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Clinical Cancer Research Vol. 11, 3280-3287, May 1, 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Shc Family Expression in Neuroblastoma: High Expression of shcC Is Associated with a Poor Prognosis in Advanced Neuroblastoma

Elena Terui1, Tadashi Matsunaga1, Hideo Yoshida1, Katsunori Kouchi1, Hiroaki Kuroda1, Tomoro Hishiki1, Takeshi Saito1, Shin-ichi Yamada1, Hiroshi Shirasawa2 and Naomi Ohnuma1

Authors' Affiliations: Departments of 1 Pediatric Surgery (E6) and 2 Molecular Virology (E2), Graduate School of Medicine, Chiba University, Chiba, Japan

Requests for reprints: Hiroshi Shirasawa, Department of Molecular Virology (E2), Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuoku, Chiba 260-8677, Japan. Phone: 81-43-226-2312; Fax: 1-043-226-2366.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The biological features and prognosis of neuroblastoma, a neural crest-derived pediatric tumor, are closely associated with expression of the Trk receptor. Because the Shc family proteins (ShcA, ShcB, and ShcC) are adaptors for various receptors, including Trk receptors, and are regulators of neuronal cell development, we speculated that they may play a role in neuroblastoma. Therefore, in this study, we used semiquantitative reverse transcription-PCR to examine the expression of these three genes in 15 neuroblastoma cell lines, an all-trans-retinoic acid–treated neuroblastoma cell line, and 52 tumor samples. In neuroblastoma cell lines and tumor samples, shcA was ubiquitously and highly expressed. Little expression of shcA was observed. Also, shcB was hardly expressed in neuroblastoma cell lines, but its expression in RT-BM-1 cells was enhanced after all-trans-retinoic acid–induced differentiation, and it was highly expressed in low-stage tumors (P = 0.0095). This suggests that ShcB participates in cellular differentiation and may correlate with a favorable prognosis in neuroblastoma. Finally, the expression of shcC was observed in most of the neuroblastoma cell lines and in some stage 4 patients. Patients with a high expression of shcC had a very poor prognosis (P < 0.0001) and amplification of MYCN, and all died within 31 months after diagnosis. Therefore, ShcC seems to be associated with an aggressive tumor phenotype, perhaps by enhancing TrkB signals. Our results suggest that the expressions of shcB and shcC are important biological factors in neuroblastoma and are useful prognostic indicators.

Key Words: Neuroblastoma • Shc family • differentiation • survival • prognostic factor

Neuroblastoma is a pediatric solid tumor derived from a sympathoadrenal cell lineage of the neural crest (1), and it is clinically and biologically heterogeneous. Neuroblastomas found in children less than 1 year of age have a favorable outcome and often show the ability to differentiate or regress spontaneously, whereas those identified after 1 year of age are frequently metastatic with a poor prognosis (2). Neurotrophins and their receptors are essential for the growth, differentiation, and regression of neural crest-derived cells and are known to play important roles in the biology and clinical behavior of neuroblastomas (1, 3, 4). Several studies have shown that a high expression of TrkA, a high affinity receptor for nerve growth factor, correlates with a favorable prognosis and may mediate differentiation and/or regression (4, 5). In contrast, tumors with unfavorable biology frequently express TrkB, a receptor for brain-derived neurotrophic factor (BDNF), which contributes to the survival of neuroblastoma cells and resistance to chemotherapy (6–8).

Signal transduction through the Trk receptors leads to the activation of downstream signaling, including the Ras/mitogen-activated protein kinase and the phosphoinositide 3-kinase pathways. Binding of neurotrophins to the Trk receptors results in the autophosphorylation of intracellular tyrosine residues. Phosphotyrosine residues on the receptor then act as docking sites for adaptor molecules (9). Shc, a direct adaptor molecule, has recently received attention because of its role in modifying the development of neural progenitor cells (10). Three shc genes have been identified in mammals: shcA, shcB, and shcC. Expression of shcA is ubiquitous, whereas shcB and shcC are predominantly expressed in neuronal cells. Shc proteins possess two phosphotyrosine binding domains (PTB and SH2) as well as a central glycine/proline-rich region (CH1) that contains tyrosine phosphorylation sites (11).

