The EMT signaling pathways in endometrial carcinoma

Endometrial cancer (EC) is the most common gynecologic malignancy of the female genital tract and the fourth most common neoplasia in women. In EC, myometrial invasion is considered one of the most important prognostic factors. For this process to occur, epithelial tumor cells need to undergo an epithelial to mesenchymal transition (EMT), either transiently or stably, and to differing degrees. This process has been extensively described in other types of cancer but has been poorly studied in EC. In this review, several features of EMT and the main molecular pathways responsible for triggering this process are investigated in relation to EC. The most common hallmarks of EMT have been found in EC, either at the level of E-cadherin loss or at the induction of its repressors, as well as other molecular alterations consistent with the mesenchymal phenotype-like L1CAM and BMI-1 up-regulation. Pathways including progesterone receptor, TGFβ, ETV5 and microRNAs are deeply related to the EMT process in EC.


Introduction
Endometrial cancer (EC) is made up of a biologically and histologically diverse group of neoplasms characterized by their distinct pathogeneses.These differential characteristics

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have been classified into a dualistic model, first proposed by Bokhman [1].Nowadays, this model is being challenged, since tumors seen in daily practice occasionally show overlapping or combined morphologic and molecular characteristics between both types, in addition to ambiguous features [2].Nevertheless, we still follow this classification as it is currently used in the clinical practice.Type I, or estrogen-dependent endometrioid ECs, represent the most common subtype.This type usually develops in periand early postmenopausal women.It is an estrogen-associated lesion often seen in conjunction with endometrial hyperplasia.The histological subtypes that correspond to endometrioid adenocarcinoma and its variants, as well as mucinous adenocarcinoma, are allocated to this group.Type II, or non-endometrioid ECs, tend to affect older, postmenopausal women and are a non-estrogen-associated lesion.These cancers are not preceded by endometrial hyperplasia, though they can occasionally arise in endometrial polyps or from precancerous lesions, endometrial intraepithelial carcinoma, or in the vicinity of atrophic endometrium [3].The most virulent histologic subtypes, such as papillary serous, clear cell carcinomas, and carcinosarcomas are to be found in this group [4].The clinicopathological differences between the two types are paralleled by specific genetic alterations, with type I showing microsatellite instability and mutations in PTEN, PIK3CA, K-RAS, and CTNNB1 (beta-catenin) and type II exhibiting TP53 mutations and chromosomal instability [5].
Epidemiologically, EC is ranked as the most common gynecologic malignancy of the female genital tract and the fourth most common neoplasia in women after breast, colorectal and lung cancer.Recent data from the US estimates that 47,130 new cases of EC will be diagnosed in 2012, but only 8,010 deaths are expected [6].According to these statistics, EC is considered to be a good prognosis cancer, since it is usually detected in its initial stages by the presentation of disease-related symptoms.In its early stages, EC is confined to the endometrium and can be treated by hysterectomy with or without adjuvant treatment resulting in survival rates around 96 % at 5 years.However, 30 % of all EC cases are still diagnosed at regional or distant stages, and are related to lower survival rates, 67 and 16 %, respectively.When comparing the epidemiologic data of both EC types, type I lesions are generally diagnosed at an earlier stage and possess a more favorable prognosis than those of type II.Generally, type II are more aggressive tumors that are commonly diagnosed in advanced stages and often recur, despite aggressive treatment [7].
