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Department of Molecular Pathology, Istituto di Ricovero e Cura a Carattere Scientifico Neuromed, Pozzilli, 86077 Italy [A. C., A. A., G. D. G., A. P., V. L., F. M. G., R. C., L. F., G. R.]; Departments of Experimental Medicine and Pathology [A. A., A. P., V. L., M. Z., G. G., A. G., L. F., G. R.] and Neurological Sciences [F. M. G., R. C.], University of Rome "La Sapienza," 00161 Rome, Italy; Sidney Kimmel Cancer Center, San Diego, California 92121 [D. M., C. L.]; and The Cancer Center, University of California at San Diego, La Jolla, California 92093 [D. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: Thirty-one human gliomas with different grades of malignancy were investigated for Egr-1 mRNA and the protein expression, frequency, and spectrum of p53 gene mutations, mdm2 gene amplification, and p16/INK4a/ARF allele loss.
Results: The amplification of Mdm2 and the deletion of the p16/INK4a gene was found in 3 and 5 cases, respectively, whereas mutations of p53, including two novel mutations, were observed in 10 other cases. The three types of changes occurred strictly mutually exclusively, emphasizing that these genes operate in a common pathway critical to glioma progression. EGR-1 mRNA was significantly down-regulated in astrocytomas (14.7 ± 5.1%) and in glioblastomas (33.6 ± 10.0%) versus normal brain. Overall, EGR-1 mRNA was strongly suppressed (average, 15.2 ± 13.9%) in 27 of 31 cases (87%), independent of changes in p16/INK4a/ARF and Mdm2; whereas 4 of 31 cases with residual EGR-1 expression as well as the highest EGR-1 variance segregated with p53 mutations. Immunohistochemical analyses confirmed the suppression of EGR-1 protein.
Conclusions: These results indicate that EGR-1 is commonly suppressed in gliomas independent of p16/INK4a/ARF and Mdm2 and that suppression is less crucial in tumors bearing p53 mutations, and these results implicate an EGR-1 growth regulatory mechanism as a target of inactivation during tumor progression.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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EGR-1 (also known as NGFI-A, TIS8, Krox-24, and Zif268) is a member of the immediate early genes family and encodes a nuclear phosphoprotein involved in the regulation of cell growth and differentiation in response to signals such as mitogens, growth factors, and stress stimuli (4, 5, 6, 7)
. However, in other circumstances, EGR-1 is induced very early in the apoptotic process (8)
, where it mediates the activation of downstream regulatory genes such as p53 and tumor necrosis factor 
(9
, 10) .
In contrast with these functions, previous studies have shown that EGR-1 is down-regulated in several types of neoplasia, suggesting a role as a tumor suppressor gene in analogy with WT-1, another family member which binds to a similar DNA motif (11 , 12) . For example, EGR-1 has been found to be decreased or undetectable in human breast and small cell lung tumors (13 , 14) as well as in an array of tumor cell lines (11 , 15 , 16) . Gene deletion or mutations have also been reported in sporadic cancer cases (17) . Moreover, reexpression of EGR-1 suppresses the growth of transformed cells both in soft agar and in athymic mice (16) . This is at least partially related to the induction of TGF-ß14 expression—a factor with an important role in the progression of gliomas (18 , 19) . Similarly, studies with antisense vectors indicated that the transformed phenotype is enhanced by the inhibition of EGR-1 expression (20) . These studies suggest a consistent growth suppression role for EGR-1 in cells of neuroectodermal origin. Here, we directly test this hypothesis by examination of the expression of EGR-1 in fresh surgical specimens of glioblastomas and normal tissue.
