Receptor Deleted In Colorectal Cancer Research Articles

A human DCC variant causing mirror movement disorder reveals that the WAVE regulatory complex mediates axon guidance by netrin-1-DCC.

The axon guidance cue netrin-1 signals through its receptor DCC (deleted in colorectal cancer) to attract commissural axons to the midline. Variants in DCC are frequently associated with congenital mirror movements (CMMs). A CMM-associated variant in the cytoplasmic tail of DCC is located in a conserved motif predicted to bind to a regulator of actin dynamics called the WAVE (Wiskott-Aldrich syndrome protein-family verprolin homologous protein) regulatory complex (WRC). Here, we explored how this variant affects DCC function and may contribute to CMM. We found that a conserved WRC-interacting receptor sequence (WIRS) motif in the cytoplasmic tail of DCC mediated the interaction between DCC and the WRC. This interaction was required for netrin-1-mediated axon guidance in cultured rodent commissural neurons. Furthermore, the WIRS motif of Fra, the Drosophila DCC ortholog, was required for attractive signaling in vivo at the Drosophila midline. The CMM-associated R1343H variant of DCC, which altered the WIRS motif, prevented the DCC-WRC interaction and impaired axon guidance in cultured commissural neurons and in Drosophila. The findings reveal the WRC as a pivotal component of netrin-1-DCC signaling and uncover a molecular mechanism explaining how a human genetic variant in the cytoplasmic tail of DCC may lead to CMM.

Netrin-1 Role in Nociceptive Neuron Sprouting through Activation of DCC Signaling in a Rat Model of Bone Cancer Pain.

Bone cancer pain (BCP) is a common primary or metastatic bone cancer complication. Netrin-1 plays an essential role in neurite elongation and pain sensitization. This study aimed to determine the role of netrin-1 from the metastatic bone microenvironment in BCP development and identify the associated signaling pathway for the strategy of BCP management. The rat BCP model was established by intratibial implantation of Walker 256 cells. Von Frey filaments measured the mechanical pain threshold. Movement-induced pain was assessed using limb use scores. Expressions of associated molecules in the affected tibias or dorsal root ganglia (DRG) were measured by immunofluorescence, immunohistochemistry, real-time quantitative polymerase chain reaction, or western blotting. Transduction of deleted in colorectal cancer (DCC) signaling was inhibited by intrathecal injection of DCC-siRNA. In BCP rats, the presence of calcitonin gene-related peptide (CGRP)-positive nerve fibers increased in the metastatic bone lesions. The metastatic site showed enrichment of well-differentiated osteoclasts and expressions of netrin-1 and its attractive receptor DCC. Upregulation of DCC and increased phosphorylation levels of focal adhesion kinase (FAK) and Rac family small GTPase 1/Cell division cycle 42 (Rac1/Cdc42) were found in the DRG. Intrathecal administration of DCC-siRNA led to a significant reduction in FAK and Rac1/Cdc42 phosphorylation levels in the DRG, decreased nociceptive nerve innervation, and improved pain behaviors. Netrin-1 may contribute to the activation of the BCP by inducing nociceptive nerve innervation and improving pain behaviors.

Adamtsl3 mediates DCC signaling to selectively promote GABAergic synapse function

The molecular code that controls synapse formation and maintenance invivo has remained quite sparse. Here, we identify that the secreted protein Adamtsl3 functions as critical hippocampal synapse organizer acting through the transmembrane receptor DCC (deleted in colorectal cancer). Traditionally, DCC function has been associated with glutamatergic synaptogenesis and plasticity in response to Netrin-1 signaling. We demonstrate that early post-natal deletion of Adamtsl3 in neurons impairs DCC protein expression, causing reduced density of both glutamatergic and GABAergic synapses. Adult deletion of Adamtsl3 in either GABAergic or glutamatergic neurons does not interfere with DCC-Netrin-1 function at glutamatergic synapses but controls DCC signaling at GABAergic synapses. The Adamtsl3-DCC signaling unit is further essential for activity-dependent adaptations at GABAergic synapses, involving DCC phosphorylation and Src kinase activation. These findings might be particularly relevant for schizophrenia because genetic variants in Adamtsl3 and DCC have been independently linked with schizophrenia in patients.

