Targeting PAK1 is effective against cutaneous squamous cell carcinoma in a syngenic mouse model.

By taking advantage of forward genetic analysis in mice, we have demonstrated that Pak1 plays a crucial role during DMBA/TPA skin carcinogenesis. Although Pak1 has been considered to promote cancer development, its overall function remains poorly understood. To clarify the functional significance of Pak1 in detail, we sought to evaluate the possible effect of an allosteric inhibitor against PAK1 (NVS-PAK1-1) on a syngeneic mouse model. To this end, we established two cell lines, 9AS1 and 19AS1, derived from DMBA/TPA-induced squamous cell carcinoma (SCC) that engrafted in FVB mice. Based on our present results, NVS-PAK1-1 treatment significantly inhibited the growth of tumors derived from 9AS1 and 19AS1 cells in vitro and in vivo. RNA-sequencing analysis on the engrafted tumors indicates that NVS-PAK1-1 markedly potentiates the epidermal cell differentiation and enhances the immune response in the engrafted tumors. Consistent with these observations, we found an expansion of Pan-keratin-positive regions and potentially elevated infiltration of CD8-positive immune cells in NVS-PAK1-1-treated tumors as examined by immunohistochemical analyses. Together, our present findings strongly suggest that PAK1 is tightly linked to the development of SCC, and that its inhibition is a promising therapeutic strategy against SCC.

Meis1 plays roles in cortical development through regulation of cellular proliferative capacity in the embryonic cerebrum.

Meis1 (myeloid ecotropic insertion site 1) is known to be related to embryonic development and cancer. In this study, to analyze the function of Meis1 in neural stem cells, we crossed Meis1fl/fl (Meis1 floxed) mice with Nestin-Cre mice. The results showed that Meis1-conditional knockout mice showed cerebral cortex malformation. The mice had a significantly thinner cortex than wildtype mice. At E14.5, BrdU incorporation and Pax6-positive radial glial cells were significantly decreased in the cerebral cortex of Meis1 knockout embryos as compared with wild-type embryos, whereas Tbr2-positive intermediate progenitors and NeuN-positive differentiated neurons were not. Cell death detected by immunostaining with cleaved caspase3 antibody showed no difference in the cortex between knockout and wild-type embryos. Furthermore, knockout of Meis1 in embryo by in utero electroporation showed that cellular migration was disturbed during cortical development. Therefore, Meis1 could play important roles during cortical development through the regulation of cell proliferation and migration in the embryonic cerebral cortex.

Functional Polymorphism in Pak1-3' Untranslated Region Alters Skin Tumor Susceptibility by Alternative Polyadenylation.

We identified a functional SNP in the 3' untranslated region of Pak1 that is responsible for the skin tumor modifier of MSM 1a locus. Candidate SNPs in the 3' untranslated region of Pak1 from resistance strain MSM/Ms were introduced into susceptible strain FVB/N using CRISPR/Cas9. The 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate skin carcinogenesis experiments revealed an SNP (Pak1-3' untranslated region-6C>T: rs31627325) that strongly suppressed skin tumors. Furthermore, MBNL1 bound more strongly to FVB-allele (6C/C) and regulated the transcript length in the 3' untranslated region of Pak1 and tumorigenesis through polyadenylation. Therefore, the alternative polyadenylation of Pak1 is cis-regulated by rs31627325.

The Japanese Wild-Derived Inbred Mouse Strain, MSM/Ms in Cancer Research.

MSM/Ms is a unique inbred mouse strain derived from the Japanese wild mouse, Mus musculus molossinus, which has been approximately 1 million years genetically distant from standard inbred mouse strains mainly derived from M. m. domesticus. Due to its genetic divergence, MSM/Ms has been broadly used in linkage studies. A bacterial artificial chromosome (BAC) library was constructed for the MSM/Ms genome, and sequence analysis of the MSM/Ms genome showed approximately 1% of nucleotides differed from those in the commonly used inbred mouse strain, C57BL/6J. Therefore, MSM/Ms mice are thought to be useful for functional genome studies. MSM/Ms mice show unique characteristics of phenotypes, including its smaller body size, resistance to high-fat-diet-induced diabetes, high locomotive activity, and resistance to age-onset hearing loss, inflammation, and tumorigenesis, which are distinct from those of common inbred mouse strains. Furthermore, ES (Embryonic Stem) cell lines established from MSM/Ms allow the MSM/Ms genome to be genetically manipulated. Therefore, genomic and phenotypic analyses of MSM/Ms reveal novel insights into gene functions that were previously not obtained from research on common laboratory strains. Tumorigenesis-related MSM/Ms-specific genetic traits have been intensively investigated in Japan. Furthermore, radiation-induced thymic lymphomas and chemically-induced skin tumors have been extensively examined using MSM/Ms.

