Background
Endometriosis is a benign gynecological disease characterized by the presence of functional endometrial glands and stroma outside the uterine cavity [
1]. The precise etiology of endometriosis is unclear. One of the more widely accepted hypotheses is that endometriosis originates from the retrograde menstruation of endometrial cells that implant on peritoneal surfaces [
2]. Although retrograde menstruation can be observed in many women, only a minority develop endometriosis. The success of the ectopic implantation seems to be dependent on several factors, including changes enabling endometrial cell migration, adhesion and invasive growth, as well as changes in anti-apoptotic signaling, angiogenesis and inflammatory response [
3,
1,
4]. Epithelial to mesenchymal transition (EMT) is a process whereby epithelial cells lose polarity and cell-to-cell contacts and acquire the migratory and invasive abilities of mesenchymal cells [
5]. These abilities might be prerequisites for the establishment of endometriotic lesions.
A hallmark of EMT is the functional loss of E-cadherin expression in epithelial cells. For endometriosis, studies on E-cadherin expression have led to contradictory results. While some studies reported a reduction of E-cadherin expression in endometriosis compared with the endometrium [
6‐
9], others found no difference in E-cadherin expression in endometriosis compared with the endometrium [
10‐
14]. Previous studies demonstrated that E-cadherin-negative epithelial cells were increased in peritoneal endometriosis compared with eutopic endometrium and that in vitro, E-cadherin-negative, N-cadherin-positive endometriotic epithelial cells showed invasive growth [
15,
16]. Loss of E-cadherin expression together with a cadherin switch, in which E-cadherin is replaced by the expression of mesenchymal cadherins such as N-cadherin, is an important feature of EMT [
5].
TWIST1, a highly conserved basic helix-loop-helix (bHLH) transcription factor that represses E-cadherin (CDH1) transcription, represents an EMT inducer and has been convincingly associated with tumor progression and the metastatic process [
17‐
19].
SNAIL, a zinc-finger transcription factor, and its close relative SLUG have similar properties to TWIST1 and thus belong to the EMT inducers [
14].
Factors that facilitate the survival and proliferation of misplaced endometrial cells may also contribute to the development of endometriosis. In endometriosis, epithelial and stromal cells show a lower number of apoptotic cells than in patients without endometriosis [
20‐
22]. It has been hypothesized that the expression of the anti-apoptotic factor BCL-2 and the reduction of the pro-apoptotic factor BAX in endometriosis lesions allows for the survival of the tissue in ectopic sites [
23,
24,
21,
22]. Increased expression of genes such as MYC, Cyclin D1, and Ki67 was shown to be upregulated in ectopic tissues, suggesting that lesions exhibit a higher proliferation rate [
25,
26,
23,
27,
28]. Several studies investigating MYC expression in endometriosis observed increased MYC mRNA and protein expression in ectopic and eutopic endometrium from endometriosis patients [
26,
29‐
31,
25].
Using qRT-PCR and immunohistochemistry (IHC), we analyzed the expression, localization and correlation of CDH1 and TWIST1, CDH1 and SNAIL, CDH1 and SLUG, and TWIST1 and MYC in more than 100 ectopic and eutopic endometrial tissues from the proliferative and secretory endometrium of women with endometriosis and in matched tissue samples from the same patients. At present, the concurrent expression of TWIST1 and MYC in the same sample and in paired analysis of eutopic and ectopic tissues of the same patient has never been evaluated.
Discussion
In the present study, we were able to demonstrate that epithelial TWIST1, SNAIL and SLUG expression was overexpressed in ectopic lesions compared to eutopic endometrium glands. Correspondingly, in paired analysis of samples from the same patient, we found that in a significant proportion of samples, TWIST1, SNAIL and SLUG expression was negative in eutopic endometrium, whereas it was positive in ectopic lesions. We further showed overexpression of MYC in the glandular epithelium of endometriotic lesions compared to eutopic endometrium. Analysis of matched tissue samples revealed a trend towards more frequent epithelial MYC expression in ectopic lesions, although this did not reach significance. Few data exist that indicate a potential role of TWIST1 in the pathogenesis of endometriosis [
14,
37]. Moreover, we showed a significant inverse expression between TWIST1 and CDH1 in controls and eutopic and ectopic tissue of patients. In addition, a significant inverse expression between SNAIL/ SLUG and CDH1 was observed in eutopic and ectopic tissue of patients. This finding suggests that TWIST1, SNAIL and SLUG might be important regulators of EMT in endometrium that is obviously upregulated in ectopic lesions. A study including patients with ovarian endometriosis analyzed mRNA expression of the stemness-related gene OCT4 and TWIST1 [
37]. They reported an increased expression of OCT4 in ectopic endometrium and a positive correlation of OCT4 with TWIST1 [
37]. The study that measured the mRNA expression of homogenized cells lacked discrimination between stromal and epithelial tissue. In our study of 110 endometriosis patients, we discriminated between epithelial and stromal protein expression by using IHC analysis for TWIST1 and MYC. We found that the epithelial expression of TWIST1 is significantly increased in ectopic endometrium while the stromal expression is reduced. Our findings suggest that the enhanced expression of TWIST1, SNAIL and SLUG in ectopic lesions plays a crucial role in the formation and maintenance of ectopic lesions in endometriosis. It can be hypothesized that the glandular epithelial cells lose polarity and cell-to-cell contacts by EMT and acquire migratory and invasive abilities to establish ectopic lesions.