The netrin-1 receptor DCC promotes the survival of a subpopulation of midbrain dopaminergic neurons: Relevance for ageing and Parkinson's disease.

Mechanisms that determine the survival of midbrain dopaminergic (mDA) neurons in the adult central nervous system (CNS) are not fully understood. Netrins are a family of secreted proteins that are essential for normal neural development. In the mature CNS, mDA neurons express particularly high levels of netrin‐1 and its receptor Deleted in Colorectal Cancer (DCC). Recent findings indicate that overexpressing netrin‐1 protects mDA neurons in animal models of Parkinson’s disease (PD), with a proposed pro‐apoptotic dependence function for DCC that triggers cell death in the absence of a ligand. Here, we sought to determine if DCC expression influences mDA neuron survival in young adult and ageing mice. To circumvent the perinatal lethality of DCC null mice, we selectively deleted DCC from mDA neurons utilizing DATcre/loxP gene‐targeting and examined neuronal survival in adult and aged animals. Reduced numbers of mDA neurons were detected in the substantia nigra pars compacta (SNc) of young adult DATcre/DCCfl/fl mice, with further reduction in aged DATcre/DCCfl/fl animals. In contrast to young adults, aged mice also exhibited a gene dosage effect, with fewer SNc mDA neurons in DCC heterozygotes (DATcre/DCCfl/wt). Notably, loss of mDA neurons in the SN was not uniform. Neuronal loss in the SN was limited to ventral tier mDA neurons, while mDA neurons in the dorsal tier of the SN, which resist degeneration in PD, were spared from the effect of DCC deletion in both young and aged mice. In the ventral tegmental area (VTA), young adult mice with conditional deletion of DCC had normal mDA neuronal numbers, while significant loss occurred in aged DATcre/DCCfl/fl and DATcre/DCCfl/wt mice compared to age‐matched wild‐type mice. Our results indicate that expression of DCC is required for the survival of subpopulations of mDA neurons and may be relevant to the degenerative processes in PD.

Netrin-1 expression and targeting in multiple myeloma

Deleted in colorectal cancer (DCC) and uncoordinated-5 (UNC5) receptors, play a key role in tumor progression of several solid tumors by inducing apoptosis when unbound to their ligand netrin-1. Netrin 1 is currently being evaluated as a therapeutic target. These receptors, known as dependence receptors, and their ligands, have not yet been extensively explored in hematological malignancies. Here, we performed a screening of various human myeloma cell lines and bone marrow samples from multiple myeloma patients for netrin-1 and its receptors to determine the expression of netrin 1 and its receptors in multiple myeloma as well as to assess the potential anti-myeloma activity of a novel anti-netrin-1 treatment (NP137). Our results showed heterogeneous expression of netrin-1 and its receptors DCC and UNC5H2(B) in six human myeloma lines. Additionally, immunohistochemistry and flow cytometry showed expression of these molecules in a majority of myeloma patient samples. In vitro NP137 did not induce apoptosis of myeloma cell lines yet enhanced the cytotoxicity of bortezomib and dexamethasone. In vivo, NP137 treatment of SCID mice with established RPMI8226 myeloma tumors led to a reduction of tumor size compared to controls. Ex vivo, NP137 lowered the plasma cells percentage in bone marrow aspirates in a fraction of the patient samples analyzed. These results suggest that netrin signaling could constitute a novel therapeutic target in multiple myeloma.

Netrin-1 and Its Receptor DCC Are Causally Implicated in Melanoma Progression.

