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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure Statement
  8. References
  9. Supporting Information

Dihydropyrimidinase-like proteins (DPYSLs) are a family of proteins developmentally regulated during maturation of the nervous system. Recently, members of the DPYSL family have been reported to be involved in cancer with low expression of DPYSL1 correlating with poor clinical outcomes in non-small cell lung cancer and functioning as a metastasis suppressor. Neuroblastoma (NB) is a tumor derived from precursor cells of the sympathetic nervous system and is the most common solid tumor in childhood. So far the biological functions of DPYSLs in NB remain elusive. Studying the potential roles of DPYSLs in NB may give us new insights into NB tumorigenesis. In the present study, using antibodies specific to different members of the DPYSL family, DPYSL1, DPYSL2 and DPYSL3, we investigated regulation of their expression and their subcellular distribution during retinoic acid (RA)-induced differentiation in NB cells. The correlation between DPYSLs and MYCN, a biomarker for poor prognosis of NB, was evaluated. We found that DPYSL3 levels increased during RA-induced cell differentiation. Downregulation of MYCN by small interfering RNA (siRNA) increased DPYSL3 levels, while upregulation of MYCN in non-MYCN NB cells decreased DPYSL3 levels. DPYSL1 and DPYSL2 expression didn't change during RA treatment or under different expression levels of MYCN. Moreover, a high level of DPYSL3 mRNA, but not that of DPYSL1 or DPYSL2 mRNA, was detected in tumors from advanced-stage NB that have a better survival. These data indicated that DPYSL3, not DPYSL1 or DPYSL2, is negatively regulated by MYCN and may be used as a potential biomarker for NB.

Neuroblastoma (NB) is the most common extracranial-solid tumor in childhood and derives from precursor cells of the sympathetic nervous system.[1, 2] Neuroblastoma is characterized by its remarkable biological heterogeneity. Some patients experience spontaneous regression or differentiation into benign ganglioneuroma, while other patients are affected by rapid and fatal tumor progression despite increasingly intensive treatment strategies.[3] MYCN is an oncogene that is critical in NB tumorigenesis and is a biomarker for poor prognosis, advanced stage and rapid tumor progression in NB. Retinoic acid (RA), a vitamin A derivative, is known to induce some NB cell differentiation and inhibit MYCN expression.[4] Retinoic acid has been incorporated into the standard treatment protocols for high-risk neuroblastoma, and has led to a significant increase of event-free survival in NB patients.[5, 6]

While retinoids are known to stimulate NB cell differentiation, the mechanisms mediating neurite extension remain to be elucidated. Dihydropyrimidinase-like protein is the mammalian homolog of the Caenorhabditis elegans (C. elegans) uncoordinated 33 (unc-33)-like phosphorylation gene. It is originally identified with different names by different groups as CRMP (Collapsin Response Mediator Protein),[7] Ulip (Unc-33-like phosphoprotein),[8] TUC,[9] DRP (dihydropyrimidineamidohydrolase-related protein)[10] and TOAD (Turned On After Division).[11, 12] There are five members in this family, DPYSL1/CRMP1/Ulip3/DRP1, DPYSL2/CRMP2/Ulip2/DRP2, DPYSL3/CRMP4/Ulip1/DRP3, DPYSL4/CRMP3/Ulip4/DRP4, DPYSL5/CRMP5/Ulip6/DRP5, that are expressed in distinct yet partially overlapping patterns of expression during the development of the nervous system in human, rat, and mice.[13-16] Although DPYSLs were originally found to be expressed during the development of nervous system, different family members have been shown to be expressed in tumors derived from lung, colon and prostate.[17-21] We previously reported that RA treatment increased DPYSL mRNA and protein expression in neuroblastoma cells;[22] however, the protein expression of different family members and their regulation by RA had not been reported. In the present study, we used antibodies specific to DPYSL1, DPYSL2 or DPYSL3, the most studied DPYSL family members, to study the regulation of DPYSLs protein expression after RA treatment, and to investigate the correlation between DPYSLs and MYCN in NB cells.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure Statement
  8. References
  9. Supporting Information

Cell culture and treatment

Neuroblastoma cells (MYCN amplified cell lines: KCNR, NGP and BE2; non-MYCN amplified cell lines: AS and SHEP) were cultured in RPMI 1640 supplemented with 10% FBS for 24 h, then cells were treated with 5 μM all-trans retinoic acid (RA) (Sigma-Aldrich, St. Louis, MO, USA) or control solvent for 4 days. Whole cell proteins were isolated. A tetracycline-regulated MYCN system was transfected into non-MYCN amplified SHEP cells. The MYCN expression was inhibited with the presence of tetracycline (TET).