In order to tackle the causes of mortality associated with EC, it is necessary to understand how this cancer disseminates.EC spreads by direct extension through the 89 myometrium, in an exfoliation of cells that are shed 90 through the fallopian tubes, lymphatic dissemination, and/ 91 or hematogenous dissemination [8].The most common 92 route of spread is the direct extension of a tumor to the 93 myometrium.In this review, we will focus on the mecha-94 nisms that initiate this local invasion, as this invasion is 95 considered one of the most important prognostic factors for 96 type I and type II ECs, usually correlating with lymphatic 97 spread, risk of recurrence, and overall survival rate.In the 98 multistep process that characterizes myometrial invasion, 99 the initial events are delineated by the dissociation of 100 tumor cells from the epithelial layer of the endometrial 101 glands and the penetration through the basement membrane 102 into the adjacent connective tissue, i.e., the myometrium 103 [7].This process is similar in other types of cancer, and 104 many authors [8,9] have pointed out that for this process to 105 occur, epithelial tumor cells need to undergo an epithelial 106 to mesenchymal (EMT) transition, either transiently or 107 stably, and to differing degrees.Interestingly, Stewart and Little [40] were the first to approach an immunohistochemical identification of the distinctive morphological alterations consistent with EMT features in EC.They managed to characterize the presence of microcystic, elongated and fragmented glands within EC tissues, which they named ''MELFs''.MELFs are specific tumor alterations, which, through immunohistochemical study, can be identified by strong CK7 expression, showing a reduction of E-Cadherin and hormone receptor and increased snail expression.Loss of hormone receptors and decreased E-cadherin immunoreactivity have been reported previously in EC [23,[41][42][43][44] and in breast carcinoma [45,46].Although EMT has been broadly described in EC, the molecular pathways responsible for triggering this process are still poorly delineated for this type of cancer.Here, we will review current literature pertaining to the main effectors of EMT in EC.  [57,58].Up-regulation of the ETV5 gene in EEC was described by Planaguma `282 et al. [59] with a specific and significant increase restricted to those tumor stages associated with myometrial infiltration.Recently, ETV5 has arisen as an inducer of EMT in EC through a main regulatory effect on the Zeb1 promoter, and has also been found to induce EMT in ovarian cancer cell lines [60].Along with ETV5 over-expression in EC cell lines, the main features of EMT were also observed.These included a modulation of cell adhesion, cell-cell contact and cellular junctions, and actin cytoskeleton reorganization.At the same time, main cellular functions were altered, as well as cell-to-cell signaling and interaction and cellular movement, as the principal modified biological processes [61].
Concomitant with EMT, the up-regulation of ETV5 in EC also regulated the MMP2 [62] and HEP27 [63] promoter regions.The former protein is a matrix metalloproteinase, which is primarily responsible for the degradation of the helical domains of type IV collagen, i.e., the principal collagen of basement membranes that allows tumor cells to infiltrate surrounding tissues.The latter is a member of the superfamily of short-chain dehydrogenases/ reductases that protect tumor cells against apoptosis, which is induced by oxidative stress generated during the invasive process.
In our work, we have also identified a number of proteins involved in the acquisition of invasive capabilities by ECs [64], such as lipoma-preferred partner (LPP), which acts as a novel co-regulatory partner of ETV5 in the transcriptional regulation of the EMT process.LPP is reorganized from cell-cell contacts to focal adhesions when EMT is induced by ETV5, and it translocates to the nucleus ahead of external stimuli, establishing cross-talk between the tumor cells and their surrounding microenvironments [61].
Interestingly, our group linked TGFb and progesterone receptor pathways with ETV5 by means of a proteomic analysis that compared EC cells stably over-expressing ETV5 with both control, non-transfected cells and cells that had been transfected with the empty vector [63] (Fig. 2).