EGR-1 has been implicated in the regulation of p53, thereby providing an additional explanation for a suppressor role (9
, 10 , 21
, 22)
. The function of p53, in turn, is determined in part by the Mdm2 protein and by an alternate reading frame (ARF or p14ARF) product of the p16/INK4a locus. ARF binds to Mdm2, allowing active p53 to accumulate in the nucleoplasm (23)
. Indeed, homozygous deletion of the murine ARF gene in mice leads to a similar phenotype as for inactivation of p53, further indicating that ARF and p53 function in the same biochemical pathway (reviewed in Ref. 24
). Thus, deletion of ARF or overexpression of Mdm2 may be functionally redundant means of disrupting the ARF-Mdm2-p53 pathway. In 
75% of B-cell lymphomas that develop in myc-transgenic mice, there occurs either a mutation in p53, deletion of ARF, or overexpression of Mdm2, but not more than one of these alterations (25)
. Similarly, in humans, p53 is itself mutated in >50% of cancers, whereas deletion of ARF and amplification or overexpression of Mdm2 occur in a high fraction of the remaining cases (24)
. This mutational pattern is commonly manifested in human glioblastomas as well (26, 27, 28)
. Therefore, to examine the relationship between EGR-1 and p53, we also examined the genetic status of p16INK4a, Mdm2, and p53 in the primary glioblastomas.
We find that EGR-1 expression was strongly reduced in fresh, surgically derived brain tumors compared with normal brain tissue, where the basal expression is high (reviewed in Ref. 29 ). EGR-1 was suppressed in >87% of the cases independent of whether alterations in p16/INK4a and Mdm2 were present but significantly less suppressed and more variable in tumors bearing p53 mutations. These results indicate that loss of EGR-1 expression may provide an important marker of glial cell malignancy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Regulatory Affairs.
The Institutional Ethics Board approved the experimental design and scope of this study. All subjects provided a written informed consent for this study. Only tissue excess to diagnostic and patient management needs was made available for research.
Isolation and Northern Analysis of Total RNA.
Total RNA was extracted from tumor pieces (50 mg) using the Ultraspec RNA isolation system (Biotecx Laboratories, Houston, TX). For Northern analysis, 20 µg of denaturated RNA was used for each sample and processed as described previously (15)
. Levels of EGR-1 expression in control and cancer patients were expressed as means ± SE and compared using the t test, ANOVA, and nonparametric Mann-Whitney U test. All tests were two-sided, with a critical level for significance defined as P < 0.05.
DNA Extraction and Analysis.
For Southern analysis, 20 µg of high-molecular weight DNA was digested with 50 units of TaqI, electrophoretically separated in 0.8% agarose gel, and transferred to nylon membranes, as above. Membranes were hybridized with DNA probes labeled with [32P]dATP by random priming. The GAPDH probe was a 1.3-kb, full-length cDNA clone encoding rat GAPDH (31)
. A 1.6kb BglII fragment from pCMV-EGR-1 plasmid was the probe for EGR-1 (32)
, corresponding to the coding region of the mouse cDNA. The DNA probes for MTS1/p16, a 514-bp fragment corresponding to exon 2 of the MTS1 gene, and for Mdm2, bases 53–653 of the human Mdm2 cDNA sequence, were generated by PCR amplification as described (33
, 34)
. The identity of the generated probes was corroborated by restriction-enzyme analysis. The probe for HTSHR was the 2.5kb EcoRI fragment from the pBabeTSHR plasmid (35)
corresponding to the full-length cDNA for HTSHR. The Mdm2, MTS1, and HTSHR probes identify 8.0- and 5.5-kb, 3.3-kb, and 4.2- and 1.9-kb bands, respectively.
DNA Sequencing of p53 Exons 5–9.
For analysis of p53 mutations, three DNA fragments (containing exons 5 and 6, exon 7, and exons 8 and 9, respectively) were PCR-amplified from genomic DNA by using primers described previously (36)
.
Direct sequencing was performed with a ABI PRISM 377 DNA Sequencer using the DNA Sequencing kit, Big Dye Terminator (PE Applied Biosystems, Inc., Boston, MA) according to the manufacturer’s instructions. The results were analyzed by means of the ABI sequencing analysis software.
