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Molecular Oncology, Markers, Clinical Correlates |
, Neuregulin-1 
, and Betacellulin, in Normal Endometrium and Endometrial Cancer
Receptor Biology Laboratory, Imperial Cancer Research Fund Molecular Oncology Unit [R. S., E. B., W. J. G.], Division of Investigative Sciences [F. M.], Imperial College School of Medicine, Hammersmith Campus, London W12 0NN, and Oncology Department, The Royal Surrey County Hospital, Guildford, Surrey GU2 5XX [H. T.], United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, neuregulin 
, and betacellulin, were expressed at significantly higher levels in the secretory as compared with the proliferative phase of the menstrual cycle, suggesting a role for these proteins in endometrial maturation. In endometrial adenocarcinoma, overexpression of c-erbB-3, c-erbB-4, and betacellulin with underexpression of neuregulin as compared with normal controls was observed. Neuregulin expression was not found to be significantly different in the two groups. These results suggest that signaling through the c-erbB-3 and c-erbB-4 receptors and the ligands neuregulin , neuregulin , and betacellulin are important in endometrial carcinogenesis. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The NRGs are encoded by three distinct genes called NRG1 (2)
, NRG2 (3, 4, 5)
, and NRG3 (6)
. The NRG1 gene has been studied in most detail and has been shown to encode a complex family of proteins that includes more than a dozen isoforms derived from alternative splicing (7)
. Each NRG variant, however, contains an EGF-like motif responsible for receptor binding, although the COOH-terminal third may either be derived from the or exon, which appears to determine the nature of the second receptor recruited into the dimer (8
, 9)
. c-erbB-3 and c-erbB-4 act, respectively, as the low and high affinity receptors of these ligands (10)
, and EGFR and c-erbB-2 function act as secondary receptors, the recruitment into heterodimers of which is hierarchically controlled (11)
.
Betacellulin, initially identified as a growth factor from an insulinoma cell line, is an 80-amino acid protein derived from a 177-amino acid precursor that contains an EGF-like element (12) . It has been shown to function as a ligand for EGFR (13) and c-erbB-4 but does not interact with c-erbB-2 or c-erbB-3 (14) . As yet, there is little information on the physiological role of betacellulin or its role in cancer, although it is expressed in a number of normal tissues (15) .
The human c-erbB-3 receptor, as its name suggests, is the third member of the family and is widely expressed in normal tissues (16) . The most significant difference between c-erbB-3 and the other family members is that it has little or no tyrosine kinase activity (17 , 18) . Thus, although homodimers of c-erbB-3 may be inactive, it has been shown to signal effectively by pairing with EGFR or c-erbB-4 in heterodimers and in particular with c-erbB-2, for which it appears to be the preferred partner. The c-erbB-3 protein has also been shown to be expressed at elevated levels in a range of human malignancies (19) .
c-erbB-4, the fourth and most recently described member of the EGFR family (20) , is a Mr 180,000 glycosylated receptor, the pattern of expression of which in normal tissues has been described in detail only recently (21) . There have been only a handful of reports on its expression in cancer (21) , and both underexpression (22) and overexpression (23 , 24) have been reported.