Deleted in colorectal cancer (DCC), the receptor for the multifunctional cue netrin-1, acts as a tumor suppressor in intestinal cancer and lung metastasis by triggering cancer cell death when netrin-1 is lowly expressed. Recent genomic data highlighted that DCC is the third most frequently mutated gene in melanoma; we therefore investigated whether DCC could act as a melanoma tumor suppressor. Reexpressing DCC in human melanoma cell lines promoted tumor cell death and tumor growth inhibition in xenograft mouse models. Genetic silencing of DCC prodeath activity in a BRAFV600E mouse model increased the proportion of mice with melanoma, further supporting that DCC is a melanoma tumor suppressor. Netrin-1 expression was elevated in melanoma compared with benign melanocytic lesions. Upregulation of netrin-1 in the skin cells of a BRAFV600E-mutated murine model reduced cancer cell death and promoted melanoma progression. Therapeutic antibody blockade of netrin-1 combined with dacarbazine increased overall survival in several mouse melanoma models. Together, these data support that interfering with netrin-1 could be a viable therapeutic approach in patients with netrin-1-expressing melanoma. SIGNIFICANCE: Netrin-1 and its receptor DCC regulate melanoma progression, suggesting therapeutic targeting of this signaling axis as a viable option for melanoma treatment.

Dependence and Guidance Receptors-DCC and Neogenin-In Partial EMT and the Actions of Serine Proteases.

The Epithelial-Mesenchymal Transition (EMT) is an important concept in understanding the processes of oncogenesis, especially with respect to the relationship between cell proliferation and metastatic properties such as spontaneous cell motility, chemotaxic migration and tissue invasion. EMT is now recognized as a more complex phenomenon than an all-or-nothing event, in which different components of the EMT may have distinct roles in the physio-pathological regulation of cell function and which may in turn depend on differential interactions with cell constituents and metabolic products. This mini-review summarizes recent work on the induction of cancer properties in parallel with the presence of EMT activities in the presence of serine proteases, with the focus on those tumor suppressors known as “dependence” receptors such as neogenin and Deleted in Colorectal Cancer (DCC). It is concluded that various forms of partial EMT should be given more detailed investigation and consideration as the results could have valuable implications for the development of disease-specific and patient-specific therapies.

Abstract 5291: Netrin-1 expression and targeting in multiple myeloma

Abstract Netrin-1 was first described as an axonal chemoattractant in peripheral nervous system leading to axon outgrowth. Later on, after well characterizing its receptors, deleted in colorectal cancer (DCC) and uncoordinated-5 (UNC5), and their role in apoptosis, they were classified as dependence receptors. Dependence receptors belong to a family of receptors distinguished from other classical receptors by their capability of inducing cell death in the absence of their respective ligands. Their importance in tumorigenesis suggest that this family of receptors along with their ligands could constitute a novel target in cancer therapy. Recent studies showed upregulation of netrin-1 and its receptors in lung cancer tumors after treatment with conventional chemotherapies. The aim of our study was to analyze the expression of netrin 1 and its receptors UNC5B and DCC in multiple myeloma and to determine the impact of a monoclonal antibody targeting netrin-1 on myeloma cell lines in vitro and in vivo. This antibody, NP137, produced by NetrisPharma, is now in early phase clinical trials. In the first place, we performed a screening of various human myeloma cell lines, including AMO-1, LP-1, MM1S, RPMI8226, U266 and OPM-2, for netrin-1 and its different receptors using RT-qPCR and western blotting. We also analyzed the expression of netrin 1 and its receptors in fresh bone marrow aspirates by flow cytometry and in bone marrow biopsies using immunohistochemistry obtained from patients with multiple myeloma. Our results showed that myeloma cell lines expressed netrin-1 and its receptors DCC and UNC5B as determined both by RTqPCR and by western blotting. This expression was also analyzed in patient samples with 16 bone marrow biopsies using immunohistochemistry and in 10 fresh bone marrow aspirates by flow cytometry. Netrin-1 was identified by IHC in 6/16 samples while DCC and UNC5B were identified in all and in 13 samples, respectively. In fresh samples, both netrin-1 and its receptors were found to be more highly expressed than in normal lymphocytes. The exposure of myeloma cell lines to anti-netrin-1 humanized antibody in vitro did not induce a significant decrease in cell viability in vitro, whereas treatment of SCID mice with established RPMI8226 myeloma tumors lead to a significant reduction of tumor size in comparison to controls, suggesting a possible targeting of netrin 1 in this disease. Combination of NP137 with conventional anti-myeloma agents will be presented. In conclusion, these results suggest that netrin 1 signaling could constitute a novel original target in multiple myeloma. Citation Format: David Fahed, Kamel Chettab, Patrick Mehlen, Doriane Mathe, Anne Tourette, Morgane Denis, Alexandra Traverse-Glehen, Lionel Karlin, Charles Dumontet. Netrin-1 expression and targeting in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5291.