Densitometric Analysis and Allele Dosage.
Densitometric scans of Northern and Southern results were elaborated using the NIH Image software.5
For Northern analysis, the EGR-1 signals were normalized to the expression of the GAPDH gene of each sample, probed on the same membranes. Allele dosage was measured and calculated as described (34)
. Hybridization signals were normalized to the values observed at the control locus HTSHR on the same membrane. Mdm2 amplification is defined as at least five copies of gene/cell. Loss of heterozygosity at the p16INK4a locus was defined as a >40% decrease in the densitometry value (37)
.
Immunohistochemistry.
A rabbit polyclonal antibody against EGR-1 (Santa Cruz Biotechnology, Santa Cruz, CA) and a mouse monoclonal antibody against GFAP (Sigma Chemical Co., St. Louis, MO) and p53 (PharMingen, Heidelberg, Germany) were used for staining. Peroxidase-conjugated secondary antibodies were localized with the aid of an ABC Universal Quick kit (Vector Laboratories, Burlingame, CA) following the manufacturer suggested procedure.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. As a reference value for basal EGR-1 steady-state mRNA level, the intermediate level of expression of NBA2 was taken as 100%.
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. Mean values (± SE) are 88.0 (± 9.6) in normal brain tissue, 14.7 (± 5.1) in astrocytomas (P < 0.005 versus normal), and 33.6 (± 10.0) for the 22 glioblastoma cases (P < 0.01 versus normal). Most glioblastomas, 87%, are tightly clustered within an expression level of less than one-fourth of the reference tissue. However, four glioblastoma cases exhibited mRNA levels comparable with or even greater than those of normal tissue, suggesting a bimodal distribution within the glioblastoma multiforme group. Other distinguishing features of this high expression group are noted below. Examples of Northern analysis for the various tumors and normal brain samples are shown in Fig. 2
.
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EGR-1 protein expression and localization in normal human brain tissue and in the gliomas was investigated by immunocytochemistry (Fig. 3)
. Similar to mRNA, EGR-1 was less apparent in tumors compared with normal astrocyte populations. In normal subpial and white matter astrocytes, EGR-1 expression was readily apparent and widely distributed. Within cells, EGR-1 was localized to the cell nucleus. Within nuclei, EGR-1 was either diffuse or limited to nuclear membrane in most cases. Cortical neurons were strongly positive with a nuclear localization similar to that of astrocytes (Fig. 3, c and d)
. GFAP staining was performed to identify the astrocyte population in gray and white matter (Fig. 3, a and b)
. To our knowledge, this is the first observation of EGR-1 protein in normal human brain tissue.
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stained positively for EGR-1 protein. However, the fraction of positive cells for each tumor ranged from around 13% to no more than 30%. Conversely, efforts to detect EGR-1 protein were, as a rule, negative in all sections from those tumors showing deficient mRNA expression, although the presence of a few EGR-1-positive cells, accounting for <2% of the total cell population, could be observed. Inasmuch as EGR-1 is highly expressed in normal brain and because gliomas are highly infiltrating tumors, it might not be infrequent that normal cells are found mixed within the tumor population. For these reasons, we suspect that the amount of RNA and protein found in the tumors investigated here might be overestimated. Moreover, we cannot rule out that stable EGR-1 protein derived from the normal cells that occur in low-grade astrocytomas, such as cases T15 and T36, may cause the relatively high EGR-1 protein measurements observed in these cases. In conclusion, our in situ studies provide an explanation for the variation of EGR-1 protein expression in tumors that exhibit an overall suppression of mRNA and provide support for the general correlation of low levels of EGR-1 mRNA in fresh tumor tissue and a parallel decrease in protein accumulation.
Frequency and Spectrum of p53 Gene Mutations.