The expression of EGFR and its ligands and c-erbB-2 receptor in the normal and malignant endometrium have been studied previously (25, 26, 27, 28, 29, 30, 31)
. We have, in this pilot study, looked for the first time at the expression of c-erbB-3 and c-erbB-4 growth factor receptors and their principal ligands, NRG-1 , NRG-1 , and betacellulin, in normal endometrum and compared them with the expression in endometrial adenocarcinomas. In normal endometrium, the expression of these proteins has been compared in the two phases, proliferative and secretory, where the endometrium is under different hormonal milieus and therefore may provide a clue regarding the regulation of these proteins by the sex steroid hormones, particularly estrogen. Persistent and prolonged estrogenic stimulation is a well-known risk factor in the causation of endometrial carcinoma. In particular, in the breast, c-erbB-2 and c-erbB-3 mRNA and protein expression are down-regulated by estrogen and induced by tamoxifen, but it is not established whether similar regulation of expression occurs in endometrium. This may be of more than academic interest in the light of their role in cell proliferation and the apparent paradoxical effect of tamoxifen, a widely used antiestrogenic compound, on the incidence of breast and endometrial cancer (32)
.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, NRG-1 , and betacellulin using synthetic peptides derived from the COOH-terminal third of the EGF homology region. The immunizing sequences were as follows: NRG-1 , CQPGFTGARCTENVPMK (called NRG76); NRG-1 , PNEFTGDRCQNYVMAS (called NRG102); and betacellulin, CDEGYIGARCERVDLFY (called BTC 97). Each purified peptide was coupled to keyhole limpet hemocyanin and used to raise antibodies in rabbits. The serum titers were determined by ELISA, and the antibodies were purified by affinity chromatography as described by Rajkumar and Gullick (34) . The properties of antibodies raised against NRG76 (35) and BTC97 (36) have been described previously.
The properties of the antibody raised to NRG1- were determined by Western blotting. Purified, recombinant isoforms of NRG1 and variants were a generous gift from Dr. B. Ratzkin (Amgen). The forms used in this work are rH-NRG-1 (14–249), rH-NRG-2 (14–241), rH-NRG-1 (14–246), and rH-NRG-2 (14–238) (Ref. 37
). 0.5 µg of each purified form was run on a 10% polyacrylamide gel; this was transferred to nitrocellulose, and Western blotting was performed using 5 µg/ml of affinity-purified NRG76 antibody, after which the blots were stripped and reprobed using the same concentration of antibody NRG102.
For tissue sections and immunohistochemistry, a total of 43 normal endometria (16 proliferative, 19 secretory, and 8 hyperplasia) and 41 endometrial adenocarcinoma (grading as per Fédération Internationale Gynaecologists et Obstetristes classification: 30 grade 1, 6 grade 2, and 5 grade 3) were analyzed by immunohistochemistry of formalin-fixed, paraffin-embedded tissues. The tissue blocks were retrieved from the archives of the Hammersmith and Wexham Park Hospitals. No pretreatment was required for any of the antibodies. The streptavidin-biotin immunoperoxidase technique was used. Briefly, sections were brought to water, endogenous peroxidase was blocked, and the primary antibodies were applied at 5 µg/ml for the polyclonal and 1 µg/ml for the monoclonal antibodies and incubated at 4°C overnight. Appropriate biotinylated secondary antibodies (swine antirabbit and rabbit antimouse) were used (Dako), followed by a third layer of horseradish peroxidase-labeled streptavidin (Dako). The color was developed using 3,3'-diaminobenzidine and hydrogen peroxide. Negative controls included omission of the primary antibody and blocking of reactivity by preincubation with a 50-fold excess of the corresponding immunizing peptide. The positive control included was a section of the kidney wherein the tubular cells stained positive for the five antigens tested in this study.
For scoring of immunoreactivity, we used a scoring system described previously (33 , 38 , 39) wherein both the percentage of cells positive and the overall intensity of staining were taken into account. On the basis of the percentage of tumor cells positive, a score of: 0, no cells positive; 1, up to 25% positive; 2, 26–50% positive; 3, 51–75% positive; and 4, >75% positive, was assigned. A second score for intensity of staining was assigned as follows: 0, negative; 1, weakly positive; 2, moderately positive; and 3, strongly positive.
The two individual scores were added, giving a final score that therefore could range from 0 to 7. Any evidence of membrane positivity was given an additional score of 1. In normal endometrium, the glands and stroma were evaluated separately. Scoring was performed by two independent observers (E. B. and R. S.). The rate of discordance was <5%, and all such cases were reassessed together.
Statistical Analysis.
