Expression of ephrin receptor A1 on the membrane of epitheliocyte glands of the uterine mucosa in patients with endometrial cancer and endometriosis

Endometriosis is a chronic inflammatory disease with a high prevalence and serious reproductive and health consequences [1, 2]. Endometriosis is one of the most common benign gynecologic neoplasms in premenopausal women and affects, according to various estimates, 10–15% of women of reproductive age [3].  Despite many years of research, the pathogenetic mechanisms of the disease remain unclear, and the methods of diagnosis and treatment are still ineffective. This disease is a significant public health problem, strongly affecting the quality of life of women, as well as being a significant economic burden.

Three phenotypes of the disease are distinguished according to clinical manifestation: peritoneal endometriosis (PE), ovarian endometrioma, and deep infiltrative endometriosis (DE) [4]. DE is manifested by an infiltration of endometriosis deeper than 5 mm beneath the peritoneum and is the most aggressive form of the disease. DE has all common signs of malignant diseases: increased proliferative and reduced apoptotic activity, invasiveness, adhesiveness propensity to metastasize; it can progress to malignant tumor [5, 6]. This suggests the possibility of using cancer therapies for DE treatment as well. In this regard, ephrin receptors (Eph) are of interest, which have been proven in experimental animal studies as a promising target for cancer treatment [7].

Ephrin receptors (Eph) are part of the family of receptor tyrosine kinases involved in embryogenesis, controlling organ growth by intercellular communications [8].  Eph are expressed on the surface of the cell membrane and their functional activation is realized by binding to the corresponding ligand, ephrin. Eph receptor tyrosine kinases and their ephrin ligands are critical regulators of cell contact-dependent signal signaling and pattern formation. Eph/ephrin binding can lead to activation of various biological functions such as adhesion or repulsion, or increased or decreased motility [9]. Pathologic increase or decrease in Eph expression is associated with various diseases: cancer, cardiovascular diseases, brain diseases, etc. [10]. Therefore, Ephs are of interest as therapeutic target in the treatment of various diseases [11].

The EphA1 receptor was first detected in an erythropoietin-producing human liver cancer cell in 1987, and its increased expression (overexpression) was noticed in various types of human cancer, including gastric carcinoma, colorectal cancer, ovarian, and breast cancer [12]. The receptor regulates many aspects of tumor biology, patient survival, and is considered as a therapeutic target for cancer treatment [13]. At the same time, we did not find works on EphA1 expression in EC. We also found no works on receptor expression in eutopic and ectopic endometrium in endometriosis.

In this regard, the aim of this work was to conduct a comparative study of EphA1 expression on the membrane of epitheliocytes of the glands of the uterine mucosa in patients with endometriosis and EC by immunohistochemistry.

Material and methods

The study was performed at the Academician V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia.

Four groups of 46 patients were formed for the study: Group 1 consisted of 20 women with DE (10 – in proliferative, 10 – in secretory phase of menstrual cycle (MC)), Group 2 consisted of 21 women with PE (12 – in proliferative, 8 – in secretory phase of MC), Group 3 included 6 women with EC (high-grade endometrial carcinomas stage FIGO Ia pT1aNxM0), and Group 4 (control) included 9 women without endometriosis (no pathological changes in the endometrium) and operated on for tubal-peritoneal infertility factor. The groups were additionally divided into subgroups depending on the phase of the menstrual cycle on the day before the operation. Exclusion criteria were the presence of severe extragenital diseases and other oncopathology. All patients signed informed consent for participation in this study. The study was approved by the Ethics Committee of the National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov of the Ministry of Health of the Russian Federation.

Semi-quantitative evaluation of EphA1 expression, the standard technique of immunohistochemical staining of deparaffinized preparations with antibodies was used, as described previously [14]. To detect EphA1, we used antibodies from Abcam Inc: rabbit polyclonal antibodies ab217363 at a dilution of 1:200, and goat anti-rabbit IgG H&L immunoglobulin (HRP) clone ab205718 as secondary antibodies. Slices stained without specific antibodies were used as a negative control.

Statistical analysis

Statistical analysis was performed using the MedCalc Statistical Software 11.5.0 software package. Statistical significance between the compared groups was performed using the Mann–Whitney U test. Data are presented as median (Me) and quartiles of Q1 and Q3 in Me format (Q1;Q3). The level of statistical significance in testing the null hypothesis was considered to be p<0.5.

Results

The data presented in the Table 1 show that the expression of EphA1 receptor on the membrane of epithelial cells of the glands of the uterine mucosa in patients without endometrial pathology is significantly higher in the secretory phase than in the proliferative phase (35.0 and 22.0 c.u., respectively, p=0.003, Figure A, B).

The expression level of the receptor on the membrane of epithelial cells of the glands in endometrial cancer cells significantly higher than that in normal endometrial cells both in the proliferative and secretory phases (46.0 c.u., p=0.003, Table 1, Figure B)), which indicates abnormally elevated (overexpressed) level of EphA1 in cancer cells (Figure E).