Subchondral bone osteoclasts induce sensory innervation and osteoarthritis pain.

Joint pain is the defining symptom of osteoarthritis (OA) but its origin and mechanisms remain unclear. Here, we investigated an unprecedented role of osteoclast-initiated subchondral bone remodeling in sensory innervation for OA pain. We show that osteoclasts secrete netrin-1 to induce sensory nerve axonal growth in subchondral bone. Reduction of osteoclast formation by knockout of receptor activator of nuclear factor kappa-B ligand (Rankl) in osteocytes inhibited the growth of sensory nerves into subchondral bone, dorsal root ganglion neuron hyperexcitability, and behavioral measures of pain hypersensitivity in OA mice. Moreover, we demonstrated a possible role for netrin-1 secreted by osteoclasts during aberrant subchondral bone remodeling in inducing sensory innervation and OA pain through its receptor DCC (deleted in colorectal cancer). Importantly, knockout of Netrin1 in tartrate-resistant acid phosphatase-positive (TRAP-positive) osteoclasts or knockdown of Dcc reduces OA pain behavior. In particular, inhibition of osteoclast activity by alendronate modifies aberrant subchondral bone remodeling and reduces innervation and pain behavior at the early stage of OA. These results suggest that intervention of the axonal guidance molecules (e.g., netrin-1) derived from aberrant subchondral bone remodeling may have therapeutic potential for OA pain.

Mesocorticolimbic Connectivity and Volumetric Alterations in DCC Mutation Carriers.

The axon guidance cue receptor DCC (deleted in colorectal cancer) plays a critical role in the organization of mesocorticolimbic pathways in rodents. To investigate whether this occurs in humans, we measured (1) anatomical connectivity between the substantia nigra/ventral tegmental area (SN/VTA) and forebrain targets, (2) striatal and cortical volumes, and (3) putatively associated traits and behaviors. To assess translatability, morphometric data were also collected in Dcc-haploinsufficient mice. The human volunteers were 20 DCC+/- mutation carriers, 16 DCC+/+ relatives, and 20 DCC+/+ unrelated healthy volunteers (UHVs; 28 females). The mice were 11 Dcc+/- and 16 wild-type C57BL/6J animals assessed during adolescence and adulthood. Compared with both control groups, the human DCC+/- carriers exhibited the following: (1) reduced anatomical connectivity from the SN/VTA to the ventral striatum [DCC+/+: p = 0.0005, r(effect size) = 0.60; UHV: p = 0.0029, r = 0.48] and ventral medial prefrontal cortex (DCC+/+: p = 0.0031, r = 0.53; UHV: p = 0.034, r = 0.35); (2) lower novelty-seeking scores (DCC+/+: p = 0.034, d = 0.82; UHV: p = 0.019, d = 0.84); and (3) reduced striatal volume (DCC+/+: p = 0.0009, d = 1.37; UHV: p = 0.0054, d = 0.93). Striatal volumetric reductions were also present in Dcc+/- mice, and these were seen during adolescence (p = 0.0058, d = 1.09) and adulthood (p = 0.003, d = 1.26). Together these findings provide the first evidence in humans that an axon guidance gene is involved in the formation of mesocorticolimbic circuitry and related behavioral traits, providing mechanisms through which DCC mutations might affect susceptibility to diverse neuropsychiatric disorders.SIGNIFICANCE STATEMENT Opportunities to study the effects of axon guidance molecules on human brain development have been rare. Here, the identification of a large four-generational family that carries a mutation to the axon guidance molecule receptor gene, DCC, enabled us to demonstrate effects on mesocorticolimbic anatomical connectivity, striatal volumes, and personality traits. Reductions in striatal volumes were replicated in DCC-haploinsufficient mice. Together, these processes might influence mesocorticolimbic function and susceptibility to diverse neuropsychiatric disorders.

