1 Department of Obstetrics and Gynecology, University Hospital, Berne; 2 Institute of Pathology and 3 Department of Clinical Research, University of Berne, Berne, Switzerland
Received 29 May 2002; revised 1 October 2002; accepted 22 October 2002
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Abstract |
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Members of the Eph family of tyrosine kinases have been implicated in embryonic pattern formation and vascular development; however, little is known about their role in the adult organism. We have observed estrogen-dependent EphB4 expression in the normal breast suggesting its implication in the hormone-controlled homeostasis of this organ. Since the endometrium is a similarly hormone dependent organ and endometrial carcinoma is thought to result from estrogenic stimulation, we have investigated EphB4 expression in normal human endometrium and during its carcinogenesis.
Patients and methods:
EphB4 expression was analyzed immunohistochemically in 26 normal endometrium specimens, 15 hyperplasias and 102 endometrioid adenocarcinomas and correlated with clinical and prognostic tumor characteristics.
Results:
In normal endometrial tissue no EphB4 protein was detected. Strikingly, we observed a drastic increase (P <0.0001) in the number of EphB4 protein-expressing glandular epithelial cells in the majority of hyperplasias and carcinomas. Moreover, we found a statistically highly significant positive correlation between EphB4 expression and post-menopausal stage of the patient (P = 0.007).
Conclusions:
These findings indicate that in the endometrium, EphB4 is an early indicator of malignant development and, thus, EphB4 may represent a potent tool for diagnosis and therapeutic intervention.
Key words: endometrial cancer, immunohistochemistry, tyrosine kinase
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Introduction |
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Receptor tyrosine kinases (RPTKs) compromise 14 distinct members, the largest of which is the Eph family group [4]. The Eph receptors are grouped into two major subclasses, EphA and EphB, based on sequence homologies and ligand binding specificities [5]. Their ligands, the ephrins, are membrane anchored by either a GPI (glycosylphosphatidylinositol) linkage (ephrin-A) or a transmembrane region (ephrin-B). Ephrin-A ligands interact preferentially with receptors of the EphA subclass and ephrin-B ligands with receptors of the EphB subclass [6]. Given the membrane bound localization of both the ligands and the receptors, signaling must function in the cell-to-cell range rather than in long-range communication [7]. Furthermore, interaction between EphB receptors and their ligands can provoke bi-directional signaling and mutual cellcell communication, since ephrin-B ligands have been shown to undergo phosphorylation on conserved intracellular tyrosines upon receptor interaction [8, 9].
Eph receptors and ephrin ligands play a pivotal role in pattern formation, cell aggregation and migration, as well as segmentation during embryonic development [10]. In particular, Eph family members, including EphB4, are instrumental in the development of the embryonic capillary network [10]. Little is known about Eph functions in adult organisms; however, it is conceivable that this receptorligand family is involved in morphogenic processes persisting in adult organs, such as those occurring in the endometrium.
We have previously described hormone-dependent and cell type-specific expression of the EphB4 receptor during normal mouse mammary gland development [11]. In the human breast, EphB4 was expressed in the luminal epithelial cells of normal breast cells [12] and a drastic reduction of EphB4 expression was observed in almost all invasive carcinomas analyzed. Furthermore, we found a highly significant correlation between EphB4 positivity and low histological grade of the tumors [13]. Like in the breast, hormonal changes throughout the menstrual cycle induce the endometrium epithelium into proliferation, differentiation and cell death by apoptosis [14]. Any escape from this tightly regulated equilibrium of growth and regression may eventually lead to malignant transformation.
In search of putative regulatory mechanisms affecting the pathogenesis of endometrial cancer, we investigated by immunohistochemistry the expression of EphB4 in normal endometrium, in endometrial hyperplasia and in a series of endometrioid adenocarcinomas. Furthermore, the level of EphB4 expression in cancer as defined by semiquantitative analyses was correlated to patient survival.
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Patients and methods |
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Evaluation procedures
The number of immunoreactive cells was semiquantitatively estimated by two independent investigators (E.K. and G.B.) who were blinded with regard to the results of tumor stage, clinical variables and follow-up data. First, areas with intense staining were selected at low magnification with a 4x objective. Then semiquantitative analyses were performed with the 40x objective in these preselected areas: + corresponds to <10% positive cells, ++ to 1050% positive cells and +++ to >50% positive cells. For every sample, at least 100, and usually >1000 cells were analyzed.
