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Tumor Biology |
Departments of Neurology [A. L., A. I.] and Developmental and Molecular Biology [A. I.], Albert Einstein College of Medicine, Bronx, New York 10461; Departments of Pathology [R. B.], Pediatric Oncology [A. D., C. D.], and Surgery [A. I.], Bambino Gesu’ Children Hospital, Rome 00165, Italy; Department of Pediatrics, La Sapienza University, Rome 00161, Italy [C. D.]; and Department of Biochemistry, Fukui Medical University, Fukui 910-1193, Japan [Y. Y.]
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ABSTRACT |
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INTRODUCTION |
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Previous studies proposed that the different biological properties of Rb and Rb family members result from their ability to regulate E2F transcription factors (6, 7, 8) . Our recent results from genetic intercrosses of Rb and Id2 knockout mice indicated that negative control of Id2 activity is an additional requirement for Rb function in vivo (5) . Here we analyzed the expression of N-Myc and Id2 during normal development and in primary neuroblastoma. Because of the crucial importance ascribed to loss of Rb function in human cancer, we investigated whether overexpression of Id2 induced cellular transformation and predicted clinical outcome in children with neuroblastoma. Having found that Id2 expression is a prognostic determinant in neuroblastoma, we asked whether inhibition of Id2 could serve as a point of intervention in cancer by studying the consequences of eliminating Id2 from primary and tumor cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 test. Survival analyses were performed according to the method of Kaplan and Meier, and comparisons of outcome between subgroups were performed by the log-rank test for univariate comparisons. For multivariate analysis, Cox’s proportional hazard regression model was applied. Statistical analyses were performed using StatView 4.1 (Abacus Concepts, Inc., Berkeley, CA).
Immunohistochemistry.
Antibodies used for immunohistochemistry included a previously characterized anti-N-Myc monoclonal antibody at concentration of 10 µg/ml (Ref. 9
; Oncogene Research Products, Boston, MA) and the anti-Id2 polyclonal antibody C-20 at concentration of 2 µg/ml (Santa Cruz Biotechnology, Santa Cruz, CA). Specificity of Id2 immunostaining was assessed by preabsorption of the antibody with the peptide from which the antibody was raised. This, but not preabsorption with a nonrelevant peptide, abolished Id2 staining. Controls using polyclonal rabbit immunoglobulin instead of primary antibody showed no evidence of staining. Also, tissues from Id2 null mice provided a negative control for Id2 antibody (Fig.1c)
. Immunohistochemical results of N-Myc and Id2 staining in neuroblastoma were scored independently by two investigators (A. L. and A. I.). At least 600 cells from 5 to 10 high-power fields were scored for each tumor sample.
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Growth Experiments and Antisense Oligonucleotide Transfections.
Primary MEFs were obtained from 13.5-day-old embryos and cultured as described (5)
. Passage 4 cells were plated at density of 3 x 104/35-mm dish and counted at the indicated days. Unless specified otherwise, cells were cultured in DMEM supplemented with 10% fetal bovine serum (Sigma Chemical Co.). Retroviral infection of NIH 3T3 was done as described (5)
. For saturation density experiments, cells were plated in DMEM containing 5% fetal bovine serum and cultivated for up to 7 days. Cells were counted daily with a hemocytometer. For growth in soft agar of NIH 3T3 derivatives and LAN1, 5 x 104 and 1 x 105 cells, respectively, were plated in 35-mm dishes in 0.33% agar solution (BiTek; Difco) in DMEM containing 5% fetal bovine serum. The bottom layer was prepared using 0.6% agar in growth medium. After 7 days, 1.5 ml of agar mixture were added. Colonies were scored in triplicate wells after incubation for 14 days. Phosphorothioate oligonucleotides complementary to human Id2 and the mismatched control were obtained from Life Technologies, Inc. The sequences of oligonucleotides were as follows: Id2-AS, 5'-AGGCTTTCATGCTGACCGC-3'; and Id2-MSM, 5'-GCGAGTTGTCGCACGGTCT-3'. Oligonucleotides were mixed with Superfect (Qiagen) according to the manufacturer’s instructions and used to treat LAN1 cells at the final concentration of 0.8 µM. After incubation for 24 h, cells were analyzed for the ability to incorporate BrdUrd and form colonies in soft agar.