Structural Basis for Draxin-Modulated Axon Guidance and Fasciculation by Netrin-1 through DCC

SummaryAxon guidance involves the spatiotemporal interplay between guidance cues and membrane-bound cell-surface receptors, present on the growth cone of the axon. Netrin-1 is a prototypical guidance cue that binds to deleted in colorectal cancer (DCC), and it has been proposed that the guidance cue Draxin modulates this interaction. Here, we present structural snapshots of Draxin/DCC and Draxin/Netrin-1 complexes, revealing a triangular relationship that affects Netrin-mediated haptotaxis and fasciculation. Draxin interacts with DCC through the N-terminal four immunoglobulin domains, and Netrin-1 through the EGF-3 domain, in the same region where DCC binds. Netrin-1 and DCC bind to adjacent sites on Draxin, which appears to capture Netrin-1 and tether it to the DCC receptor. We propose the conformational flexibility of the single-pass membrane receptor DCC is used to promote fasciculation and regulate axon guidance through concerted Netrin-1/Draxin binding.Video

Draxin regulates hippocampal neurogenesis in the postnatal dentate gyrus by inhibiting DCC-induced apoptosis

Hippocampal neurogenesis in the dentate gyrus (DG) is controlled by diffusible molecules that modulate neurogenic processes, including cell proliferation, differentiation and survival. To elucidate the mechanisms underlying hippocampal neurogenesis, we investigated the function of draxin, originally identified as a neural chemorepellent, in the regulation of neuronal survival in the DG. Draxin was expressed in Tbr2 (+) late progenitors and NeuroD1 (+) neuroblasts in the dentate granule cell lineage, whereas expression of its receptor DCC (deleted in colorectal cancer) was mainly detectable in neuroblasts. Our phenotypic analysis revealed that draxin deficiency led to enhanced apoptosis of DCC-expressing neuroblasts in the neurogenic areas. Furthermore, in vitro assays using a hippocampal neural stem/progenitor cell (HNSPC) line indicated that draxin inhibited apoptosis in differentiating HNSPCs, which express DCC. Taken together, we postulate that draxin plays a pivotal role in postnatal DG neurogenesis as a dependence receptor ligand for DCC to maintain and promote survival of neuroblasts.

DCCmutation update: Congenital mirror movements, isolated agenesis of the corpus callosum, and developmental split brain syndrome

The deleted in colorectal cancer (DCC) gene encodes the netrin-1 (NTN1) receptor DCC, a transmembrane protein required for the guidance of commissural axons. Germline DCC mutations disrupt the development of predominantly commissural tracts in the central nervous system (CNS) and cause a spectrum of neurological disorders. Monoallelic, missense, and predicted loss-of-function DCC mutations cause congenital mirror movements, isolated agenesis of the corpus callosum (ACC), or both. Biallelic, predicted loss-of-function DCC mutations cause developmental split brain syndrome (DSBS). Although the underlying molecular mechanisms leading to disease remain poorly understood, they are thought to stem from reduced or perturbed NTN1 signaling. Here, we review the 26 reported DCC mutations associated with abnormal CNS development in humans, including 14 missense and 12 predicted loss-of-function mutations, and discuss their associated clinical characteristics and diagnostic features. We provide an update on the observed genotype-phenotype relationships of congenital mirror movements, isolated ACC and DSBS, and correlate this to our current understanding of the biological function of DCC in the development of the CNS. All mutations and their associated phenotypes were deposited into a locus-specific LOVD (https://databases.lovd.nl/shared/genes/DCC).