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Epithelial to mesenchymal transition (EMT) 109 EMT is a well-described process whereby epithelial cells 110 lose their polarity and cell-cell contacts, undergoing a 111 dramatic remodeling of the cytoskeleton and acquiring a 112 migratory phenotype, which activates a mesenchymal-like 113 gene expression program.The epithelial cells form a sheet 114 or layer of cells that are tightly connected laterally by 115 specialized junction structures, including adherens junc-116 tions, desmosomes, hemidesmosomes, tight junctions and 117 gap junctions.Among these, adherens junctions play a 118 particularly important role in assembling and constructing 119 lateral epithelial cell-cell adhesions.The epithelial cells 120 also establish an aligned, apical-basal polarity through 121 their association with a lamina layer at their basal surface, 122 which ensures that the cells are only able to migrate lat-123 erally, maintaining their position within the epithelium 124 [10].Upon receiving specific signals, EMT occurs.Mes-125 enchymal cells are defined by three major characteristics in 126 their cellular phenotype and their behavior (Fig. 1): (1) a 127 loss of strong epithelial adhesive cell-cell contacts and the 128 acquisition of a dispersed, spindle-shaped morphology with 129 migratory protrusions, (2) changes in the differentiation 130 markers from cell-cell junction proteins, i.e., E-cadherin 131 and cytokeratin intermediate filaments (specific to epithe-132 lial cells) to vimentin filaments and fibronectin, and (3) an 133 increased motility for invasion through the extracellular 134 matrices.All three changes are not necessarily observable 135 during an EMT; however, the single cell acquisition of the 136 ability to migrate and invade the extracellular matrices is 137 considered to be a functional hallmark of EMT.Hence, 138 mesenchymal cells are able to detach, penetrate through the Article No. : 866 h LE h TYPESET MS Code : CLAT-D-12-00875 h CP h DISK 4 Author Proof U N C O R R E C T E D P R O O F 139 basement membrane and infiltrate surrounding tissues to 140 then metastasize at secondary sites [11, 12].To reach 141 successful dissemination, EMT must be triggered in epi-142 thelial cells through an appropriate signaling pathway.143 Cancer cells must also develop complex interactions to 144 integrate stimuli from their surrounding microenviron-145 ments [13, 14].146 Several transcription factors have emerged in recent 147 years that trigger the down-regulation of E-cadherin and, 148 consequently, produce EMT in different types of cancer.149 These transcription factors include the transforming growth 150 factor-b (TGFb), epidermal growth factor (EGF), insulin 151 growth factor 1 (IGF-1), interleukin, vascular endothelial 152 growth factor (VEGF), platelet-derived growth factor 153 (PDGF), integrin/integrin-linked kinase (ILK), notch, 154 fibroblast growth factor (FGF) and Wnt/b-catenin signaling 155 pathways [15-18].Most of these signals exert their action 156 on E-cadherin repression through the modulation of a set of 157 pleiotropically acting transcription factors, including 158 members of Snail (Snail and Slug) and basic helix-loop-159 helix (E47 and Twist) families, as well as two double zinc 160 finger and homeodomain (Zeb1 and Zeb2) factors [9].161 These repressors are expressed in various combinations in a 162 number of malignant tumor types and have been shown in 163 experimental models of carcinoma formation to be causally 164 important for programming invasion [10, 19].Hence, these 165 repressors are recognized as key inducers of EMT.166 EMT in EC invasion 167 In EC, hallmarks of EMT have been reported either at 168 the level of E-cadherin loss or at the induction of its repressors.On one side, Hurt et al. [20] and Singh et al. [21] have described high levels of Zeb1 in relation to gynecologic carcinoma progression, and Kyo et al. [22] have observed a pattern of twist expression preferentially at the 'marginal regions' of ECs, which was inversely correlated with E-cadherin expression.Snail was also proposed to play a role in EC progression and was correlated with reduced estrogen-receptor a expression [23].On the other side, several reports [24, 25] have associated negative E-cadherin expression, increased nuclear b-catenin, and loss of a-catenin with the acquisition of aggressive biological behavior, especially in high-grade tumors.Furthermore, some studies have demonstrated a correlation between reduced E-cadherin and the presence of lymph node metastasis and/or adverse patient outcomes [26-30].Independently of the most common features of EMT described above, other molecular alterations have been consistent with this phenotype.For example, L1CAM expression was inversely correlated to the expression of hormone receptors and E-Cadherin and was found to be associated with poor prognosis [31].L1CAM up-regulation was mimicked in EC cell lines treated with the EMT inducer TGFb1 and blocked when Slug was depleted.In line with this result, L1CAM presents two functionally active promoter sites containing b-catenin/TCF-LEF and E-boxes, respectively, indicating that its expression can be regulated by TGFb1, but also by Wnt/b-catenin [32].Conversely, the over-expression of a self-renewal gene, called BMI-1, has been found in multiple human cancers, including gastric cancer [33], lung cancer [34], breast cancer [35], prostate cancer [36], ovarian cancer [37], and EC [38].BMI-1 up-regulation has been found to contribute to the EMT-derived invasive phenotype in EC cells, and its silencing reverts EMT and reduces EC cell invasion [39].

Fig. 1
Fig. 1 General features of epithelial to mesenchymal transition (EMT).Schematic diagram of the main molecular, morphological and phenotypical changes occurring in EMT

Fig. 2 6
Fig.2Epithelial to mesenchymal transition (EMT) networks in endometrial carcinoma (EC).To obtain the interaction network, we selected genes differentially expressed in EC that have been related to EMT, and we used Ingenuity Pathways Analysis software to plot direct and indirect interactions