Spontaneous regression of neuroblastoma (NB) resembles the developmentally regulated programmed cell death (PCD) of sympathetic neurons. with E2F1 to selectively transactivate the proapoptotic target gene. The cleavage of UNC5D and its induction of apoptosis were strongly inhibited by addition of netrin-1. mice consistently exhibited a significant increase in dorsal root ganglia neurons and resistance to NGF depletion-induced apoptosis in sympathetic neurons compared with wild-type cells. Our data suggest that UNC5D forms a positive feedback loop with p53 and E2F1 to promote NGF dependence-mediated PCD during NB regression. Introduction Neuroblastoma (NB) is one of the most common solid tumors in children and arises from the sympathoadrenal lineage of the neural crest. The enigma of NB is that many tumors found in infants less than Biricodar 1 year of age frequently regress spontaneously even though the tumor metastasizes to the liver skin and/or bone marrow designated as stage Biricodar 4s (1). Accumulating evidence suggests that both genetic and epigenetic changes may affect the clinical behavior of NBs. However the molecular and biological bases of NB spontaneous regression and aggressiveness remain elusive. One of the breakthroughs for understanding how NB regresses was the discovery that both TRKA and p75NTR high- and low-affinity receptors respectively for nerve growth factor (NGF) are expressed at significantly high levels in favorable NBs (2 3 whereas TRKB and its ligands brain-derived neurotrophic factor (BDNF) and neurotrophin 4/5 (NT4/5) are highly expressed in aggressive NBs in an autocrine and/or paracrine manner (4). It has been hypothesized that the quantitative relationship between NGF and its receptor complexes within tumor tissue as well as the acquisition of NGF dependence may be crucial to inducing NB regression (5). Recent progress in developmental neurobiology has shown that some important molecules such as p53 Biricodar (6) p63 (7) and E2F1 (8 9 as well as the c-JUN/EGLN3/KIF1Bβ pathway (10-12) are involved in the regulation or induction of NGF depletion-induced programmed cell death (PCD) of sympathetic neurons. Recently Bredesen and colleagues proposed a new “dependence receptor” concept which was originally initiated from the idea of the NGF dependence of developing neuronal cells: some receptors display 2 completely opposing actions depending on the availability of their ligands (13). In the presence of their ligands the receptors transduce a “positive” signal for differentiation migration or survival; conversely those receptors conduct a “negative” signal to trigger apoptosis in the absence of any ligand (13). To date a growing number of dependence receptors have been identified including deleted in colorectal cancer (DCC; ref. 14) Ret (15) UNC5 Biricodar (16 17 Patched (18) neogenin (19) ALK (20) and integrins such as αvβ3 and α5β1 (21). To identify the genes Biricodar that play a key role in both developmentally regulated neuronal PCD and spontaneous regression of NB we previously generated cDNA libraries from primary NB tissues and identified UNC5D as one of the genes highly expressed in the favorable subset (22). UNC5D is the fourth member of the human dependence receptor UNC5 family (17) and shares the same ligand netrin-1 (encoded by is a direct transcriptional target of the tumor suppressor p53 and mediates p53-induced cell death (24). We also previously found that is directly targeted by p53 for its expression (17). In addition knockout mice c-COT of have shown different phenotypes (25-27). However the functional role of UNC5D remains unclear. In the present study we explored the functional role of UNC5D in NGF depletion-induced PCD using primary NB cells as well as mouse-derived neuronal cells. Our results showed that NGF depletion-inducible UNC5D formed a positive feedback loop with p53 E2F1 and caspases to accelerate NGF deficiency-mediated cell death. Results High-level expression of UNC5D but not netrin-1 is associated with favorable prognosis of NB. was selected from our NB cDNA project as a differentially expressed gene between favorable and unfavorable subsets (Supplemental Figure 1A; supplemental material available online with this article; doi: 10.1172 Quantitative RT-PCR analysis using 108 primary NBs revealed that expression levels of mRNA were significantly higher in favorable NBs than unfavorable ones (Figure ?(Figure1A).1A). Immunohistochemical analysis also showed that similar to TRKA UNC5D was positive in tumor cells from favorable NBs with a tendency to.