Effects of intra-arterial transplantation of adipose-derived stem cells on the expression of netrin-1 and its receptor DCC in the peri-infarct cortex after experimental stroke

BackgroundStem cell transplantation has been documented to promote functional recovery in animal models of stroke; however, the underlying mechanisms are not yet fully understood. As netrin-1 and its receptor deleted in colorectal cancer (DCC) are important regulators in neuronal and vascular activities, the present study attempted to explore whether netrin-1 and DCC are involved in the neuroprotection of stem cell-based therapies in a rat ischemic stroke model.MethodsAdult male Sprague–Dawley rats were subjected to a transient middle cerebral artery occlusion (MCAO) and subsequently received an intra-arterial injection of 2 × 106 PKH26-labeled adipose-derived stem cells (ADSCs) or saline 24 h later. Neurological function was evaluated by behavioral tests before the rats were sacrificed at days 7 and 14 after MCAO. The migration of ADSCs and regeneration of neuronal fibers and blood vessels were determined by immunofluorescence staining. The expression of netrin-1 and DCC was analyzed by Western blot and immunofluorescence staining.ResultsADSC transplantation significantly improved the neurological recovery at days 7 and 14, and noticeably promoted the regeneration of neuronal fibers and blood vessels in the peri-infarct cortex at day 14. PKH26-labeled ADSCs located mainly in the peri-infarct area at days 7 and 14. In ADSC-treated rats, the expression of netrin-1 and DCC significantly increased in the peri-infarct cortex at days 7 and 14. Immunofluorescence staining showed that netrin-1 was mainly expressed by neuronal perikaryal in the peri-infarct cortex, and DCC was mainly expressed by neuronal fibers and was present around the blood vessels in the peri-infarct cortex.ConclusionsThese findings suggest that ADSC transplantation facilitates the regeneration of neuronal fibers and blood vessels in the peri-infarct cortex and improves neurological functions, which may be attributed, at least in part, to the involvement of upregulated netrin-1 and DCC in the remodeling of neuronal and vascular networks in the peri-infarct cortex.

Dependence receptor involvement in subtilisin-induced long-term depression and in long-term potentiation

The serine protease subtilisin induces a form of long-term depression (LTD) which is accompanied by a reduced expression of the axo-dendritic guidance molecule Unco-ordinated-5C (Unc-5C). One objective of the present work was to determine whether a loss of Unc-5C function contributed to subtilisin-induced LTD by using Unc-5C antibodies in combination with the pore-forming agents Triton X-100 (0.005%) or streptolysin O in rat hippocampal slices. In addition we have assessed the effect of subtilisin on the related dependence receptor Deleted in Colorectal Cancer (DCC) and used antibodies to this protein for functional studies. Field excitatory postsynaptic potentials (fEPSPs) were analyzed in rat hippocampal slices and protein extracts were used for Western blotting. Subtilisin produced a greater loss of DCC than of Unc-5C, but the antibodies had no effect on resting excitability or fEPSPs and did not modify subtilisin-induced LTD. However, antibodies to DCC but not Unc-5C did reduce the amplitude of theta-burst long-term potentiation (LTP). In addition, two inhibitors of endocytosis – dynasore and tat-gluR2(3Y) – were tested and, although the former compound had no effect on neurophysiological responses, tat-gluR2(3Y) did reduce the amplitude of subtilisin-induced LTD without affecting the expression of DCC or Unc-5C but with some loss of PostSynaptic Density Protein-95. The results support the view that the dependence receptor DCC may be involved in LTP and suggest that the endocytotic removal of a membrane protein or proteins may contribute to subtilisin-induced LTD, although it appears that neither Unc-5C nor DCC are involved in this process.