The shcA gene was originally identified as a proto-oncogene involved in growth factor signaling (11). There are three isoforms of the ShcA protein, p46, p52, and p66, which are generated from the shcA transcript by RNA splicing or alternative translational initiation (11, 12). The mRNA for shcA is broadly expressed in adult human tissue except for the brain, and its protein is a downstream target and effector for many types of cell surface receptors. One of the most important roles of ShcA protein is in the activation of Ras during mitogenesis (11). Recently, ShcA was found to be highly expressed in the developing brain, suggesting that it plays a role in neuronal cell proliferation (10). In addition, constitutive phosphorylation of ShcA has been found in various human tumor cell lines (13).

The shcB mRNA is expressed in a wide range of human adult tissues. In the adult mouse, the ShcB protein expression is limited to the neuronal system. In rat and mouse embryos, the expression of shcB mRNA is much higher in dorsal root ganglia and superior cervical ganglia than in the brain (14, 15).

ShcC is expressed mainly in human adult brain (16). Expression of shcC mRNA is observed in migrating neural crest cells in mouse embryos and also in the superior cervical ganglia of 8-week-old mice (15). In the mouse brain, the level of ShcC protein increases after birth, and its level is maintained in the mature brain, suggesting that it participates in the differentiation and survival of neuronal cells (10, 15). In shcB/shcC double mutant mice, there is a significant loss of postmitotic superior cervical ganglia neurons, suggesting that ShcB and ShcC have functionally redundant roles in the survival of superior cervical ganglia neurons (15).

The Shc family proteins seem to act in developing neuronal cells as molecular switches from proliferation to survival and/or differentiation. ShcA is primarily expressed in proliferating neuronal cells (10). In postmitotic neurons, the level of ShcB protein increases slightly from early development until postnatal day 1, whereas ShcA expression decreases (14, 15). Furthermore, the level of ShcC protein increases after birth and is maintained in the mature brain, suggesting that ShcC contributes to survival of postmitotic neurons (10, 15).

Collectively, these findings indicate that the Shc family plays a significant role in neuronal development. Therefore, the expression of these proteins could correlate with neuroblastoma biology. There is only one report discussing the role of Shc family expression in neuroblastoma cells in which ShcC protein was found to be highly expressed in neuroblastoma cell lines and was associated with a poor prognosis (17). Neurotrophin downstream signaling through Trk receptor in neuroblastoma cells has been well studied but no clear association with biological features of neuroblastoma has been observed (6, 18, 19).

In this study, we examined 15 neuroblastoma cell lines for the expression of the three shc transcripts. Furthermore, we examined the role of shc family in neuroblastoma differentiation by examining the effect of all-trans-retinoic acid (RA) on the expression of shc mRNA in RT-BM-1 cells. Finally, we examined the expression of the shc family mRNA in 52 clinical neuroblastoma tumor samples and the association with tumor stage, patient age, MYCN amplification, and outcome.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Neuroblastoma cell lines. In these studies, we used 15 human neuroblastoma cell lines (NB69, SK-N-SH, SH-SY5Y, NB-1, cNBI, CHP134, IMR32, GOTO, NMB, NLF, NGP, SMS-KAN, SMS-KCN, LAN-5, and RT-BM-1). All cell lines were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum and 100 µg/mL kanamycin (Sigma, St. Louis, MO) at 37°C in an atmosphere of 5% CO2. RT-BM-1 cells (2 x 106 to 3 x 106 cells in a 90-cm tissue culture dish) were grown for 2 days, after which neuronal differentiation was induced with 5 µmol/L all-trans-retinoic acid (Sigma).

Tumor specimens. We studied 52 neuroblastoma patients who were treated at Chiba University Hospital, Chiba Children's Hospital, or Matsudo Municipal Hospital between 1988 and 2000. Untreated tumor tissues were immediately frozen after surgical resection or biopsy and stored in liquid nitrogen. Before gene analyses, pathologic examination confirmed that the specimens consisted of neuroblastoma cells. Pathologic classification was done by a pathologist (Dr. Hiroshi Horie, Pathologic Department of Chiba Children's Hospital) according to the International Neuroblastoma Pathology Committee classification (20). Ganglioneuromas and ganglioneuroblastoma intermixed types were not included in this study. The tumors were staged according to the International Neuroblastoma Staging System (2). Twenty-six of the patients were found by a mass screening program, and the other 26 were identified based on their clinical symptoms. The patients' characteristics are summarized in Table 1.