CENP-50 is required for papilloma development in the two-stage skin carcinogenesis model.

CENP-50/U is a component of the CENP-O complex (CENP-O/P/Q/R/U) and localizes to the centromere throughout the cell cycle. Aberrant expression of CENP-50/U has been reported in many types of cancers. However, as Cenp-50/U-deficient mice die during early embryogenesis, its functions remain poorly understood in vivo. To investigate the role of Cenp-50/U in skin carcinogenesis, we generated Cenp-50/U conditional knockout (K14CreER -Cenp-50/Ufl/fl ) mice and subjected them to the 7,12-dimethylbenz(a)anthracene (DMBA)/terephthalic acid (TPA) chemical carcinogenesis protocol. As a result, early-stage papillomas decreased in Cenp-50/U-deficient mice. In contrast, Cenp-50/U-deficient mice demonstrated almost the same carcinoma incidence as control mice. Furthermore, mRNA expression analysis using DMBA/TPA-induced papillomas and carcinomas revealed that Cenp-50/U expression levels in papillomas were significantly higher than in carcinomas. These results suggest that Cenp-50/U functions mainly in early papilloma development and it has little effect on malignant conversion.

Pak1 maintains epidermal stem cells by regulating Langerhans cells and is required for skin carcinogenesis.

Pak1 (serine/threonine p21-activated kinases) was previously reported to have oncogenic activity in several cancers. However, its roles in the cancer microenvironment are poorly understood. We demonstrated that Pak1 expression in Langerhans cells (LCs) is essential for the maintenance of epidermal stem cells and skin tumor development. We found that PAK1 is localized in LCs by immunohistochemistry. Furthermore, the number of LCs significantly decreased in MSM/Ms Pak1 homozygous knockout mice (MSM/Ms-Pak1-/-). F1 hybrid (FVB/N×MSM/Ms) Pak1 heterozygous knockout mice (F1-Pak1+/-) had increased numbers of Th17 cells in the skin. Therefore, Pak1 knockdown cells were prepared using LC-derived XS52 cells (XS52-Pak1KD) and co-cultured with keratinocyte-derived C5N cells. As a result, XS52-Pak1KD cell supernatants promoted C5N cell proliferation. We then carried out DMBA/TPA skin carcinogenesis experiments using F1-Pak1+/- mice. Of note, F1-Pak1+/- mice exhibited stronger resistance to skin tumors than control mice. F1-Pak1+/- mice had fewer epidermal stem cells in the skin bulge. Our study suggested that Pak1 regulates the epidermal stem cell number by changing the properties of LCs and functions in skin carcinogenesis. We clarified a novel role of Pak1 in regulating LCs as a potential therapeutic target in skin immune disease and carcinogenesis.

A Polymorphic Variant in p19Arf Confers Resistance to Chemically Induced Skin Tumors by Activating the p53 Pathway.

Identification of the specific genetic variants responsible for the increased susceptibility to familial or sporadic cancers is important. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified a strong genetic locus, Stmm3, conferring resistance to chemically induced skin papillomas on chromosome 4. Here, we report the cyclin-dependent kinase inhibitor gene Cdkn2a/p19Arf as a major responsible gene for the Stmm3 locus. We provide evidence that the function of Stmm3 is dependent on p53 and that p19ArfMSM confers stronger resistance to papillomas than p16Ink4aMSMin vivo. In addition, we found that genetic polymorphism in p19Arf between a resistant strain, MSM/Ms (Val), and a susceptible strain, FVB/N (Leu), alters the susceptibility to papilloma development, malignant conversion, and the epithelial-mesenchymal transition. Moreover, we demonstrated that the p19ArfMSM allele more efficiently activates the p53 pathway than the p19ArfFVB allele in vitro and in vivo. Furthermore, we found polymorphisms in CDKN2A in the vicinity of a polymorphism in mouse Cdkn2a associated with the risk of human cancers in the Japanese population. Genetic polymorphisms in Cdkn2a and CDKN2A may affect the cancer risk in both mice and humans.