Recent reports suggest that EGR-1 can induce the transcription of the p53 gene leading to increased apoptosis of tumor cells and that, conversely, EGR-1 expression may be influenced by the level of p53 (9)
. These observations suggest, as one hypothesis, that suppression of EGR-1 may serve to augment the survival properties of astrocytic tumors by blocking, at least in part, the induction of p53 or by blocking a p53-related function. Therefore, we sought to determine whether p53 gene status correlates with the EGR-1 expression level within the same samples.
The p53 gene was characterized by examining the nucleotide sequences of exons 5–9 for the presence of mutations. Among the 31 patients examined, 10 mutations were found (2 astrocytomas and 8 glioblastomas), as summarized in Table 1
. Briefly, two mutations were located in exon 5, three in exon 6, three in exon 7, and two in exon 8, all mutations resulting in an amino acid substitution. Three mutations were found in the L domain, and two in the LH domain. The L (codons 236–251) and LH (codons 163–195) domains are two loop-based elements that bind to DNA. Another mutation (T33) is located at LSH, a third loop-based p53 domain. As reported in the literature, for most tumors, more than two-thirds of missense mutations are found in L, LH, or LSH (38)
.
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as "I." All mutations observed here were already found in tumors of different histological type,6
except for T26 and T40, which bear mutations reported here for the first time.
Loss of EGR-1Expression Is Frequent in Tumors Carrying a Conserved p53 Pathway.
By comparing the levels of EGR-1 mRNA in the 21 tumors bearing wild-type copies of the p53 gene with those present in samples with the mutated gene (Table 1)
, we noticed that EGR-1 expression is significantly higher in p53-mutated tumors. The average values of EGR-1 mRNA in the two groups were 11.9% (± 2.9) and 62.2% (± 18.1) compared with normal values, respectively (P = 0.001, according to Student’s t test; and P = 0.0002, as evaluated by Mann-Whitney nonparametric U test). Moreover, in the subset of four tumors with mutated p53 responsible for the bimodal expression of EGR-1 mRNA of the glioblastoma multiforme (Fig. 1)
, the average RNA expression is 116%, or very similar to that of normal tissue. Thus, the pattern of results is consistent with marked suppression of EGR-1 mRNA levels in tumors bearing wild-type p53, whereas EGR-1 is significantly less suppressed in the group bearing a mutated p53 gene or not suppressed at all as in a subset of the tumors with a mutated p53 gene.
These results were also confirmed by the evaluation of p53 intracellular protein levels, which are known to accumulate in cells carrying the mutated gene. Indeed, we examined both the expression of EGR-1 (Fig. 3e)
and the accumulation of p53 protein by immunohistochemistry on contiguous tumor sections from tumor biopsies carrying mutations in the p53 gene (Fig. 3f)
. The expression of EGR-1 and p53 was also examined in tumors carrying wild-type copies of the tumor suppressor gene as a control (Fig. 3, g and h)
. Labeled p53 protein was demonstrated in all sections from the tumors with mutated p53 but not in sections from wild-type p53. The frequency of cells found in glioma specimens that were expressing p53 and/or EGR-1 is graphically reported in Fig. 4
. EGR-1 expression significantly overlapped with p53 accumulation in all three examined tumors with mutated p53 (correlation coefficient rPearson = 0.86; P = 0.0067). All of the four tumors that showed <10% EGR-1 positive cells had undetectable levels for EGR-1 mRNA expression. Thus both the mRNA analysis and immunohistochemical analysis indicated a consistent pattern of significantly elevated EGR-1 mRNA and protein expression in tumors lacking wild-type p53.
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. The allele dosage ratios ranged from 25-fold to 50-fold in the case of T27. All three of these tumors expressed a wild-type p53 gene (Table 1)
.
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Analysis of Alterations of p16 Gene.