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  		Table 1. Characteristics of 52 neuroblastoma samples

 
Semiquantitative reverse transcription-PCR. Total RNA was extracted either by using ISOGEN (Nippon gene, Tokyo, Japan) or according to the method of Chomczynski and Sacchi (21). For cDNA synthesis, total RNA was denatured at 65°C for 10 minutes and immediately chilled on ice for 2 minutes. Five micrograms of total RNA in a volume of 30 µL with pd(N)6 random hexamers were added to First-Strand Reaction Mix Beads (Ready-To-Go You-Prime First-Strand Beads, Amersham Biosciences Corp., Piscataway, NJ) containing moloney murine leukemia virus reverse transcriptase. First strand cDNAs were obtained after a 1-hour incubation at 37°C. For PCR amplification, oligonucleotide primers for shcA, shcB, and shcC were designed as follows. Because these three genes have a high degree of identity, primers were designed based on RNA encoding the CH1 domain, which has a lower degree of identity (30-40%) than the other regions. Primers for shcA were 5'-CAGCCACTTGGGAGCTACAT-3' (sense) and 5'-GGGCTTCATGTCAAACAGGT-3' (antisense); for shcB, 5'-AGGGCCCATCTCCTTCTCTA-3' (sense) and 5'-CCACTGAGCACTCATGCAAC-3' (antisense); for shcC, 5'-CTCCTGACACAGCCCAGTTT-3' (sense) and 5'-GAGGCTGCCTTGCTTAACAC-3' (antisense); for trkA, 5'-TGTTCAGGTCAACGTCTCCT-3' (sense) and 5'-GCAGCGTGTAGTTGCCGTTG-3' (antisense); and for full-length trkB, 5'-GGACACCACGAACAGAAGTA-3' (sense) and 5'-CTGGGGATTTTCAATGACAG-3' (antisense). The predicted sizes of the PCR products using these primers were 229 bp for shcA, 243 bp for shcB, 325 bp for shcC, 254 bp for trkA, and 314 bp for full-length trkB. PCR was carried out in a final volume of 25 µL containing aliquots of cDNA corresponding to 50 ng of RNA along with 1 unit of AmplyTaq Gold Polymerase (Applied Biosystems, Foster City, CA), 200 µmol/L deoxynucleotide triphosphates, and 0.2 µmol/L of each primer in 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, and 1.5 mmol/L MgCl2. All PCR amplifications were done using a GeneAmp PCR system 2400 (Perkin-Elmer, Wellesley, MA). The samples were denatured at 95°C for 5 minutes, followed by 33 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds. The final cycle was followed by a 7-minute extension at 72°C. The housekeeping gene ß2-microglobulin was coamplified as an internal standard. The primers for ß2-microglobulin were 5'-ACCCCCCACTGAAAAAGATGA-3' (sense) and 5'-ATCTTCAAACCTCCATGATG-3' (antisense), producing a PCR product of 120 bp. According to previous reports by Matsunaga et al. (22), these reaction conditions result in linear amplification of the PCR products of both target genes and controls.

Analysis and quantification of amplified products. PCR products were separated by electrophoresis on 2.5% agarose gels containing SYBER Gold (Molecular Probes, Eugene, OR). The gels were visualized by UV transillumination and recorded as digital images using a Kodak Digital Science DC40 camera. The intensity of each band was measured using the 1D Image Analysis Application (Eastman Kodak, Rochester, NY) and was normalized according to the ß2-microglobulin signal.

Statistical analysis. The Mann-Whitney U test was used to evaluate the significance of the expression of each analyzed gene in the tumor samples. The probability of cumulative survival was calculated by the product limit method of Kaplan and Meier. Cox regression models were used to evaluate the prognostic significance of each clinical and biological factor. Statistical analysis was done using StatView version 4.5 (Abacus Concepts, Piscataway, NJ).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Shc family expression in human neuroblastoma cell lines. Figure 1 shows the expression of shcA, shcB, and shcC in 15 human neuroblastoma cell lines as analyzed by semiquantitative reverse transcription-PCR (RT-PCR). All cell lines expressed high levels of shcA mRNA, whereas very little expression of shcB was detected. All 13 cell lines also expressed relatively high levels of shcC. There was no association between the expression of these genes and MYCN status in these 15 cell lines. The expression of trkA was clearly observed in almost all cell lines except for NMB, NLF, and SMS-KAN, which showed very low expression. The expression of full-length trkB was detected only in SMS-KCN, CHP-134, and RT-BM-1 cells (data not shown).