In endometriosis, EMT is induced by multiple signals. For example, 17ß-estradiol (E2), which is known to be high in endometriotic tissue, has been shown to induce EMT in human endometrial epithelial cells through upregulation of the hepatocyte growth factor [
38‐
40]. EMT can be induced by proinflammatory cytokines. One inflammatory mediator relevant in EMT is TGF-ß which is increased in peritoneal fluid of women with endometriosis [
41]. TNF-α and IL-6 may synergistically nudge the TGF-ß signaling pathway towards EMT progression [
42]. A significantly increased secretion of TNF-α and IL-6 in the culture media of peritoneal macrophages of endometriosis patients was found in response to E2 compared to nontreated macrophages [
39]. Moreover, levels of IL-6 are higher in human endometrial stromal cells derived from the endometrial biopsies of women with endometriosis when compared with women without the disease [
43]. TNF-α and IL-6 but also oxidative stress can promote NF-κB activation, which regulates the expression of Snail1, Slug, Twist, ZEB1, and ZEB2 [
44,
42]. A recent study showed that iron overload leads to NF-κB activation in human endometrial stromal cells [
44].
MYC is a well-known oncogene, and its function in tumor formation has been intensively studied. In endometriosis, the overexpression of MYC is also well established. Nevertheless, the role in pathogenesis is still unclear. MYC is upregulated in the ectopic and eutopic endometrium of patients with endometriosis when analyzed by reverse transcription PCR and IHC [
26,
29‐
31,
25]. In the present study, we observed higher MYC expression in the glandular epithelium of endometriotic lesions compared to eutopic tissues, in concordance with Pellegrini et al. MYC overexpression suggests a higher proliferation rate in lesions than in eutopic tissues. In contrast to the epithelial expression of MYC, the stromal expression of MYC was predominantly negative in the ectopic endometrium.
Our observation of overexpressed MYC and TWIST1 in the epithelial cells of ectopic lesions prompted us to investigate a putative correlation of these two markers. In the present study, we were not able to detect the concurrent regulation of epithelial MYC and TWIST1 in the same sample in either eutopic or ectopic tissues. Actually, the exact opposite was true. We found a significant inverse expression of MYC and TWIST1 in paired samples. Thus, we excluded a simultaneous upregulation of TWIST1 and MYC that may orchestrate the cellular changes associated with invasion and proliferation in endometriosis. It seems that a high expression of TWIST1, which was shown to be associated with the stemness marker OCT4 in endometriosis, excludes a high expression of MYC, which is associated with proliferation [
37] in endometriosis. TWIST1 has also been shown to be an important regulator of stemness in epithelial ovarian cancer [
45,
46]. Thus, in ectopic lesions with high epithelial MYC expression, the additional upregulation of TWIST1 appears to provide no further advantage for the cell and vice versa. There are several other theories concerning the pathogenesis of endometriosis. Some studies revealed the presence of adult stem cells in the basalis but also functionalis layers of the human endometrium [
47‐
50]. These endometrium-derived stem cells, which are distributed by retrograde menstrual efflux, may also contribute to the establishment of ectopic endometriotic lesions [
50‐
54]. The monoclonal origin of some endometriotic lesions, long-time culture properties of cell clones established from endometriotic lesions, and the isolation of progenitor cells from menstrual blood support this hypothesis [
55‐
60]. Another theory concerning the pathogenesis of endometriosis is the coelomic metaplasia theory, which proposes that mesothelial cells on the ovary or pelvis change to endometriotic gland cells [
61,
62].
The observation of increased MYC expression during the proliferative cycle phase has already been reported [
35,
26,
36]. In utero, MYC is upregulated by estrogens by an estrogen response cis-acting element (ERE) in its promotor [
63‐
65]. Endometriotic lesions show increased production of estradiol and higher concentrations of estradiol have been detected in the peritoneal fluid of women with endometriosis than in that of normal controls [
66]. Compared to estrogen receptor (ER) α, ERß is expressed in markedly higher levels in ectopic lesions than in eutopic tissue. [
67]. Deficient methylation of the ERß gene promotor has been suggested to result in pathological overexpression of ERß in endometriosis, which in turn represses ERα expression [
67]. Previous studies showed a remarkably elevation of ERß mRNA and protein expression relative to the normal endometrium [
68,
25]. In addition, the eutopic endometrium of women with endometriosis have elevated ERß expression when compared with the endometrium of healthy women [
69‐
71], suggesting that high levels of ERß in the endometrium may predispose women to endometriosis. Increased MYC expression might be due to enhanced local estrogen levels in endometriotic lesions [
72,
63,
65]. In this study, we showed a significant correlation between MYC expression and the proliferative menstrual cycle phase of the women with high estrogen levels. In contrast, we confirmed previous findings that the expression of TWIST1 was not associated with cycle phase [
14].
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
KP and BS made substantial contributions to conception, design, acquisition of data, analysis and interpretation of data. KP and BS were involved in drafting the manuscript and revising it critically for intellectual content. BP assisted in concept development and manuscript preparation, recruitment of patients, acquisition, and analysis and interpretation of data. SG made contributions to the acquisition of data, performed statistical analysis, and reviewed the manuscript. NN and EM made contributions to the acquisition of data, performed statistical analysis, and reviewed the manuscript. RW, GY and LK made substantial contributions in the recruitment of patients, provided clinical information (staging) and assisted in manuscript preparation. HH made substantial contributions to concept development and study design and revised it critically for important intellectual content. All authors read and approved the final manuscript.