DCC Confers Susceptibility to Depression-like Behaviors in Humans and Mice and Is Regulated by miR-218

BackgroudVariations in the expression of the Netrin-1 guidance cue receptor DCC (deleted in colorectal cancer) appear to confer resilience or susceptibility to psychopathologies involving prefrontal cortex (PFC) dysfunction. MethodsWith the use of postmortem brain tissue, mouse models of defeat stress, and in vitro analysis, we assessed microRNA (miRNA) regulation of DCC and whether changes in DCC levels in the PFC lead to vulnerability to depression-like behaviors. ResultsWe identified miR-218 as a posttranscriptional repressor of DCC and detected coexpression of DCC and miR-218 in pyramidal neurons of human and mouse PFC. We found that exaggerated expression of DCC and reduced levels of miR-218 in the PFC are consistent traits of mice susceptible to chronic stress and of major depressive disorder in humans. Remarkably, upregulation of Dcc in mouse PFC pyramidal neurons causes vulnerability to stress-induced social avoidance and anhedonia. ConclusionsThese data are the first demonstration of microRNA regulation of DCC and suggest that, by regulating DCC, miR-218 may be a switch of susceptibility versus resilience to stress-related disorders.

Anchoring of Protein Kinase A by ERM (Ezrin-Radixin-Moesin) Proteins Is Required for Proper Netrin Signaling through DCC (Deleted in Colorectal Cancer)

Netrin-1, acting through its principal receptor DCC (deleted in colorectal cancer), serves as an axon guidance cue during neural development and also contributes to vascular morphogenesis, epithelial migration, and the pathogenesis of some tumors. Several lines of evidence suggest that netrin-DCC signaling can regulate and be regulated by the cAMP-dependent protein kinase, PKA, although the molecular details of this relationship are poorly understood. Specificity in PKA signaling is often achieved through differential subcellular localization of the enzyme by interaction with protein kinase A anchoring proteins (AKAPs). Here, we show that AKAP function is required for DCC-mediated activation of PKA and phosphorylation of cytoskeletal regulatory proteins of the Mena/VASP (vasodilator-stimulated phosphoprotein) family. Moreover, we show that DCC and PKA physically interact and that this association is mediated by the ezrin-radixin-moesin (ERM) family of plasma membrane-actin cytoskeleton cross-linking proteins. Silencing of ERM protein expression inhibits DCC-PKA interaction, DCC-mediated PKA activation, and phosphorylation of Mena/VASP proteins as well as growth cone morphology and neurite outgrowth. Finally, although expression of wild-type radixin partially rescued growth cone morphology and tropism toward netrin in ERM-knockdown cells, expression of an AKAP-deficient mutant of radixin did not fully rescue growth cone morphology and switched netrin tropism from attraction to repulsion. These data support a model in which ERM-mediated anchoring of PKA activity to DCC is required for proper netrin/DCC-mediated signaling.

Netrin-1 improves post-injury cardiac function in vivo via DCC/NO-dependent preservation of mitochondrial integrity, while attenuating autophagy

Reperfusion injury of the heart is a severe complication of angioplasty treatment of acute myocardial ischemia, for which no therapeutics are currently available. The present study aimed to identify whether and how a novel protein, netrin-1, induces cardioprotection in vivo during ischemia/reperfusion (I/R) injury. Wild type (WT) C57BL6/J mice were subjected to a 30min coronary occlusion followed by a 24h reperfusion with vehicle (normal saline), netrin-1, UO126 (MEK1/2 inhibitor), PTIO (nitric oxide/NO scavenger), netrin-1/UO126 or netrin-1/PTIO intraventricularly. Some were injected of netrin-1 via tail vein. Netrin-1 at 5μg/kg induced a substantial reduction in infarct size (19.7±5.0% from 41.3±1.8% in the controls), and markedly improved cardiac function as measured by ejection fraction and fractional shortening from echocardiography. Experiments with mice deficient in netrin-1 receptor DCC (deleted in colorectal cancer, DCC+/−), or reperfusion with netrin-1/UO126 or netrin-1/PTIO, attenuated the protective effects of netrin-1, implicating intermediate roles of DCC, ERK1/2 and NO. Netrin-1 induced phosphorylation of ERK1/2 and eNOS was abolished in DCC+/−mice. Electron spin resonance (ESR) determination of NO production from isolated left ventricles demonstrated that netrin-1 improves NO bioavailability, which was attenuated by UO126 or in DCC+/−mice, suggesting upstream roles of DCC and ERK1/2 in NO production. Netrin-1 further reduced mitochondrial swelling and mitochondrial superoxide production, which was absent when co-treated with PTIO or UO126, or in DCC+/−mice, indicating critical roles of DCC, ERK1/2 and NO in preserving mitochondrial integrity. In a permanent coronary ligation model of myocardial infarction (MI) to assess post-MI remodeling, netrin-1 abolished the marked increase in autophagy. In summary, our data demonstrate robust cardioprotective effect of netrin-1 in vivo, as shown by reduced infarct size and improved cardiac function. Mechanistically, this protection is mediated by netrin-1 receptor DCC, and NO dependent preservation of mitochondria. This work clearly establishes a therapeutic potential of netrin-1 for acute treatment of MI, perhaps also for chronic post-MI remodeling. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