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  		Fig. 1. Expression of shc family in human neuroblastoma cell lines. Expression of mRNA was assessed by semiquantitative RT-PCR. Lanes 1 to 3, neuroblastoma cell lines with single-copy MYCN; lanes 4 to 15, neuroblastoma cell lines with MYCN amplification. Expression of the housekeeping gene ß2-microglobulin was used as the internal control.

 
Expression of shcB in RT-BM-1 cells increases after all-trans-retinoic acid–induced differentiation. RT-BM-1 cells were cultured for 2 days and then treated with RA for 1 week. Short neurites started to grow 3 days after RA treatment and continued to extend in length thereafter. After the week of RA treatment, the cells gathered into clusters with a complex network of interconnected neurites. The cells were harvested on days 0, 1, 3, 5, and 7, and total RNA was extracted. The expression levels of shcA, shcB, and shcC mRNA were assessed by semiquantitative RT-PCR. We found that the level of shcA expression increased slightly, whereas that of shcC decreased slightly following the week of RA treatment (data not shown). The expression of shcB mRNA gradually increased to ~5-fold after 1 week of RA treatment (Fig. 2).



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  		Fig. 2. Expression of shcB in RA-induced RT-BM-1 cells. RT-BM-1 cells were cultured for 2 days and then treated with 5 µmol/L RA. Cells were harvested after 0, 1, 3, 5, and 7 days of RA treatment, and total RNA was extracted. The expression of shcB was determined by semiquantitative RT-PCR. The results shown are the averages of two experiments.

 
Expression of shc family genes in neuroblastoma tumor samples. We analyzed the expression of shc family genes in 52 tumor samples by semiquantitative RT-PCR. A representative example of the RT-PCR is shown in Fig. 3, and quantitation of each shc gene mRNA is shown in Fig. 4. The expression of each of shc family member along with trkA and trkB expression is listed in Table 1. The Mann-Whitney U test was done to evaluate the association between clinical stages (stages 1, 2, and 4S versus 3 and 4) and the expression of the three shc genes. We found that shcA was expressed in all neuroblastoma samples, with higher expression at earlier stages (1, 2, and 4S; P = 0.0002; Fig. 4A). A relatively low expression of shcB was detected mainly in earlier stages (P = 0.0095; Fig. 4B). Ten of fourteen stage 1 patients expressed a high level of shcB, whereas three stage 4 patients showed a high expression of shcB. The two surviving patients had an amplification of MYCN. We could not detect shcC in earlier stage samples, although six stage 4 tumors expressed substantially higher levels of shcC than the rest of the tumor samples (Fig. 4C). Only one patient under 1 year of age expressed a high level of shcC, and this patient died of the disease. Finally, there was a very significant association (P = 0.0001) between a high level of shcC expression and an advanced tumor stage (stages 3 and 4).



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  		Fig. 3. Expression of shcA, shcB, and shcC in primary neuroblastoma samples. Expression of mRNAs was assessed by semiquantitative RT-PCR. Representative results are shown. Lanes 1 to 5, stage 1; lanes 6 and 7, stage 2; lanes 8 to 10, stage 3; lanes 11 to 20, stage 4.

 


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  		Fig. 4. Expression of shc family in neuroblastoma samples. The expression of shcA, shcB, and shcC was determined by semiquantitative RT-PCR. Levels were normalized to the level of ß2-microglobulin mRNA. Y axis, PCR ratio; X axis, tumor stages. The Mann-Whitney U test was done to evaluate the association between clinical stages (stages 1, 2, and 4S versus 3 and 4) and the expression of the three genes. The mean expression values of each PCR ratio (shcA, 0.946; shcB, 0.111) are shown by dotted horizontal lines. {circ}, less than 1 year; {bigtriangleup}, older than 1 year; {bullet} or {blacktriangleup}, died of the disease. A, shcA expression; B, shcB expression; C, shcC expression.

 
Correlation between shc expression and survival rate. We compared the event-free survival rate with the expression of each of the shc transcripts using the Kaplan-Meier method. The differential expressions of shcA and shcB were divided into two groups (high versus low) according to their mean expression ratio from PCR (shcA, 0.946; shcB, 0.111). The differential expression of shcC was divided into three groups: one group with high expression (PCR ratio > 0.8), a second group with low expression (PCR ratio = 0.008-0.12), and the third group with no expression. The P value for each survival curve was determined by the log-rank test (Table 2).