The parathyroid hormone regulates skin tumour susceptibility in mice.

Using a forward genetics approach to map loci in a mouse skin cancer model, we previously identified a genetic locus, Skin tumour modifier of MSM 1 (Stmm1) on chromosome 7, conferring strong tumour resistance. Sub-congenic mapping localized Parathyroid hormone (Pth) in Stmm1b. Here, we report that serum intact-PTH (iPTH) and a genetic polymorphism in Pth are important for skin tumour resistance. We identified higher iPTH levels in sera from cancer-resistant MSM/Ms mice compared with susceptible FVB/NJ mice. Therefore, we performed skin carcinogenesis experiments with MSM-BAC transgenic mice (Pth MSM-Tg) and Pth knockout heterozygous mice (Pth +/-). As a result, the higher amounts of iPTH in sera conferred stronger resistance to skin tumours. Furthermore, we found that the coding SNP (rs51104087, Val28Met) localizes in the mouse Pro-PTH encoding region, which is linked to processing efficacy and increased PTH secretion. Finally, we report that PTH increases intracellular calcium in keratinocytes and promotes their terminal differentiation. Taken together, our data suggest that Pth is one of the genes responsible for Stmm1, and serum iPTH could serve as a prevention marker of skin cancer and a target for new therapies.

CENP-R acts bilaterally as a tumor suppressor and as an oncogene in the two-stage skin carcinogenesis model.

CENP-R is a component of the CENP-O complex, including CENP-O, CENP-P, CENP-Q, CENP-R, and CENP-U and is constitutively localized to kinetochores throughout the cell cycle in vertebrates. CENP-R-deficient chicken DT40 cells are viable and show a very minor effect on mitosis. To investigate the functional roles of CENP-R in vivo, we generated CENP-R-deficient mice (Cenp-r-/- ). Mice heterozygous or homozygous for Cenp-r null mutation are viable and healthy, with no apparent defect in growth and morphology, indicating Cenp-r is not essential for normal development. Accordingly, to investigate the role of the Cenp-r gene in skin carcinogenesis, we subjected Cenp-r-/- mice to the 7,12-dimethylbenz(a)anthracene (DMBA)/TPA chemical carcinogenesis protocol and monitored tumor development. As a result, Cenp-r-/- mice initially developed significantly more papillomas than control wild-type mice. However, papillomas in Cenp-r-/- mice showed a decrease of proliferative cells and an increase of apoptotic cells. As a result, they did not grow bigger and some papillomas showed substantial regression. Furthermore, papillomas in Cenp-r-/- mice showed lower frequency of malignant conversion to squamous cell carcinomas. These results indicate Cenp-r functions bilaterally in cancer development: during early developmental stages, Cenp-r functions as a tumor suppressor, but during the expansion and progression of papillomas it functions as a tumor-promoting factor.

Oncogenic Lmo3 cooperates with Hen2 to induce hydrocephalus in mice.

We previously reported that LMO3 and HEN2 act as oncogenes in neuroblastoma development through up-regulating MASH1 transcription by interfering with HES1. To confirm these results in vivo, we generated transgenic mice of these genes. Lmo3 or Hen2 was expressed under the control of Wnt1 promoter, which is expressed in the central nervous system and neural crest of the sympathoadrenal lineage from which neuroblastoma develops. Heterozygous Lmo3 and Hen2 transgenic mice (Tg (Lmo3) and Tg (Hen2)) developed hydrocephalus at higher frequency than for the wild type mice, and all heterozygous double-transgenic mice (Tg (Lmo3; Hen2)) developed hydrocephalus. Therefore, Lmo3 and Hen2 may be involved in and have synergistic effects on hydrocephalus development. Although aqueduct stenosis occurred in all genotypes, it was mild in Tg (Lmo3; Hen2) mice. Furthermore, hydrocephalus was detected at E18.5 in Tg (Lmo3; Hen2). These results suggest that the causes of hydrocephalus are not only aqueduct stenosis but also disorder of neocortical development. A similar phenotype was reported in Robo1/2(-/-) mice, in which Hes1 expression level was decreased in ventricular zone progenitors. Thus, it is suggested that the expression levels of Lmo3 and/or Hen2 could determine the fate of stem cells by inhibiting Hes1 function during nervous system development and might be a trigger of aberrant neurogenesis in vivo.