Statistical analysis
SPSS statistical software (SPSS, Chicago, IL, USA) was used for statistical analysis. The significance of differences between individual groups was analyzed using Fishers exact test or the Pearson chi-square test. The relationship between the dependent variable EphB4 and the other independent variables was examined using the multiple logistic regression model for polytomous data with ordinal scale [17]. The full model was reduced by a backward elimination procedure to get to the final model. Analyses of survival were performed using the KaplanMeier method [18]. Survival distributions were compared with the log-rank test. Values of P <0.05 were considered statistically significant.
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Results |
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Discussion |
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Overexpression of EphB4 has been described in colon cancer [20] and in a variety of human lung and breast cancer cell lines [12, 21], but only a few studies have addressed the expression of EphB4 during carcinogenesis. Nevertheless, in experimentally-induced invasive mouse mammary tumors, overexpression of EphB4 suggested its involvement in the carcinogenic process [22]. In endometrial carcinoma we demonstrated a significant increase of EphB4 expression compared with normal tissue. Takai et al. [23] have recently analyzed 20 endometrial carcinomas, and in analogy to our results, induction of EphB4 expression was found in all samples. In these studies, EphB4 expression correlated with poor prognostic factors, such as grade and myometrial invasion. Although our study included a higher number of patients, this association could not be confirmed.
It has been suggested that the roles of Eph receptors and their ephrin ligands include the promotion of angiogenesis and the modulation of cellmatrix attachment [24]. Interactions between Eph receptors and ephrin ligands may result in destabilization, which can also affect cellmatrix attachment, and thereby promote invasion and metastasis [25]. Indeed, in endometrial carcinoma, ephrin-B2, the EphB4 ligand, is almost always expressed in cancer cells neighboring EphB4-positive cells, which suggests autocrine and/or paracrine activation [23].
The implication of EphB4 in embryonic vascular network formation is demonstrated by the fact that in the developing vasculature EphB4 is exclusively expressed in embryonic veins, whereas expression of one of its ligands, ephrin-B2, is limited to arteries [26]. Moreover, in tumor angiogenesis expression of EphA2 and its ligand ephrin-A1 was found to be up-regulated not only in tumor blood vessels but also in tumor cells, suggesting interaction between the endothelium with the surrounding tumor cells [25]. In accordance with this hypothesis, we have observed that EphB4 expression in endometrial carcinoma was stronger at the interface between cancer cells and stroma where possibly angiogenesis is most critical for progression and survival of endometrial carcinoma. It remains to be further clarified whether EphB4 expression results in the promotion of angiogenesis during endometrial carcinogenesis.
In the mouse mammary gland, the expression of EphB4 and its ligand ephrin-B2 is induced by estrogens, whereas no receptor or ligand proteins are detected in ovariectomized mice [27]. In the normal human breast epithelium, the highest portion of EphB4-positive epithelial cells is found during the estrogen dominated follicular phase of the cycle and EphB4 expression is drastically reduced in breast cancer [13]. The endometrium and the breast epithelium both undergo cyclic proliferation and differentiation, and exposure to estrogen has been identified as one of the major risk factors for the development of both cancers. Surprisingly, the opposite was true in the endometrium, where no EphB4 expression could be detected in normal tissue. Furthermore, in endometrial carcinoma the highest portion of cells expressing EphB4 is found in post-menopausal patients with low estrogen levels. These results suggest that estrogen down-regulates EphB4 protein in normal and malignant endometrium. Tamoxifen is a well-known estrogen agonist on the endometrium and an antagonist on breast tissue [3]. These opposite effects are mainly explained by its action on the two estrogen receptor subtypes, ER and ERß, which are differentially expressed in breast and endometrial epithelial cells [28]. It has recently been demonstrated that ER
is able to bind to insulin-like growth factor 1 receptor (IGF-1R) leading to its autophosphorylation and thus activation [29]. Conceivably, regulation of EphB4 expression is affected by a similar ER subtype-specific mechanism.
In summary, we have shown that EphB4 is expressed early during endometrial carcinogenesis and therefore could represent an indicator for malignant transformation. EphB4 expression, however, does not appear to have prognostic significance in endometrioid carcinomas. Additional studies are clearly needed to clarify this issue.
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Acknowledgements |
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Footnotes |
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References |
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