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RESULTS |
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. Lung is an example of abundant expression of N-Myc and Id2 proteins in the epithelium without any detectable signal in the adjacent mesenchyme (Fig. 1, a–c)
. A similar pattern of expression is present in the small intestine (data not shown). During early neurogenesis (before day E12), N-Myc and Id2 are expressed in the telencephalon throughout the proliferating ventricular zone (12, 13, 14)
. However, at E15, the two proteins appear in the intermediate zone, which predominantly contains postmitotic neurons (Fig. 1, d and e)
. Positive staining in the ventricular and subventricular layers is detectable only in small areas of the olfactory lobe and the hippocampus (data not shown). N-Myc and Id2 are also abundant in large, differentiated neurons of dorsal root and trigeminal ganglia, which fail to incorporate BrdUrd at this stage of development (Fig. 1, f and g
, and data not shown). To determine at which time active Rb appears in the brain and how it relates with N-Myc and Id2, we compared expression of N-Myc, Id2, and Rb in embryonic and postnatal brains by Western immunoblot analysis (Fig. 1h)
. Although expression of N-Myc and Id2 decreases with development, significant amounts of the two proteins are found at E15, and they are still detectable after 9 days of postnatal life. Rb undergoes a change from hyperphosphorylated (abundant at E10) to hypophosphorylated, active forms by E15. Thus, expression of N-Myc and Id2 is detectable at midgestation in predominantly postmitotic neuroectodermal tissues and is paralleled by a shift of Rb from inactive to active forms.
Id2 Expression Is Predictive of Poor Survival of Neuroblastoma Patients.
To determine whether the "N-Myc-Id2 pathway" is activated in neuroblastoma cells before tissue culture and whether Id2 expression correlates with clinical behavior, we conducted an immunohistochemical analysis for the proteins N-Myc and Id2 in 47 primary neuroblastomas. N-Myc and Id2 were not expressed in postnatal adrenal medulla, the most common site of origin of neuroblastoma in humans (data not shown). N-Myc and Id2 were either absent ("negative" samples; Fig. 2, a and b
) or detectable in the nuclei of 25% or more tumor cells ("positive" samples; Fig. 2, c–f
). In our series there were 8 tumors with N-myc gene amplification (Table 1)
. All of them showed positive staining for N-Myc and Id2. Among the 37 tumors without N-myc gene amplification, 19 had N-Myc protein expression. These findings confirm results from previous studies, which indicated that mechanisms other than gene amplification lead to deregulated expression of N-Myc in a significant number of neuroblastomas (9
, 15
, 16)
. With the exception of one case (N-Myc negative/Id2 positive), we found an invariable correlation between the expression of N-Myc and Id2 (28 tumors were N-Myc positive/Id2 positive; 18 tumors were N-Myc negative/Id2 negative). We scored the percentage of neuroblastoma cells in each tumor that stained positive for N-Myc and Id2 (LI) and found that the LIs for N-Myc and Id2 were highly correlated (Pearson correlation coefficient, 0.868; P < 0.0001; Fig. 2g
). Next, we examined the correlation between Id2 expression and clinical behavior of neuroblastoma. On all patients, the cumulative overall and event-free survival of Id2-negative tumors were 0.877 ± 0.082 and 0.889 ± 0.074 versus 0.417 ± 0.103 and 0.472 ± 0.094 for Id2-positive tumors (Fig. 3, a and b)
. The Kaplan-Meier analysis showed that Id2 expression was predictive of increased mortality (Fig. 3, a and b
; log-rank P = 0.0046 and 0.0065 for overall and event-free survival, respectively). Outcome of neuroblastoma patients of ages <1 year is good, regardless of other prognostic variables (17)
. Conversely, patients with N-myc gene amplification display an invariably poor prognosis (18)
. We therefore asked whether Id2 remained a predictive indicator for patients older than 1 year of age and/or lacking N-myc gene amplification. Kaplan-Meier plots of event-free survival showed that Id2 expression was associated with increased mortality in each subgroup (Fig. 3, c–e)
. 2 analysis demonstrated a positive correlation between Id2 expression and unfavorable clinical stages (Table 1
; P = 0.0051). When outcome was evaluated in this subgroup of patients, there was a trend toward a correlation between Id2 expression and low event-free survival (P = 0.12; Fig. 3f
). In a multivariate Cox proportional hazard model that included Id2 expression (positive), age (<1 year), and N-myc copy number (>3 copies), Id2 expression was the strongest independent predictor of disease-free survival (P = 0.0264; relative hazard, 10.996; Table 2
).
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. NIH 3T3-Id2 cells overcame the serum requirement of vector-infected NIH 3T3 for entry into S-phase and long-term proliferation (Fig. 4b
and data not shown). In the presence of high levels of Id2, cells overgrew a monolayer doubling the saturation density of control cells (Fig. 4c)
. As a further sign of transformation, when cultured in soft agar, NIH 3T3-Id2 formed distinct foci (29 ± 5 colonies/well), which were absent in cultures of vector-infected NIH 3T3 (Fig. 4d)
. However, NIH 3T3-Id2 were not tumorigenic when injected in nude mice (data not shown).