Meis1 regulates epidermal stem cells and is required for skin tumorigenesis.

Previous studies have shown that Meis1 plays an important role in blood development and vascular homeostasis, and can induce blood cancers, such as leukemia. However, its role in epithelia remains largely unknown. Here, we uncover two roles for Meis1 in the epidermis: as a critical regulator of epidermal homeostasis in normal tissues and as a proto-oncogenic factor in neoplastic tissues. In normal epidermis, we show that Meis1 is predominantly expressed in the bulge region of the hair follicles where multipotent adult stem cells reside, and that the number of these stem cells is reduced when Meis1 is deleted in the epidermal tissue of mice. Mice with epidermal deletion of Meis1 developed significantly fewer DMBA/TPA-induced benign and malignant tumors compared with wild-type mice, suggesting that Meis1 plays a role in both tumor development and malignant progression. This is consistent with the observation that Meis1 expression increases as tumors progress from benign papillomas to malignant carcinomas. Interestingly, we found that Meis1 localization was altered to neoplasia development. Instead of being localized to the stem cell region, Meis1 is localized to more differentiated cells in tumor tissues. These findings suggest that, during the transformation from normal to neoplastic tissues, a functional switch occurs in Meis1.

Congenic mapping and allele-specific alteration analysis of Stmm1 locus conferring resistance to early-stage chemically induced skin papillomas.

Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to early-stage chemically induced skin papillomas on chromosome 7 with a large number of [(FVB/N×MSM/Ms)×FVB/N] F1 backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 7. We used linkage analysis and congenic mouse strains to refine the location of Stmm1 (Skin tumor modifier of MSM 1) locus within a genetic interval of about 3 cM on proximal chromosome 7. In addition, we used patterns of allele-specific imbalances in tumors from F1 backcross and N10 congenic mice to narrow down further the region of Stmm1 locus to a physical distance of about 5.4 Mb. To gain the insight into the function of Stmm1 locus, we carried out a long term BrdU labelling experiments with congenic mice containing Stmm1 locus. Interestingly, we observed a decrease of BrdU-LRCs (Label Retaining Cells) in a congenic strain heterozygous or homozygous for MSM allele of Stmm1. These results suggest that Stmm1 responsible genes may have an influence on papillomagenesis in the two-stage skin carcinogenesis by regulating epidermal quiescent stem cells.

Dependence receptor UNC5D mediates nerve growth factor depletion-induced neuroblastoma regression.

Spontaneous regression of neuroblastoma (NB) resembles the developmentally regulated programmed cell death (PCD) of sympathetic neurons. Regressing tumor cells express high levels of the nerve growth factor (NGF) receptors TRKA and p75NTR and are dependent on NGF for survival; however, the underlying molecular mechanism remains elusive. Here, we show that UNC5D, a dependence receptor that is directly targeted by p53 family members, is highly expressed in favorable NBs. NGF withdrawal strongly upregulated UNC5D, E2F1, and p53 in human primary favorable NBs. The induced UNC5D was cleaved by caspases 2/3, and the released intracellular fragment translocated into the nucleus and interacted 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. Unc5d(-/-) 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.

Oncogenic LMO3 collaborates with HEN2 to enhance neuroblastoma cell growth through transactivation of Mash1.

Expression of Mash1 is dysregulated in human neuroblastoma. We have also reported that LMO3 (LIM-only protein 3) has an oncogenic potential in collaboration with neuronal transcription factor HEN2 in neuroblastoma. However, the precise molecular mechanisms of its transcriptional regulation remain elusive. Here we found that LMO3 forms a complex with HEN2 and acts as an upstream mediator for transcription of Mash1 in neuroblastoma. The high levels of LMO3 or Mash1 mRNA expression were significantly associated with poor prognosis in 100 primary neuroblastomas. The up-regulation of Mash1 remarkably accelerated the proliferation of SH-SY5Y neuroblastoma cells, while siRNA-mediated knockdown of LMO3 induced inhibition of growth of SH-SY5Y cells in association with a significant down-regulation of Mash1. Additionally, overexpression of both LMO3 and HEN2 induced expression of Mash1, suggesting that they might function as a transcriptional activator for Mash1. Luciferase reporter assay demonstrated that the co-expression of LMO3 and HEN2 attenuates HES1 (a negative regulator for Mash1)-dependent reduction of luciferase activity driven by the Mash1 promoter. Chromatin immunoprecipitation assay revealed that LMO3 and HEN2 reduce the amount of HES1 recruited onto putative HES1-binding sites and E-box within the Mash1 promoter. Furthermore, both LMO3 and HEN2 are physically associated with HES1 by immunoprecipitation assay. Thus, our present results suggest that a transcriptional complex of LMO3 and HEN2 may contribute to the genesis and malignant phenotype of neuroblastoma by inhibiting HES1 which suppresses the transactivation of Mash1.