Small interfering RNA and plasmids

Small interfering RNA (siRNA) targeting MYCN was designed and purchased from Qiagen (Valencia, CA, USA) as described previously.[23] MYCN plasmid (PIRV-MYCN) and empty vector were kindly provided by Dr Geoff Krystal, McGuire VA Medical Center (Richmond, VA, USA).

Transfection of MYCN siRNA and MYCN plasmid

MYCN or control siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were transfected into KCNR, NGP and BE2 cells. PIRV-MYCN plasmid and empty vector were transfected into AS cells. Transfection was performed using the Nucleofector from Amaxa Biosystems and the cell line Nucleofector Kit V with conditions according to program A-030.

Antibody production

Polyclonal DPYSL antibodies were obtained from Zymed (San Francisco, CA, USA) by immunizing rabbits with the following synthesized peptides: DPYSL1 peptide, corresponding to amino acid region 409–423 (VVIWDPDKLKTITAK) of human DPYSL1; DPYSL 2 peptide, corresponding to amino acid region 140–153 (ISEWHKGIQEEME) of the human DPYSL 2; DPYSL3 peptide, corresponding to amino acid region 501–514 (GPVFDLTTTPKGGT) of the human DPYSL3 sequence.

Immunofluorescence staining

KCNR cells were plated onto collagen (BD Biosciences, San Jose, CA, USA, working concentration 50 μg/mL)-coated 24-well plates containing coverslips at a density of 9000/well. Cells were treated with 5 μM retinoic acid (Sigma) or control solvent for 4 days. The cells were fixed with 4% paraformaldehyde, rinsed in PBS and incubated with the appropriate primary antibodies (anti-DPYSLs antibodies [1:500], and anti-α-tubulin antibody [1:200]) at room temperature for 2 h. Subsequently the cells were rinsed in PBS and incubated with secondary antibodies (FITC-conjugated goat anti-mouse IgG (1:100) or TRITC-conjugated goat-anti rabbit IgG (1:100) (Jackson Immuno Research, Westgrove, PA, USA) at room temperature for 45 min. After incubation with secondary antibodies, the coverslips containing cells were sealed using Vectashield Mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and stored at 4ºC.

Western blotting

Western blotting was performed as described previously.[24] Briefly, protein lysate (30 μg) was loaded onto SDS-PAGE gels, transferred to nitrocellulose and probed with the indicated specific anti-DPYSLs antibodies (1:1000 dilution) or anti-actin antibody (1:2000 dilution). Signals were detected using enhanced chemiluminescence reagents (Amersham Life Science, Arlington Heights, IL, USA).

Cell cycle analysis

To assess cell cycle changes, KCNR cells were seeded into 150-mm dishes and treated with RA at 5 μM for 4 days. Cells were washed in cold PBS, incubated with RNase at 100 μg/mL and propidium iodide (PI) at 50 μg/mL for 30 min in a dark environment at room temperature. The stained cells were analyzed for DNA content by fluorescence activated cell sorting (FACS) in a FACSscan (Becton-Dickinson, Franklin Lakes, NJ, USA). CELLQUEST software (BD Biosciences) was used to quantify the percentage of cells in different stages of the cell cycle.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure Statement
  8. References
  9. Supporting Information

DPYSL protein expression in KCNR cells after RA treatment

To evaluate expression patterns of DPYSL1, -2 and -3 proteins in response to RA treatment, KCNR cells were treated with RA for 4 days. We previously reported that RA-treatment induced neuroblastoma cell differentiation, and the morphologic hallmark of neural differentiation is neurite extension.[4] Figure 1(a) demonstrates morphologic changes of KCNR cells after RA treatment. There were marked neurite extensions in RA-treated cells and cell cycle analysis revealed that there was an increase of cells in the G1 phase of the cell cycle in RA-treated cells compared to control cells (Fig. 1b). Proteins were isolated from RA-treated cells and evaluated for expression of DPYSL1, DPYSL2 and DPYSL3. A basal level expression of each of the DPYSLs was detected in non-treated control cells as evidenced by the 62 kDa and a 72 kDa isoforms of DPYSL1, the 62 kDa DPYSL2, and the 62 kDa and a 72 kDa isoforms of DPYSL3 (Fig. 1c). The expression of both DPYSL3 isoforms increased after RA treatment, while expression of DPYSL1 and DPYSL2 was unchanged (Fig. 1c). There was also an increase in a 65 kDa DPYSL3 protein, which we have previously shown is an underphosphorylated form of the 72 kDa DPYSL3 isoform.[22] We also found that transfection of DPYSL3 into NB cells increased the expression of neural differentiation marker MAP2 and induced neurite extension (Fig. S1). These data indicate that protein levels of DPYSL3, but not DPYSL1 or DPYSL2, increased during RA-induced KCNR cell differentiation.