The five proteins examined (c-erbB-3, c-erbB-4, NRG-1 , NRG-1 , and betacellulin) were analyzed for association with the menstrual phase in normal endometrial samples using the Mann-Whitney and Kruskal-Wallis test. The expression in the endometrial glands was compared with the stromal expression using the Wilcoxon matched pairs signed rank test. The endometrial adenocarcinoma samples were compared as a group with the normal endometrium for the expression of each of the proteins and analyzed using the Mann-Whitney U test. Within the endometrial carcinoma group, association with the grade/differentiation was sought by the Mann-Whitney and Kruskal-Wallis test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. form of NRG, antibodies were raised to a synthetic peptide from the unique COOH terminal sequence of the EGF-like domain. These were evaluated for their ability to recognize the isoforms and not to cross-react with the variants by Western blotting. The NRG102 antiserum reacted specifically with the 1 and 2 (Fig. 1
, Lanes 3 and 4) proteins but did not recognize the forms (Fig. 1
, Lanes 1 and 2). Conversely, antibody NRG76 recognized the 1 and 2 forms and not the variants (Fig. 1)
. The antibodies were then evaluated for their ability to react with formalin-fixed, parraffin-embedded tissues. The staining obtained with each antibody was blocked by the immunizing peptide, confirming the specificity of their reaction. The characterization of the other antibodies used in this study has been referred to earlier in "Materials and Methods."
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, NRG-1 , and betacellulin by immunocytochemical staining. Predominantly cytoplasmic staining was seen with all of the antibodies used, which was diffuse with the exception of NRG1-, which showed both a diffuse and a granular pattern of staining. Membrane staining for the c-erbB-3, c-erbB-4 receptors and the ligand betacellulin was seen only in the context of endometrial cancers in 12, 4, and 11 cases (30, 8, and 28% of the total). Nuclear immunoreactivity was observed in the normal endometrial stromal nuclei in a small number of cases for NRG1- and , betacellulin, and c-erbB4. No nuclear staining was seen in endometrial cancers.
The levels of expression of each of the receptor or ligand were compared among: (a) the glandular versus stromal compartments in normal endometrial samples; (b) glands in the proliferative versus secretory phases; (c) the entire group of normal endometrial samples versus endometrial adenocarcinomas, and (d) in the endometrial carcinoma group, correlation to histological tumor grade, i.e., grade 1 versus grades 2 and 3 combined (in view of the small number of cases in each group). The results obtained for each of the proteins examined are described below. The hyperplastic endometrium, in general, showed expression of each of the proteins analyzed at levels similar to that seen in the proliferative endometrium. The histograms obtained by plotting the glandular reactivity score against the number of cases in the two phases of the menstrual cycle and for hyperplastic endometrium for each protein analyzed are shown in Fig. 2
. The histograms derived similarly by comparing the normal group versus the endometrial carcinoma are displayed in Fig. 3
.
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in 30% (12 of 40) cases. When analyzed as a group, there was significant overexpression in the carcinomas as compared with the normals (Mann Whitney U test; P = 0.00001). In addition, there was a correlation with tumor grade, with the well-differentiated tumors showing higher levels of c-erbB-3 as compared with the moderately and poorly differentiated tumors analyzed as a group (Kruskal-Wallis test, P = 0.0001).
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). The endometrial carcinomas generally showed modest levels, and using a cutoff score of 7 and above, there was evidence for overexpression in six cases (15%). Furthermore, when the carcinomas were analyzed as a group and compared with the normals, significant overexpression was observed in the endometrial carcinomas (Fig. 4D
; Mann-Whitney U test, P = 0.0002).
NRG-1 .