LMO3 interacts with p53 and inhibits its transcriptional activity.

High expression of LMO3 contributes to the development and aggressiveness of neuroblastoma. LMO3 belongs to the LIM-only protein family, in which de-regulation of its members is implicated in human carcinogenesis. However, the molecular mechanism of LMO3 activity in oncogenesis remained poorly characterized. We found that LMO3 is a direct interacting partner of p53 both in vitro and in vivo. The DNA-binding domain of p53 is required for this interaction. Furthermore, expression of LMO3 repressed p53-dependent mRNA expression of its target genes by suppressing promoter activation. Interestingly, chromatin immunoprecipitation assay showed that LMO3 facilitated p53 binding to its response elements. This suggests that LMO3 acts as a co-repressor of p53, suppressing p53-dependent transcriptional regulation without inhibition of its DNA-binding activity.

High expression of ncRAN, a novel non-coding RNA mapped to chromosome 17q25.1, is associated with poor prognosis in neuroblastoma.

Neuroblastoma shows complex patterns of genetic aberrations including MYCN amplification, deletion of chromosome 1p or 11q, and gain of chromosome 17q. The 17q gain is frequently observed in high-risk neuroblastomas, however, the candidate genes still remain elusive. In the present study, we integrated the data of comparative genomic hybridization of 236 tumors by BAC array and expression profiling of 136 tumors by using the in-house cDNA microarray carrying 5,340 genes derived from primary neuroblastomas. A novel candidate gene mapped to chromosome 17q25.1 with two splicing variants, Nbla10727 and Nbla12061, was identified. The transcript size appeared to be 2.3 kb by Northern blot, however, the cDNA sequences had no obvious open reading frame. The protein product was undetectable by both in vivo and in vitro translation assays, suggesting that the transcript might not encode any protein product. Therefore, we named it as ncRAN (non-coding RNA expressed in aggressive neuroblastoma). In analysis of 70 patients with sporadic neuroblastoma, the high levels of ncRAN mRNA expression were significantly associated with poor outcome of the patients (p<0.001). The multivariate analysis showed that expression of ncRAN mRNA was an independent prognostic factor among age, stage, origin and MYCN expression. Ectopic expression of ncRAN induced transformation of NIH3T3 cells in soft agar, while knockdown of endogenous ncRAN with RNA interference significantly inhibited cell growth in SH-SY5Y cells. Collectively, our results suggest that ncRAN may be a novel non-coding RNA mapped to the region of 17q gain and act like an oncogene in aggressive neuroblastomas.

Relationship of DDX1 and NAG gene amplification/overexpression to the prognosis of patients with MYCN-amplified neuroblastoma.

PURPOSE: Amplification of the MYCN gene strongly correlates with advanced stage, rapid tumor progression and poor prognosis in neuroblastoma (NB). Several genes in the MYCN amplicon, including the DEAD box polypeptide 1 (DDX1) gene, and neuroblastoma-amplified gene (NAG gene), have been found to be frequently co-amplified with MYCN in NB. The aim of this study was to clarify the prognostic significance of the co-amplification or overexpression of DDX1 and NAG with MYCN. PROCEDURE: The gene copy numbers and mRNA expression levels of MYCN, DDX1, and NAG in 113 primary NBs were determined by the real-time quantitative polymerase chain reaction or quantitative reverse transcriptase/polymerase chain reaction assay. The relationships between gene co-amplification/overexpression status and stage, age at diagnosis, and overall survival were analyzed. RESULTS: For evaluating the frequency of DDX1 and NAG co-amplification, it proved appropriate to discriminate NBs with <40 copies of MYCN amplification from those with > or =40 copies of MYCN (DDX1, p = 0.00058; NAG, p = 0.0242, chi(2) for independence test). In patients with MYCN-amplified NB aged > or =18 months, those with tumor with enhanced DDX1 expression and low-NAG expression showed a significantly better outcome than those with low-DDX1 expression or enhanced NAG expression (p = 0.0245, log-rank test). None of the gene expression statuses had a significant relation to disease stage or survival for patients <18 months old. No relationship between any gene co-amplification status and disease stage, age at diagnosis, or overall survival was found. CONCLUSIONS: Our findings suggest that there may be a subset of NB in which enhanced DDX1 and low-NAG expression consequent to DDX1 co-amplification without NAG amplification contributes to susceptibility to intensive therapy. A larger study using an age cut-off of 18 months will be required.