image

Figure 1. Expressions of dihydropyrimidinase-like proteins (DPYSLs) during retinoic acid (RA)-induced differentiation in KCNR cells. KCNR cells were treated with RA for 4 days. (a) Morphological changes after RA treatment under microscopy (20× magnification). (b) Cell cycle analysis after RA treatment. (c) Whole cell lysate was extracted from KCNR cells. Thirty micrograms of protein was analyzed for the expression of DPYSL1, DPYSL2 and DPYSL3 by Western blotting. Actin was used as loading control.

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Subcellular distribution of DPYSLs in NB cells

Although DPYSLs were originally found to be cytosolic proteins, nuclear distribution of DPYSLs was reported recently and associated with the clinical outcome of patients with cancers.[25] To study the subcellular distribution of these three DPYSLs in NB cells, and assess whether RA treatment altered cellular localization, we performed immunofluorescence staining in KCNR cells with anti-DPYSLs antibodies, anti-α-tubulin antibody and nuclear staining reagent DAPI. As shown in Figure 2(a), DPYSL1 was found mainly in the cytoplasm, but there was a punctate staining in nucleus in both control and RA treated cells. DPYSL2 was predominately located in cytoplasm in control and RA-treated cells with no nuclear staining detected (Fig. 2b). No changes of DPYSL1 and DPYSL2 levels were observed. DPYSL3 is also predominately distributed in cytoplasm in control cells. After 4 days of RA treatment although DPYSL3 expression increased, consistent with the Western blot analysis in Figure 1(c), nuclear DPYSL3 was not detected (Fig. 2c). These data indicated that there was no readily detectable subcellular translocation of DPYSLs in response to RA treatment although DPYSL1 was detected in both cytoplasm and nucleus.

image

Figure 2. The subcellular distribution of dihydropyrimidinase-like proteins (DPYSLs) in KCNR cells. KCNR cells were treated with retinoic acid (RA) or control solvent for 4 days, fixed with paraformaldehyde. The cells were incubated with anti-DPYSLs and anti-α-tubulin antibodies, and detected with fluorescein-conjugated secondary antibodies (TRITC-red for DPYSLs, FITC-green for α-tubulin). DAPI (blue) was used to stain the nuclei. Arrow: the distribution of DPYSL1 in nuclei. (a) DPYSL1 staining. (b) DPYSL2 staining. (c) DPYSL3 staining. The magnification of the pictures: 60×.

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MYCN regulates DPYSL3 expression

An important aspect of RA-induced neural differentiation in NB cells is the downregulation of MYCN.[26-28] We find a more than 90% decrease in MYCN expression after 4 days of RA treatment in KCNR cells (Fig. 3a). As RA treatment induced an increase of DPYSL3 protein expression (Fig. 1c) and a decrease of MYCN protein expression (Fig. 3a), we evaluated whether there was any association between MYCN and DPYSLs expression. We downregulated MYCN expression by transfecting a MYCN targeted siRNA into KCNR cells, which resulted in a 70% decrease of MYCN protein expression. This was accompanied by increased expression of both DPYSL3 isoforms (Fig. 3b). There was no detectable change in DPYSL1 and DPYSL2 expression in MYCN siRNA transfected cells compared to control siRNA transfected cells. This result indicated that downregulation of MYCN results in an increase in DPYSL3 expression in KCNR cells.