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. The defective ability to grow persisted when we compared 3T3 derivatives from Id2-/- MEFs with their wild-type counterparts, indicating that immortalization was not sufficient to overcome the requirement for Id2 (data not shown). We next sought to determine whether opposing the uncontrolled expression of Id2 in neuroblastoma cells could affect their malignant behavior. For these experiments, we used the neuroblastoma cell line LAN1 that carries N-myc amplification and Id2 overexpression (5)
. Treatment of LAN1 with a phosphothioate Id2 antisense oligonucleotide for 24 h led to a decrease of endogenous Id2 by 
60% compared with a mismatch oligonucleotide (Fig. 4f)
. Consistent with reduction of Id2, BrdUrd incorporation showed decreased S-phase entry (15% versus 28% in mismatched treated cells; Fig. 4g
). Interestingly, reduction of Id2 protein levels in LAN1 led to an even stronger effect on the ability of these cells to form colonies in soft agar (from 41 ± 3 colonies/well for mismatched treated LAN1 to 12 ± 2 colonies/well for anti-Id2-treated cells; Fig. 4h
). These results suggest that lowering Id2 in neuroblastoma may generate antitumor mechanisms involving more than antiproliferative effects. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To our knowledge, Id2 is the first transcriptional target of N-Myc, the expression of which correlates with the N-Myc protein during development and in neuroblastoma. The immunohistochemical analysis of Id2 in neuroblastoma shows that tumor aggressiveness depends, at least in part, on activation of the N-Myc-Id2 pathway. In our series, among the 17 patients who relapsed and/or died, 15 (88%) overexpressed Id2 and 2 (12%) did not. Therefore, the vast majority of prognostically unfavorable neuroblastoma deregulates Id2 expression. Although N-myc gene amplification is a well-established adverse prognostic indicator in neuroblastoma, the significance of N-Myc protein expression is still debated (9
, 15
, 16
, 21) . In our series, expression of Id2 correlates with N-Myc (Fig. 2g)
. However, the unique feature of assaying Id2 in neuroblastoma is that its expression integrates the effects of N-myc activation and possibly other upstream signals to overcome the crucial tumor suppressor function of the Rb pathway. A number of studies suggested that inactivation of the Rb pathway is a determinant of poor prognosis for cancer patients (22, 23, 24)
. We propose that neuroblastoma with favorable outcome may retain a functional Rb pathway, whereas Id2-independent mechanisms for Rb inactivation may be present in the small subgroup of unfavorable, Id2-negative neuroblastoma. The analysis of Id2 expression has considerable potential to be of practical use in the routine assessment of neuroblastoma patients. This is strengthened by the fact that immunohistochemical tests for Id2 expression are inexpensive and could easily be available to most medical centers.
Our studies on the role of Id2 in cellular proliferation show that loss of Id2 significantly reduces the rate of cellular proliferation of primary and immortalized embryonic fibroblasts. These data are consistent with previous reports showing impaired proliferation rate of other cell types from Id2-/- mice (25 , 26) . It is likely that a critical threshold of Id2 determines the rate of proliferation in primary cells. Specular of these findings are our results showing that overexpression of Id2 at levels comparable with those found in neuroblastoma cells renders cells insensitive to extracellular antimitogenic signals such as serum deprivation and contact inhibition, thus conferring oncogenic potential. The role of Id2 as an oncogenic factor in neuroblastoma is specifically supported by reduced entry into S-phase and severely compromised anchorage-independent growth of neuroblastoma cells, where Id2 expression has been forcibly reduced by antisense oligonucleotides.
In human tumors, Rb is functionally inactivated by genetic alterations of the "Rb pathway" or by constitutive activation of the "N-Myc-Id2 pathway." We have now shown that Id2 expression determines the rate of proliferation of primary, immortalized, and tumor cell lines. These observations suggest that Id2 will make a major contribution to the inappropriate cell proliferation that results from loss of the negative control of Rb upon Id2 in tumors with genetic alterations of the "Rb pathway." Anti-Id2 therapeutic approaches might be attractive new tools, even in tumors where inactivation of Rb results from mechanisms different from the activation of the "Myc-Id2 pathway."
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Grant RO1-CA85628 from the NIH (to A. I.). A. I. is recipient of a Sinsheimer Scholar award. 
2 To whom requests for reprints should be addressed, at Department of Neurology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: (718) 430-3347; Fax: (718) 430-8785; E-mail: iavarone{at}aecom.yu.edu
3 The abbreviations used are: Rb, retinoblastoma; MEF, mouse embryo fibroblast; BrdUrd, 5-bromodeoxyuridine; LI, labeling index; GFP, green fluorescent protein.
Received 8/14/01. Accepted 11/15/01.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin in the mouse brain during development. pRb, hypophosphorylated Rb; ppRb, hyperphosphorylated Rb. ppRb:pRb ratios from a quantitative densitometric analysis of the Rb blot are: E10, 7.0; E15, 0.31; E16, 0.26; P0, 0.10; and P9, 0.04.
) and NIH 3T3-Id2 (
). Cell number at each time point represents the average of duplicate samples; bars, SD. d, anchorage-independent growth of NIH 3T3-vector and NIH 3T3-Id2. Cells were assayed for their ability to grow in soft agar and photographed after 14 days in culture. e, passage 4 primary MEFs from two independent litters containing wild-type (
) and Id2-/- littermates (