Expression profiling using a tumor-specific cDNA microarray predicts the prognosis of intermediate risk neuroblastomas.

To predict the prognosis of neuroblastoma patients and choose a better therapeutic protocol, we developed a cDNA microarray carrying 5340 genes obtained from primary neuroblastomas and examined 136 tumor samples. We made a probabilistic output statistical classifier that provided a high accuracy in prognosis prediction (89% at 5 years) and a highly reliable method to validate it. Kaplan-Meier analysis indicated that the patients in an intermediate group defined by existing markers are divided by microarray into two further groups with 5 year survivals for 36% and 89% of patients (p < 10(-4)), i.e., with unfavorably and favorably predicted neuroblastomas, respectively. According to these results, we developed a gene subset chip for a clinical tool, for which our classifier exhibited 88% prediction accuracy.

Identification of novel human neuronal leucine-rich repeat (hNLRR) family genes and inverse association of expression of Nbla10449/hNLRR-1 and Nbla10677/hNLRR-3 with the prognosis of primary neuroblastomas.

To search for novel prognostic indicators, we previously cloned >2,000 novel genes from primary neuroblastoma (NBL) cDNA libraries and screened for differential expression between the subsets with favorable (stage 1 or 2 with a single copy of MYCN) and unfavorable (stage 3 or 4 with amplification of MYCN) prognosis. From them, we have identified 3 genes of human neuronal leucine-rich repeat protein (NLRR) family: Nbla10449/hNLRR-1, Nbla00061/hNLRR-2/GAC1 and Nbla10677/hNLRR-3. An additional family member, hNLRR-5, was also found by homology search against public database. NLRR family proteins have been proposed to function as a neuronal adhesion molecule or soluble ligand binding receptor like Drosophila toll and slit with multiple domains including 11 sets of extracellular leucine-rich repeat (LRR)-motifs. However, the functional role of the NLRR protein family has been elusive. Our present study shows that hNLRR mRNAs are preferentially expressed in nervous system and/or adrenal gland. In cancer cell lines, hNLRR-1, hNLRR-3 and hNLRR-5 are expressed at high levels in the neural crest-derived cells. Most remarkably, in primary NBLs, hNLRR-1 is significantly expressed at high levels in unfavorable subsets as compared to favorable ones, whereas the expression pattern of hNLRR-3 and hNLRR-5 is the opposite. In order to understand the function of these receptors, we have used newborn mouse superior cervical ganglion (SCG) cells which are dependent on nerve growth factor (NGF) for their survival. Expression of the mouse counterparts of hNLRR-2 and hNLRR-3 is up-regulated after NGF-induced differentiation and down-regulated after NGF depletion-induced apoptosis. On the other hand, expression of hNLRR-1 and hNLRR-5 is inversely regulated in the same system. These results have suggested that the regulation of the hNLRR family genes may be associated with NGF signaling pathway in both SCG cells and neuroblastoma. Our quantitative real-time RT-PCR analysis using 99 primary NBLs has revealed that high levels of hNLRR-1 expression are significantly associated with older age (>1 year, p=0.0001), advanced stages (p=0.0007), low expression of TrkA (p=0.011), and MYCN amplification (p=0.0001), while those of hNLRR-3 expression are significantly correlated with the favorable prognostic indicators. Furthermore, multivariate analysis reveals that expression of hNLRR-1 is an independent prognostic indicator in human neuroblastoma. Thus, our results demonstrate that, despite being members of the same family, hNLRR-1 and hNLRR-3 may share different biological function among the NBL subsets, and that their expression level becomes novel prognostic indicators of NBL.