Down-Regulation of UNC5D in Bladder Cancer: UNC5D as a Possible Mediator of Cisplatin Induced Apoptosis in Bladder Cancer Cells

Identifying potential targets would improve therapeutic planning anddisease management. Therefore, we investigated whether the novel identified dependence receptor UNC5D acts as a tumor suppressor in bladder malignancies. We assessed the UNC5D level in a panel of 15 primary bladder carcinomas and 6 cell lines using real-time reverse transcriptase-polymerase chain reaction and Western blot. MTT assay, TUNEL staining, colony formation assay and Western blot were done in cells untransfected and transfected with UNC5D vector, siUNC5D or siDAPK. UNC5D was dramatically down-regulated in bladder cancer tissue samples and malignant cell lines. Restoration of UNC5D expression in bladder cancer cells lacking endogenous UNC5D expression suppressed cell proliferation and survival. Cisplatin treatment significantly induced UNC5D expression and DAPK dephosphorylation while UNC5D knockdown decreased bladder cancer cell sensitivity to cisplatin. DAPK silencing significantly inhibited the effect of UNC5D on apoptosis induced by cisplatin. Our study suggests that UNC5D may have important roles as a novel suppressor in bladder cancer via the UNC5D/DAPK pathway.

Netrin-1 Induces Epithelial–Mesenchymal Transition and Promotes Hepatocellular Carcinoma Invasiveness

Hypoxia is often found in solid tumors and is associated with tumor progression and poor clinical outcomes. We elucidated the mechanism by which netrin-1 released under hypoxic stress can induce epithelial-mesenchymal transition (EMT) to promote invasion in hepatocellular carcinoma (HCC) cells. The expression of netrin-1 and the dependent receptors UNC5H and deleted in colorectal cancer (DCC) in HCC was examined by immunohistochemistry or western blot. The HepG2 cells were cultured in 21% O2 (normoxia) or 1% O2 (hypoxia) for 24 h. The release of netrin-1 from hypoxic cells was detected by ELISA. Expression of E-cadherin and vimentin were examined by western blot. Inverted microscopy or confocal microscopy was used to show the cell morphology or cytoskeletal rearrangements. Cell invasion induced by hypoxia was analyzed by Transwell chamber. Cytokine IL-8 and IL-10 mRNA levels were assessed by real-time PCR. The expression of netrin-1 was increased in HCC tissue and cell lines. The dependent receptors UNC5H and DCC were decreased in most HCC cell lines. Hypoxia induced netrin-1 release in a time-dependent manner. EMT induction was found to occur in hypoxic HCC cells in a process that was dependent on the extracellular release of netrin-1. Moreover, overexpression of netrin-1 resulted in EMT induction in normoxic tumor cells. Cytoskeletal rearrangements were found to occur and cell invasion was increased in cells with netrin-1 overexpression. Lastly, mRNA of IL-8 and IL-10 were also increased after recombinant human netrin-1 treatment. These results suggest that in hypoxic HCC cells, netrin-1 activates downstream signaling pathways to induce EMT activation with subsequent production of multiple inflammatory mediators which in turn promotes cancer invasion.