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  		Table 2. Univariate and multivariate analysis of 52 neuroblastoma patients affecting event-free survival

 
We found that there was no statistically significant difference (P = 0.1023) in the survival rates between high (80.3%) and low (60.7%) expressions of shcA (data not shown). In contrast, we found that the expression of shcB was high in 15 patients (P = 0.0295) and that their survival rate was 93.3% (Fig. 5A).



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  		Fig. 5. Correlation between event-free survival rate and expression of shc genes. Correlations between the expression of each shc genes and the event-free survival rate were analyzed by the Kaplan-Meier method. A, the differential expression of shcB was divided into two groups (high versus low) according to the mean expression ratio from PCR (0.111). High (n = 15), 93.3%; low (n = 37), 60.3%. B, the differential expression of shcC was divided into three groups: one group with high expression (PCR ratio > 0.8), a second with low expression (PCR ratio = 0.008-0.12), and the third group with no expression. High (n = 6), 0%; low (n = 6), 33.3%; no expression (n = 40), 87.5%.

 
Comparison of the survival rate with shcC expression is shown in Fig. 5B. We detected shcC expression in 12 tumors. Six of these cases had a high level of shcC expression, whereas the other six had a relatively low level. The remaining 40 patients showed no expression of shcC. Of the 52 patients with high shcC expression, six died within 31 months. The survival rate for patients with low shcC expression was 33.3%, whereas it was 87.5% for patients with no expression. These correlations between shcC expression and survival rate were highly significant (P < 0.0001).

Among 52 tumor samples, 15 had amplified MYCN. The mean expression levels of shcA and shcB were higher in patients with a single copy of MYCN (P = 0.0133 and P = 0.3978, respectively; data not shown). As shown in Fig. 6, five of six patients with high shcC expression had an amplification of MYCN. There was a highly significant correlation between MYCN amplification and a higher mean expression level of shcC.



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  		Fig. 6. Association between shcC expression and MYCN status. The Mann-Whitney U test was done to evaluate the association between the expression of shcC and MYCN status. Y axis, PCR ratio of shcC determined as described in Fig. 4; X axis, MYCN status. The association between high expression of shcC and MYCN amplification was statistically significant (P < 0.0001).

 
Expression of shcC is an independent prognostic factor for neuroblastoma. Univariate and multivariate analyses were applied to evaluate the significance of each prognostic factor (Table 2). We found that the age at diagnosis, MYCN status, and trkA expression, widely recognized as prognostic factors, were closely associated with event-free survival. Matsunaga et al. (23, 24) reported that c-srcN2, an isoform of neuronal src mRNA normally expressed in human brain, was expressed in terminally differentiated neuroblastoma cells and correlated with a favorable prognosis. Univariate analyses indicated that not only c-srcN2 but also shcB and shcC expressions were associated with event-free survival. Finally, comparison of shcC expression with other biological markers, including MYCN status, trkA expression, and c-srcN2 expression, using a Cox regression model, showed that shcC was the only strong and independent factor associated with survival (P = 0.0258). Multivariate analysis including age was also done, but shcC was still the strongest prognostic factor (data not shown).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have shown that shc family expression is involved in both clinical and biological aspects of neuroblastoma. Various levels of shc family mRNAs were expressed in all cell lines and tumor samples, suggesting that they have a diverse influence on neuronal differentiation and prognosis of neuroblastoma.

The expression of shcA mRNA expression was clearly detected in all cell lines and tumor samples. The fact that ShcA has been found in many types of human primary tumors and cell lines suggests that it contributes to maintaining the transformed phenotype in neuroblastoma cells (16). In our clinical study, although a high expression level of shcA corresponded to a slightly better prognosis, shcA expression failed to be a prognostic factor. Although TrkA is a good prognostic factor for neuroblastoma and ShcA/TrkA signaling may be responsible for survival and differentiation of PC12 cells (25, 26), ShcA is also involved in the BDNF/TrkB pathway, which is a poor prognostic factor for neuroblastoma (27). In addition, different isoforms of ShcA (generated by alternative splicing of the transcript) are reported to have opposite effects on the epidermal growth factor receptor/mitogen-activated protein kinase/Fos signaling pathway (12). This, together with the ubiquitous expression of ShcA and its involvement in a variety of receptor activities, suggests that its role in neuroblastoma cells is complex and influenced by other factors, such as Trks or the other two Shc family members. Clearly, further studies are needed to elucidate the true function of ShcA in neuroblastoma.