image

Figure 3. MYCN regulation of DPYSL3. (a) KCNR cells were treated with retinoic acid (RA) for 4 days, whole cell lysates were extracted and analyzed with anti-MYCN antibody. (b) KCNR cells were transfected with MYCN siRNA. Whole cell lysate was extracted 48 h after transfection, and 30 μg protein was used to analyze the expression of DPYSL1, DPYSL2, DPYSL3. (c). Expression of DPYSL family members in AS NB cells. Five micrograms of MYCN or empty vector plasmid were transfected into AS cells, 48 h after transfection, cells were harvested and whole cell lysate was extracted. Thirty micrograms of protein was used to analyze the expression of DPYSL1, DPYSL2, DPYSL3 and MYCN. (d) NGP and BE2 cells were either treated with RA for 4 days or transfected with MYCN siRNA for 48 h, then whole cell lysate was extracted. Thirty micrograms of protein was used to analyze the expression of MYCN and DPYSL3. (e) SHEP cells were cultured with tetracycline for 24 h or 48 h, whole cell lysate was extracted and used to analyze the expression of MYCN and DPYSL3. Actin was used as a loading control for all the Western blotting.

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To further investigate the regulation of MYCN on DPYSLs, we transfected MYCN plasmid into the AS NB cell line, which contains normal MYCN gene copy number and does not express MYCN protein. AS cells only express the 62 kDa DPYSL2 and 72 kDa DPYSL3, but not DPYSL1 by Western blot analysis (Fig. 3c). Transfection of MYCN plasmid into AS cells increased MYCN expression and this was accompanied by a decrease in DPYSL3 expression, but there was no change in DPYSL2 expression (Fig. 3c). The expression of DPYSL1 was also not altered by changes in MYCN expression. This result indicated that overexpression of MYCN selectively inhibits the expression of DPYSL3 in NB cells.

To confirm the association between DPYSL3 and MYCN in other NB cell lines, we treated two additional MYCN amplified NGP and BE2 NB cell lines with RA for 4 days or downregulated MYCN expression using MYCN siRNA. We found that the expression of 62 kDa isoform of DPYSL3 increased in both NGP and BE2 cells after RA treatment. Transfection of MYCN targeted siRNA into either BE2 or NGP cells resulted in decreases in MYCN expression and this was accompanied by increases in the 62 kDa isoform of DPYSL3. The RA-induced changes in expression of the 72 kDa isoform of DPYSL3 were cell line specific with no change in NGP cells and a decrease in BE2 cells. Downregulation of MYCN by siRNA did not affect the expression of 72 kDa isoform of DPYSL3 in either NGP or BE2 cells (Fig. 3d). Using a tetracycline (TET)-regulated MYCN system (tetracycline-off system), we also studied the regulation of DPYSL3 by MYCN in non-MYCN amplified SHEP cells. As shown in Figure 3(e), with the absence of TET, MYCN was expressed, and the expression of MYCN was inhibited in the presence of TET at 24 h and 48 h. The DPYSL3 (72 kDa isoform) expression increased at 24 h treatment with TET and continued to increase at 48 h. These data indicate that DPYSL3 is negatively regulated by MYCN in neuroblastoma cells.

Low level of DPYSL3 expression marks NB patients with a poor survival

The differential regulation of DPYSLs proteins in NB cells suggested that expression of these genes may be differentially expressed in the primary tumors of NB patients. We queried the R2 database (http://hgserver1.amc.nl/cgi-bin/r2/main.cgi), which contains expression of 88 primary tumors from patients with NB that are representative of the clinical presentation of NB. The analysis of DPYSLs mRNA expression in NB tumors and the survival of patients was performed, and the Kaplan–Meier survival graphs in Figure 4(a–c) show that when the relative levels of DPYSL1, DPYSL2 or DPYSL3 are stratified into high or low expression, only DPYSL3 has a statistically significant association with overall survival (Bonferroni corrected P-value [Bonfp] = 4.6xe-07). Low levels of DPYSL3 are found in patients who have shorter overall survival. While low levels of DPYSL1 and DPYSL2 also tend to be detected in patients with a poor survival, they didn't reach statistical significance, using a Bonferroni corrected P-value that takes into account multiple comparisons. To understand whether DPYSL3 levels were prognostically significant in advanced-stage NB patients, we only evaluated tumors from INSS Stage 3 and 4. Figure 4(d) shows that relatively higher levels of DPYSL3 are found in the tumors of patients who have a better overall survival. We also evaluated the relative levels of DPYSL3 in INSS Stage 4 patients. Even in high risk cohort, all of whom do poorly, the patients whose tumors have the lowest levels of DPYSL3 expression have the shortest overall survival (Fig. 4e). Since in vitro studies indicated that the level of DPYSL3 was regulated by MYCN, we compared the relative level of expression of DPYSL3 in the tumors based on MYCN copy status. When one stratifies the NB patients' tumors into those that contain MYCN amplification compared to those that have normal MYCN copy number, lower levels of DPYSL3 are found in tumors with MYCN amplification. A one-way anova indicates the P-value is 2.7xe-5 (Fig. 4f). There was not a significant difference in DPYSL1 or DPYSL 2 expression between the MYCN amplified or non-MYCN amplified tumors (data not shown). These data support the in vitro findings that DPYSL genes are differentially expressed and regulated in NB tumor cells, with high levels of DPYSL3 being associated with a better overall survival.