This ligand was expressed in both the glands and stroma with scores ranging from 2 to 7, with significantly higher levels of expression in the stroma than in the glands (Wilcoxon signed rank test, P = 0.0007). Incidentally, the vascular smooth muscle and the uterine smooth muscle also expressed modest levels of NRG-1 . There was also a weak association with the menstrual phase, with the secretory glands showing higher levels of expression than the proliferative glands (P = 0.0064). In endometrial carcinomas, variable levels were noted; the scores ranged from 0 to 7. Overall, there was a decrease in the level of expression in endometrial cancers (Fig. 4F)
as compared with normals, which was statistically significant (Mann Whitney test, P = 0.0035). Furthermore, a significant decrease in the levels of expression in the moderately and poorly differentiated tumors as compared with the well-differentiated tumors was also observed (Mann-Whitney test, P = 0.0001).
NRG-1 .
In normal endometria, both the glands and stroma showed weak to moderate levels of expression, with the scores ranging from 0 to 7. The glands in the secretory phase showed significantly higher levels of expression as compared with the proliferative phase (P = 0.0065). Endometrial cancers showed variable levels of expression, with the scores ranging from 0 to 8, and these were not significantly different from normal levels. There was no correlation of NRG-1 expression to the tumor grade.
Betacellulin.
Both the endometrial glands and stroma showed levels of expression ranging from weak to high with the scores from 0 to 6. The glands in the secretory phase showed significantly higher levels of expression than those in the proliferative phase (P = 0.0011). In endometrial cancers, the scores ranged from 2 to 8, and using a cutoff score of 7, there was clear evidence of overexpression in 33% of cases (13 of 40). Furthermore, overexpression was observed when endometrial carcinomas (Fig. 4E)
were analyzed as a group and compared with normal controls (Mann-Whitney test, P < 0.00001).
Simultaneous Overexpression.
In endometrial carcinomas, there was evidence for the simultaneous overexpression of more than one factor analyzed as follows: c-erbB-3 and c-erbB-4 in three cases; c-erbB-3 and c-erbB-4 and betacellulin in two cases; c-erbB4 and betacellulin in two cases; and for c-erbB-3 and betacellulin in seven cases.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, NRG-1 , and betacellulin in the normal endometrium during the two principal phases of the menstrual cycle, the proliferative and the secretory phases, which served as a baseline against which the expression in endometrial adenocarcinoma was assessed.
The heterogeneity of immunoreactivity for the proteins tested in this study necessitated the use of the scoring system detailed in "Materials and Methods." This system has been used in previous reports from this laboratory (33
, 38
, 39)
. The secretory endometrial glands showed significantly increased levels relative to the proliferative phase of the c-erbB-4 receptor and all its ligands examined including NRG-1 , NRG-1 , and betacellulin. In contrast, c-erbB-3 was expressed weakly in both phases without any differential distribution. This would imply that c-erbB-4 and its ligands play a role in the differentiation and maturation of the endometrium. It is also possible that this group of ligands and the c-erbB-4 receptor may be regulated by estrogen, and future experiments on endometrial cancer-derived cell lines should clarify this issue. The endometrial stroma showed immunoreactivity for c-erbB-3, c-erbB-4, and all of the ligands; however, the levels of expression were variable. When the levels of glandular versus stromal expression were compared, c-erbB-4 and NRG-1 showed relatively higher levels in the stroma. In previous studies, EGFR has been reported to be expressed in the normal endometrial glands (25
, 26)
, but a correlation to the menstrual phase was not observed (25)
. In another study, EGFR was reported to be present in the endometrial stromal cells but not in the glands with increasing levels in the secretory phase (28)
. The c-erbB-2 receptor, on the contrary, is reported to be expressed only in the glands and not in the stroma and is also apparently up-regulated in the secretory phase (28)
.