Examination of shcB mRNA expression revealed only very low levels in the 15 neuroblastoma cell lines. Similarly, in another study of 10 neuroblastoma cell lines, only low levels of ShcB protein were detected (17). In addition, mouse knockout experiments suggest that ShcB plays a survival role in postmitotic superior cervical ganglia neurons. Most importantly, ShcB is expressed from mitosis to postmitosis in developing neuronal cells, supporting a role in neuronal cell survival and differentiation (15). Based on these facts, we speculate that ShcB may also play a role in neuroblastoma survival and differentiation. Consistent with this idea, we found that shcB expression was enhanced following RA-induced neuronal differentiation of RT-BM-1 cells.

Clinical studies of shcB expression also support our in vitro data. The expression of shcB is higher in neuroblastoma tumor samples compared with neuroblastoma cell lines. Furthermore, the mean expression level of shcB was higher in low-stage than in advance-stage tumors, and patients with high shcB expression had a better prognosis. In addition, two of three patients alive with MYCN amplification had enhanced shcB expression. The expression of shcB might not reflect the pathologic phenotype because almost all tumors showed a poorly differentiated neuroblastoma subtype. However, this might imply that shcB has a role in later spontaneous regression and/or differentiation because it has a significant association with a good outcome despite the fact that all of the samples had the same degree of pathologic phenotype. Previous reports by Matsunaga et al. (23) showed that neuron-specific src mRNA expression is associated with neuronal differentiation in RA-induced RT-BM-1 cells and neuroblastoma samples. Others reported that ShcB is less associated with Trk receptors but is instead associated with a protein that is specifically phosphorylated by Src tyrosine kinase (14). Neuronal Src might therefore be a direct regulator of ShcB.

Of the three shc genes, shcC mRNA expression showed the most striking correlation with neuroblastoma progression. We found that shcC was highly expressed in 13 of 15 cell lines, whereas it was undetectable in low-stage tumor samples. Among 21 stage 4 patients, 12 expressed a high level of shcC, and six died within 31 months. Of these six patients, five had an amplification of MYCN, which is known to be an indicator of poor prognosis in neuroblastoma (28). Moreover, we showed that shcC expression is associated with MYCN amplification. Given these results, it is clear that shcC expression is associated with a poor prognosis in neuroblastoma. Indeed, our multivariate analysis showed that shcC is an independent prognostic factor for neuroblastoma. Why shcC expression is associated with a malignant feature remains to be determined. The only previous report on ShcC protein expression in neuroblastoma cells showed that anaplastic lymphoma kinase (ALK) is associated with hyperphosphorylated ShcC and that both the ALK and MYCN genes are amplified in two neuroblastoma cell lines (17). The authors concluded that ALK-ShcC activation is possibly caused by a coamplification with the MYCN gene (17). Our results strongly support their hypothesis, but more clinical data are needed to establish this conclusively.

ShcC binds preferably to TrkB receptors (29, 30), and, in transformed PC12 cells, the BDNF-stimulated interaction between TrkB and ShcC is stronger than that of the nerve growth factor–stimulated interaction between TrkA and ShcA (31). Furthermore, TrkB is associated with an unfavorable biology in neuroblastoma, and BDNF signaling contributes to chemoresistance in neuroblastoma cells (5–8, 32, 33). For this reason, we also examined TrkB expression in stage 4 patients. We found that the patients expressing both high shcC and trkB all died of the disease within 9 months and responded poorly to chemotherapy. These results suggest that shcC overexpression enhances TrkB function in neuroblastoma, worsening the outcome and promoting chemoresistance. ShcC might play an aggressive role in neuroblastoma by accelerating some pathway to chemoresistance through BDNF-TrkB signaling.

In conclusion, in this study, we showed that ShcB expression is a favorable factor in neuroblastoma, whereas ShcC is a strong and independent negative prognostic factor associated with an unfavorable outcome. Although further investigations are needed, our findings suggest that the Shc family, especially ShcB and ShcC, are important biological factors in neuroblastoma and useful for predicting the phenotype of clinical neuroblastoma.


   Acknowledgments
 
The authors thank Dr. Hiroshi Horie for pathologic review of all samples.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/19/04; revised 1/25/05; accepted 2/ 9/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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