Deletions of INK4a, also known as p16, are commonly associated with deletion of the alternate reading frame product, ARF, an Mdm2 antagonist (39
, 40)
. We investigated the p16 gene in our panel of glioma biopsies by Southern blot hybridization and determined gene dosage, as done above for Mdm2. Single allele or homozygous deletions were found in 1 (T16) and 4 (T1, T3, T11, and T36) cases, respectively (Fig. 4B)
. All occurred in glioblastomas that expressed a wild-type p53 and had no Mdm2 abnormalities (Table 1)
, indicating a strictly mutually exclusive pattern of change among these three types of gene changes. Moreover, the cases with deleted p16 sequences and, therefore, with normal p53 gene sequences, exhibited little or no EGR-1 mRNA expression, with an average of 3.4%. Furthermore, these data suggest that reduced EGR-1 expression is a new molecular marker of astrocytic tumors without genetic alterations impairing the p53 pathway.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ARF-Mdm2-p53 Pathway Plays a Critical Role in Glioblastomas.
Previous studies of gliomas have shown that inactivation alterations of the ARF-Mdm2-p53 pathway commonly occur in a mutually exclusive pattern with a combined frequency ranging to >80% (25
, 27
, 28 , 33
, 39
, 41)
. Indeed, we find that 13 cases exhibited either p53 mutations or Mdm2 amplification but not both. Five cases exhibited a deletion of p16, all with wild-type p53 and Mdm2 genotypes. Thus, the changes in the ARF-Mdm2-p53 pathway occurred in a strictly mutually exclusive pattern with a combined frequency of 58%. It is important to note that we examined the presence or absence of exon 2 of the p16 gene, which does not exclude the possibility that a truncated ARF protein encoded by an intact exon 1 leading to functional exon 1 or exon 1ß products may be present. However, there seems to be no evidence that deletion of exon 2 with expression of a functional truncated form occurs in vivo. Moreover, in those cases where p16INK4a and ARF deletion have been examined together in vivo, both are almost always deleted together (26, 27, 28
, 39, 40, 41)
. Thus, although it is not possible to be unequivocal about the deletion of all forms of the ARF protein, our observations are entirely consistent with the conclusion that for gliomas, as for other major human neoplasms, disruption of ARF or p53 or overexpression of Mdm2 is a major mechanism of tumor progression.
EGR-1 Levels in Vivo Are High Only When p53 Is Mutant.
The major hypothesis of this study is that EGR-1 is a tumor suppression factor and, therefore, would be inhibited in expression in fresh brain tumors, as inferred from glioblastoma cell line studies (16)
. Two major corollaries are that inhibition of expression of EGR-1 would occur regardless of the state of the ARF-Mdm2-p53 pathway if EGR-1 worked through a p53-independent mechanism (i.e., EGR-1 mRNA absent in most cases), but that inhibition of expression would occur only exclusive of other changes in the ARF-Mdm2-p53 pathway if EGR-1 acted as an a member of the pathway (i.e., EGR-1 mRNA absent only exclusive of other p53 pathway inactivating changes). Surprisingly both corollaries are manifested. EGR-1 mRNA expression is reduced or absent in 87% of all cases, and the extent of reduction, to 15% of normal, was shown to be significant even when the average for all 31 tumor cases is compared with the normal value. This conclusion is supported by direct immunohistochemical analysis of protein expression. Thus significant inhibition of expression regardless of the state of the ARF-Mdm2-p53 pathway is observed. On the other hand, EGR-1 mRNA expression is notably higher for the cluster of cases with p53 mutations (average, 66% versus 19% and 3.4% for the cases with altered Mdm2 and p16 genes, respectively), and the values are much more variable, ranging to 200% of the normal value (Table 1)
. Indeed, the average level of expression for this group was shown to be significantly increased compared with all other cases. Thus, strong inhibition of expression of EGR-1 is exclusive of the group of cases with mutations in p53. Because most mutations observed here are thought to inactivate p53 (compare Table 1
), this observation suggests that the mechanism of action of EGR-1 is not entirely independent of that for inactivation of p53 itself. Thus, during tumor progression, there appears to be reduced pressure to inhibit expression of EGR-1 when p53 has been inactivated. A model that provides an explanation of these effects is considered below.