Both the receptors c-erbB-3 and c-erbB-4 showed evidence of overexpression when the group of endometrial carcinomas was compared with the normal as a whole. Such an analysis of the two groups using the Mann-Whitney test is, perhaps, more realistic rather than giving arbitrary cutoff values to determine overexpression. For instance, using a cutoff score of 6, the proportion of c-erbB-3 and c-erbB-4 "overexpressing" tumors was 39 and 72%, respectively. If a cutoff score of 7 were used, the rates would be 32 and 17%, respectively. The situation with these two receptors is complicated because of heterogeneity of expression, unlike c-erbB-2 positivity where "overexpression" is generally homogeneous and thus more easily defined. However, from the frequency histograms depicted in Fig. 3
, it is seen that a score of 5 or more in the case of c-erbB-3 and a score of 7 or more in the case of c-erbB-4 are indicative of expression above normal levels. There have been earlier studies on the expression of EGFR and c-erbB-2 in endometrial carcinomas. It is worth mentioning here that whereas some studies report "expression," others report "overexpression." Thus, EGFR is expressed in 24% (29)
to 67% (26)
of endometrial carcinomas and is overexpressed in 49% of cases in a study where a correlation to the occurrence of metastases was noted (27)
. c-erbB-2 overexpression is variably reported in different series and ranges from as low as 12% (30)
to 52% of cases (31)
. The ERBB2 gene is also amplified in about 21% of cases (31)
. In general, c-erbB-2 expression has correlated to more aggressive disease and shortened overall survival (29, 30, 31)
.
Among the ligands, betacellulin showed evidence of overexpression. Betacellulin is a ligand for c-erbB-4 and EGFR and in the light of this finding, the relatively mild overexpression of c-erbB-4 noted in this study assumes greater importance. With respect to the NRGs, underexpression of NRG-1 in endometrial cancers was significant. A similar observation of loss of NRG-1 expression in prostatic adenocarcinoma has been made in one study (22)
, whereas in another study on prostatic carcinoma, up-regulation of the protein was noted (35)
. We did not observe significant difference in the levels of NRG-1 expression in malignant and normal endometria. The role of NRGs in cancer is very complex, not only because of the numerous isoforms that are present that may possess different functions, but also in the wide range of receptor combinations that they may stimulate. Experiments with breast cancer-derived cell lines have suggested that this large family of proteins plays a dual role, promoting mitogenesis as well as a differentiation in different settings (40
, 41)
The occasional staining of the nuclei of endometrial stromal cells with the antibodies against NRG-1 , NRG-1 , betacellulin, and c-erbB-4 is intriguing and merits a more detailed analysis. The NRGs possess nuclear localization signals in their sequence and have been shown to undergo rapid nuclear translocation in the breast cancer-derived SK-BR-3 cell line (42)
. The c-erbB-4 receptor also possesses nuclear localization signals in its sequence, and previously we have reported the observation of occasional nuclear staining seen in the epithelial lining of the distal tubules of the kidney and the ducts of the breast (21)
.
To summarize, this pilot study highlights that c-erbB-4 receptor and its ligands, the NRGs and , and betacellulin play a role in normal endometrial maturation. In endometrial adenocarcinoma, overexpression of the receptors c-erbB-3 and c-erbB-4 and the ligand betacellulin and underexpression of NRG-1 occur. It is hoped that this study will form the basis for analyses of these proteins in a larger series of endometrial cancers, which will enable us to identify useful molecular markers and identify targets for new therapies.
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FOOTNOTES |
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1 Present address: Department of Histopathology, Derriford Hospital, Plymouth, Devon, PL6 8DH. 
2 To whom requests for reprints should be addressed, at Hammersmith Hospital, MRC Cyclotron Building, Third Floor, Imperial Cancer Research Fund Oncology Group, Du Cane Road, London W12 OHS, United Kingdom.
3 The abbreviations used are: EGFR, epidermal growth factor receptor; NRG, neuregulin.
Received 3/17/99; revised 7/15/99; accepted 7/30/99.
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and erbB-3 receptor in human prostatic adenocarcinoma. Br. J. Urol., 79: 212-216, 1997.[Medline]
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, proliferative endometrium; 
, secretory endometrium; 
, cases of endometrial hyperplasia.