In PE, the expression of EphA1 on the membrane of gland epitheliocytes in eutopic endometrium did not significantly differ from that in the control group in the corresponding phases of the cycle. At the same time, increased expression of the receptor on superficial peritoneal lesions was detected in the proliferative phases of the cycle (Table).

In patients with DE on the membrane of epithelial glands in the eutopic endometrium, increased EphA1 expression was detected only in the proliferative phase: 32.5 c.u. compared to 22.0 c.u. norm (p=0.009). In the secretory phase of the cycle, EphA1 expression on the membrane of the epithelial cells of the glands of the eutopic endometrium was at the same level as in the healthy endometrium – 36.0 c.u. In the same patients, on the membrane of epithelial cells of glands in ectopic foci on the peritoneum, representing a superficial form of endometriosis, increased EphA1 expression was also detected only in the proliferative phase (41.0 c.u., p=0.003), and not in the secretory phase (Table).

Significantly increased expression of EphA1 both in the proliferative and secretory phases was detected on glandular cells of the endometrium in deeply infiltrative foci in DE (48.0, p=0.002 and 52.0, p=0.001, respectively). At the same time, the expression in both phases of the cycle was at the level of expression on the surface of gland epitheliocytes affected by EC, which is shown in Figure E.

Discussion

Ephrin receptors and their ephrin ligands are located on the cell surface, and their interaction through intercellular contact activates a wide range of signaling pathways aimed at proliferation, apoptosis, differentiation, cell adhesion, and migration [15]. EphA1, together with its main ligand EphrinA1, plays an important role in reproductive function by regulating folliculogenesis, ovulation, embryo transport, implantation, and placenta formation [16]. EphA1 is expressed on the outer membrane of endometrial epithelial cells, while its ligand EphrinA1 is seen on the outer membrane of blastocyst cells, which mediates the uterine implantation and ensures the proliferation and invasion of trophoblast into the thickness of decidual tissue [17]. The process of embryo attachment happens in the secretory phase of the menstrual cycle, which explains the found increased expression of EphA1 in this particular phase. In the secretory phase, the endometrial receptivity is formed, an important factor of which is the overgrowth of the blood vascular system in the endometrium, provided by the increased expression of angiogenic factors during this period [18]. Chinese researchers from Wenzhou showed that EphA1 activates angiogenesis by direct stimulation of the SDF-1/CXCR4 signaling system that controls the growth of the vascular system in the tissue [19].

Increased expression of EphA1 has been detected in many types of cancer, including ovarian and cervical cancer [16, 20]. Targeted inhibition of this expression in experimental models is often accompanied by inhibition of various stages of the cancer process. In this regard, ephrin receptors have recently been considered as promising targets for the therapy of various cancers [21]. We did not find any publications on EphA1 expression in malignant endometrial lesions. Our study has shown that in EC, in particular in early stage of cancer (high-grade endometrial carcinomas stage FIGO Ia pT1aNxM0), the expression of EphA1 is significantly increased compared to norm. This finding can be used for the development of therapy for the disease.

Endometriosis, especially DE, has a number of biological processes that are common with malignant diseases where ephrin receptors are involved [5]. We have previously shown that EphA2 in DE overexpressed on the surface of the epithelial cells of the glands of the ectopic endometrium similar to the level in endometrial cancer cells [22]. Similarly, EphB4 has been shown to be overexpressed in ectopic endometrium compared to eutopic [23]. Moreover, experiments with transplanted mice have shown that the suppression of EphB4 expression leads to inhibition of the growth of endometrioid cells by inhibiting angiogenesis [24].

Our study revealed an increased expression of EphA1 in the eutopic endometrium in the proliferative phase of the cycle in DE, while it was not found in the control (healthy) group or in patients with PE, which may be due to the increased level of systemic inflammatory response in DE. However, we found no increase in receptor expression in the eutopic endometrium of patients with DE during the secretory phase. To date, there are no publications on EphA1 expression in endometrial tissues, and further studies are needed to understand its function. However, the revealed overexpression of EphA1 in the endometrium of women with DE in the proliferative phase can be used to differentiate severity of endometriosis.

In ectopic lesions on the peritoneum in PE and DE, increased expression of the receptor was revealed only in the proliferative phase, but not in the secretory one. At the same time, on the membrane of epithelial cells of the glands in the infiltrative foci of the endometrium, a significantly increased expression of EphA1 was revealed in both phases of the cycle, which was similar to the expression in the same tissues affected by EC. This overexpression confirms the presence of common mechanisms in cancer and endometriosis.

Conclusion

The findings provide new insights into the molecular processes involved in the pathogenesis of endometriosis and EC. The overexpression of EphA1 makes it a promising target for the treatment of DE and EC. Further research is needed to understand the mechanism of pathologic processes underlying infertility and pain symptoms in women with endometriosis.