What Is the Mechanism of Tumor Growth Suppression Operated by EGR-1?
Much of the previous experimental evidence of the oncosuppressive activity of EGR-1 is based on the effects of constitutive reexpression of the gene in several tumor cell lines, including the glioblastoma U251 cell line (11
, 16
, 18)
, and by the suppression of expression of EGR-1 in human tumor cell lines (13
, 42)
. The reexpression of EGR-1 leads to restoration of contact inhibition, morphological reversion, and the reduction in tumorigenicity (16)
. Most of these changes are referable to up-regulation of TGF-ß1 because of direct transactivation of TGF-ß1 by EGR-1. In fact, TGF-ß1 is expressed and secreted in direct proportion to the level of expression of EGR-1 (16)
, and the effects of EGR-1 are reversed by the addition of anti-TGF-ß1 antibodies (18)
. Indeed, TGF-ß1 is recognized as a major growth regulator of naturally occurring gliomas, and inactivation of the TGF-ß1 mechanism, especially by loss or mutation of the TGF-ß1 receptor type II subunit, increases in frequency with the increasing grade of gliomas (19)
. This view is supported also by preliminary studies conducted in vitro by our group on an extended series of freshly explanted gliomas in which we observed a strong correlation between TGF-ß1 secretion and EGR-1 expression.
These observations suggest a model for the suppressor role of EGR-1 and how this factor may be related to the p53 mechanism. Both p53 and the TGF-ß1 signal transduction pathways regulate the same target gene, the cell-cycle regulator p21Cip/1Waf1 (43
, 44)
. Therefore, by directly transactivating the TGF-ß1 promoter (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)
and stimulating the secretion and subsequent activation of TGF-ß1, EGR-1 may regulate p21Cip/1Waf11 via an autocrine loop. Thus, EGR-1 itself may be a target of suppression during tumor progression, as for p53 and/or the TGF-ß1 receptor. However, if p53 is the major regulator of p21Cip/1Waf1 expression, tumor cells that first undergo complete inactivation of p53 may be relatively insensitive to the inhibition of EGR-1 expression. This mechanism provides an explanation for the bimodal distribution of EGR-1 mRNA, i.e., that relatively high mRNA values segregate with p53 mutations (compare Table 1
). In summary, we propose that EGR-1 acts in human glioma as a tumor suppressor factor.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work has been supported by grants from the Ministero della Sanità and Ministero dell’Università e Ricerca Scientifica e Tecnologica (to L. F., G. R., and A. G.). A. A. and V. L. are the recipients of a fellowship from University of Rome "La Sapienza" granted by the Regione Molise. G. D. G. is the recipient of a fellowship from Ministero dell’Università e Ricerca Scientifica e Tecnologica. D. M. and C. L. were supported by NIH Grant CA76173. 
2 To whom requests for reprints should addressed, at IRCCS Istituto Neurologico Mediterraneo Neuromed, Via Atinense 18, 86077 Pozzilli, Italy. Phone: (0865) 915243; Fax: (0865) 927575; E-mail: calogant{at}neuromed.it
3 Present address: Invitrogen Incorporated, Carlsbad, CA, 92008.
4 The abbreviations used are: TGF-ß1, transforming growth factor ß1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HTSHR, human thyrotropin receptor; NBA, astrocytes from newborn rat brain; WM, human white matter; TB, human total brain; GFAP, Glial fibrillary acidic protein; MTS = 1, multiple tumor suppressor = 1.
5 Available on the Internet at: http://rsb.info.nih.gov/nih-image.
6 IARC TP53 Mutation Database, Internet address: www.IARC.FRX.
Received 1/17/01; revised 6/18/01; accepted 6/18/01.
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, cases carrying mutated copies of the p53 gene. For each group of data, the mean ± SE is represented. The mean values of astrocytomas [P < 0.005 (*)] and glioblastomas [P < 0.01 (**)] differ significantly from normal tissue.
