic acid (tRA)-induced granulocytic differentiation of human leukemic HL-60 cells. We show that tRA, in addition to its effects on Hsp27 accumulation and phosphorylation, transiently increased the oligomerization state of this protein. While Hsp27 phosphorylation by tRA is an early phenomenon that takes place before cellular growth is altered, the redistribution of Hsp27 oligomers occurred later and concomitantly with the maximal accumulation of this protein. Hence, complex regulations of Hsp27 are induced by tRA which suggest that this protein plays a role in the pathway through which retinoids exert their effects. To approach Hsp27 function during HL-60 cell differentiation, experiments aimed at reducing the cellular content of this protein were performed by transiently inhibiting Hsp27 mRNA translation by a specific anti-sense oligonucleotide. This process, which decreased the basal level of Hsp27 by about 40%, did not interfere with the growth of undifferentiated HL-60 cells. In contrast, a decreased level of Hsp27 diminished the differentiation-mediated down-regulation of cell growth and altered some morphological changes induced by this retinoid. These results suggest that Hsp27 is a mediator of granulocytic differentiation.