image

Figure 4. DPYSL1, DPYSL2 and DPYSL3 expression in primary human neuroblastoma (NB) tumor tissue. (a) The R2 database (http://hgserver1.amc.nl/cgi-bin/r2/main.cgi?&species=hs) was queried to determine the relative expression of dihydropyrimidinase-like protein (DPYSL) family members in primary NB tumor tissue. Kaplan–Meier survival plots examining DPYSL1 (a), DPYSL2 (b) and DPYSL3 (c) are depicted. Multiple comparisons were made to stratify the patients. (d) From the original cohort of 88 patients, the 53 patients who were in the advanced-stage category (INSS Stage 3 & 4) were evaluated for relative DPYSL3 levels. (e) From the original cohort of 88 patients, the 40 patients who were in the advanced-stage category (INSS Stage 4) were evaluated for relative DPYSL3 levels. (f) All 88 NB patients were stratified into those whose tumors had MYCN amplification or not. Mean relative expression was shown in the box plots. There was a statistically significant difference showing that tumors with MYCN amplification had lower levels of DPYSL-3 expression.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure Statement
  8. References
  9. Supporting Information

In this study we found differential expression and regulation of the DPYSL gene family members in NB cells at a steady-state and upon induction of differentiation. Only DPYSL3 is induced during RA-induced differentiation. Studies using genetic approaches showed that the levels of DPYSL3 but not DPYSL1 or DPYSL2 were inversely altered with changes in MYCN expression. These data indicate that MYCN negatively regulates DPYSL3 expression in NB cells. Finally high levels of DPYSL3 but not DPYSL1 or DPYSL2 mark advanced-stage and even high-risk NB patients that have a better overall survival.

Dihydropyrimidinase-like proteins were first characterized as phosphoproteins that were highly expressed in the developing nervous system and DPYSL family members regulate neurite outgrowth, axonal guidance and cell differentiation.[13, 29, 30] Recently, a study on the roles of DPYSLs in vivo indicated that during neurulation in zebrafish DPYSL3 was required for proper positioning of neural crest cells in the developing spinal cord.[31] This report highlights the importance of DPYSL3 in differentiation of neural crest cells and supports our findings that DPYSL3 expression may be important during the differentiation of neural crest-derived NB tumor cells.

Our previous studies have shown that RA treatment induces NB cell differentiation that is accompanied by an increase of DPYSL mRNA expression.[22] The two major isoforms of DPYSL3 are expressed in most NB cell lines as a 72 kDa and a 62 kDa isoform that arise by differential promoter utilization and splicing. Both isoforms are phosphorylated.[22] In this study, the levels of the 62 kDa isoform of DPYSL3 protein levels increased after RA treatment in MYCN amplified NB cell lines, while there was no change or a decrease in the expression of the 72 kDa DPYSL3 isoform that was cell line dependent. There is also an increase in a 65 kDa band, which we previously have shown is an under-phosphorylated form of the 72 kDa isoform. While we did not detect changes in the expression of DPYSL1 and DPYSL2 protein after RA treatment in the KCNR cells. There is a report of retinoid-induced decreases in mRNA and protein expression of DPYSL2 (Ulip2, CRMP2) in the non-MYCN amplified SY5Y NB cell line.[32] So the changes in the expression patterns of DPYSLs in NB after RA treatment may be dependent on the cell line, as well as MYCN expression. The 62 kDa and 72 kDa DPYSL isoforms are phosphoproteins and our previous study showed the 65 kDa to be a less phosphorylated form of the 72 kDa protein.[22] That a 65 kDa protein is not detected in protein lysates from the MYCN siRNA transfected cell lines suggests that changes in phosphorylation of DPYSL3 may be dependent on kinases or phosphatases regulated by retinoids and not MYCN levels.

For the first time we provide evidence that DPYSL3 is negatively regulated by MYCN. Such a relationship between DPYSL3 and MYCN has not been described in any type of tumor to date. Reports on the regulation of DPYSLs or MYCN by GSK-3β may give a clue to an association of DPYSLs and MYCN in NB. The regulation of DPYSLs by GSK-3β in neuronal polarity or axon outgrowth has been reported.[33, 34] GSK3β may also indirectly regulate DPYSLs in NB cells via MYCN. We have previously shown that activated Akt in NB cells phosphorylate and thus inactivate GSK-3β.[35] Inactivation of GSK-3β would lead to an increase of MYCN protein expression, since the GSK-3β-mediated phosphorylation of MYCN protein leads to its degradation by proteasome.[36] Thus increased MYCN levels via amplification or as the result of Akt-mediated GSK-3β inactivation would lead to DPYSL3 suppression in NB cells. MYCN amplification in NB is a biomarker for poor prognosis, advanced stage and rapid tumor progression.[37] Recent evidence indicates that MYC may be a transcriptional amplifier, and the negative regulation of gene expression by MYC gene is most probably via indirect mechanisms.[38] The negative regulation of DPYSL3 by MYCN may be mediated via EZH2 as we have recently found that EZH2 suppresses a number of genes with tumor suppressor characteristics or differentiation-inducing activity in NB.[39] These biofunctional studies as well as the expression of DPYSL3 levels in the primary tumors of NB patients indicate that DPYSL3 may be a new biomarker of NB with differentiated phenotype and good prognosis.

It is well known that the tumors of NB patients who have a good prognosis are marked by a differentiated histology and a transcriptome enriched in differentiated genes.[1] Moreover, a recent NexGen sequencing study indicated that mutations in regulators of the Rac/Rho pathway implicated defects in neuritogenesis in neuroblastoma tumorigenesis.[40] Recent evidence indicates that Rho binds to DPYSL proteins and mediates neurite extension or retraction in different model systems.[41] Thus, the relative decrease in expression of DPYSL3 in NB tumors with a poor prognosis may be part of the dysregulation of neuritogenesis. For the biofunctional study of DPYSL3 in our model system, we didn't observe any effect of DPYSL3 on cell growth (data not shown), and have not been able to confirm the role of DPYSL3 on neurite extension in all the tested cell lines. The function of DPYSL3 in NB cells has not been clarified although based on the literature it is reasonable to assume that DPYSL3 may be involved in the implementation of a more differentiated phenotype. Recently, expression of the DPYSL family members have been reported in cancers. DPYSL1 (Ulip3, CRMP1) is found to be a metastasis suppressor in lung cancer patients[17] and connective tissue growth factor inhibits metastasis and invasion of human lung adenocarcinoma via upregulation of DPYSL1.[19] DPYSL2 (Ulip2, CRMP2) was detected in colorectal carcinoma and reported to be a novel biomarker.[20] DPYSL3 (Ulip1, CRMP4) has been shown to be significantly associated with poor prognosis in pancreatic cancer by promoting liver metastasis and may serve as a novel therapeutic target;[42] however, it has also been identified as a metastasis suppressor in prostate cancer.[21] But the role of different family members in cancer is complex, because a novel human isoform of DPYSL family protein named long form CRMP1 was found to be a cancer invasion enhancer, and associated with tumor progression and clinical outcomes in non-small cell lung cancer.[18] Our findings indicated that elevated levels of DPYSL3 occur in tumors of advanced-stage NB patients who have a good prognosis, and even in high-risk NB patients the levels of DPYSL3 mark those patients who may have a better overall survival. This study supports the continued evaluation of this DPYSL family member in NB tumors to understand its biologic involvement in differentiated NB tumors.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure Statement
  8. References
  9. Supporting Information

We would like to thank Dr Geoff Krystal for the MYCN expression vector, Dr Rogier Versteeg and Richard Volckmann (Academic Medical Center, Department of Human Genetics, Amsterdam, the Netherlands) for making the R2 bioinformatics database publicly available. This work was supported by and performed at the NCI Intramural Research Program of Center for Cancer Research at the National Cancer Institute in the National Institute of Health, National Natural Science Foundation of China (No. 81272538) and Society Development Foundation of Liaoning Province (No. 201225016).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure Statement
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure Statement
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
cas12278-sup-0001-FigS1.tifimage/tif335KFig. S1. DPYSL3 increased the expression of MAP2 and neurite extension.

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