Activated leukocyte cell adhesion molecule (ALCAM) is a marker of recurrence and promotes cell migration, invasion, and metastasis in early-stage endometrioid endometrial cancer.

Endometrial cancer is the most common gynaecological cancer in western countries, being the most common subtype of endometrioid tumours. Most patients are diagnosed at an early stage and present an excellent prognosis. However, a number of those continue to suffer recurrence, without means of identification by risk classification systems. Thus, finding a reliable marker to predict recurrence becomes an important unmet clinical issue. ALCAM is a cell-cell adhesion molecule and member of the immunoglobulin superfamily that has been associated with the genesis of many cancers. Here, we first determined the value of ALCAM as a marker of recurrence in endometrioid endometrial cancer by conducting a retrospective multicentre study of 174 primary tumours. In early-stage patients (N = 134), recurrence-free survival was poorer in patients with ALCAM-positive compared to ALCAM-negative tumours (HR 4.237; 95% CI 1.01-17.76). This difference was more significant in patients with early-stage moderately-poorly differentiated tumours (HR 9.259; 95% CI 2.12-53.47). In multivariate analysis, ALCAM positivity was an independent prognostic factor in early-stage disease (HR 6.027; 95% CI 1.41-25.74). Then we demonstrated in vitro a role for ALCAM in cell migration and invasion by using a loss-of-function model in two endometrial cancer cell lines. ALCAM depletion resulted in a reduced primary tumour size and reduced metastatic local spread in an orthotopic murine model. Gene expression analysis of ALCAM-depleted cell lines pointed to motility, invasiveness, cellular assembly, and organization as the most deregulated functions. Finally, we assessed some of the downstream effector genes that are involved in ALCAM-mediated cell migration; specifically FLNB, TXNRD1, and LAMC2 were validated at the mRNA and protein level. In conclusion, our results highlight the potential of ALCAM as a recurrent biomarker in early-stage endometrioid endometrial cancer and point to ALCAM as an important molecule in endometrial cancer dissemination by regulating cell migration, invasion, and metastasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

ALCAM shedding at the invasive front of the tumor is a marker of myometrial infiltration and promotes invasion in endometrioid endometrial cancer.

Endometrial cancer (EC) is the sixth deadliest cancer in women. The depth of myometrial invasion is one of the most important prognostic factors, being directly associated with tumor recurrence and mortality. In this study, ALCAM, a previously described marker of EC recurrence, was studied by immunohistochemistry at the superficial and the invasive tumor areas from 116 EC patients with different degree of myometrial invasion and related to a set of relevant epithelial and mesenchymal markers. ALCAM expression presented a heterogeneous functionality depending on its localization, it correlated with epithelial markers (E-cadherin/ß-catenin) at the superficial area, and with mesenchymal markers at the invasive front (COX-2, SNAIL, ETV5, and MMP-9). At the invasive front, ALCAM-negativity was an independent marker of myometrial invasion. This negativity, together with an increase of soluble ALCAM in uterine aspirates from patients with an invasive EC, and its positive correlation with MMP-9 levels, suggested that ALCAM shedding by MMP-9 occurs at the invasive front. In vivo and in vitro models of invasive EC were generated by ETV5-overexpression. In those, we demonstrated that ALCAM shedding was related to a more invasive pattern and that full-ALCAM recovery reverted most of the ETV5-cells mesenchymal abilities, partially through a p-ERK dependent-manner.

Targeted Proteomics Identifies Proteomic Signatures in Liquid Biopsies of the Endometrium to Diagnose Endometrial Cancer and Assist in the Prediction of the Optimal Surgical Treatment.

Purpose: Endometrial cancer (EC) diagnosis relies on the observation of tumor cells in endometrial biopsies obtained by aspiration (i.e., uterine aspirates), but it is associated with 22% undiagnosed patients and up to 50% of incorrectly assigned EC histotype and grade. We aimed to identify biomarker signatures in the fluid fraction of these biopsies to overcome these limitations.Experimental Design: The levels of 52 proteins were measured in the fluid fraction of uterine aspirates from 116 patients by LC-PRM, the latest generation of targeted mass-spectrometry acquisition. A logistic regression model was used to assess the power of protein panels to differentiate between EC and non-EC patients and between EC histologic subtypes. The robustness of the panels was assessed by the "leave-one-out" cross-validation procedure performed within the same cohort of patients and an independent cohort of 38 patients.Results: The levels of 28 proteins were significantly higher in patients with EC (n = 69) compared with controls (n = 47). The combination of MMP9 and KPYM exhibited 94% sensitivity and 87% specificity for detecting EC cases. This panel perfectly complemented the standard diagnosis, achieving 100% of correct diagnosis in this dataset. Nine proteins were significantly increased in endometrioid EC (n = 49) compared with serous EC (n = 20). The combination of CTNB1, XPO2, and CAPG achieved 95% sensitivity and 96% specificity for the discrimination of these subtypes.Conclusions: We developed two uterine aspirate-based signatures to diagnose EC and classify tumors in the most prevalent histologic subtypes. This will improve diagnosis and assist in the prediction of the optimal surgical treatment. Clin Cancer Res; 23(21); 6458-67. ©2017 AACR.

E-cadherin: A determinant molecule associated with ovarian cancer progression, dissemination and aggressiveness.

Ovarian cancer (OC) is the fifth cancer death cause in women worldwide. The malignant nature of this disease stems from its unique dissemination pattern. Epithelial-to-mesenchymal transition (EMT) has been reported in OC and downregulation of Epithelial cadherin (E-cadherin) is a hallmark of this process. However, findings on the relationship between E-cadherin levels and OC progression, dissemination and aggressiveness are controversial. In this study, the evaluation of E-cadherin expression in an OC tissue microarray revealed its prognostic value to discriminate between advanced- and early-stage tumors, as well as serous tumors from other histologies. Moreover, E-cadherin, Neural cadherin (N-cadherin), cytokeratins and vimentin expression was assessed in TOV-112, SKOV-3, OAW-42 and OV-90 OC cell lines grown in monolayers and under anchorage-independent conditions to mimic ovarian tumor cell dissemination, and results were associated with cell aggressiveness. According to these EMT-related markers, cell lines were classified as mesenchymal (M; TOV-112), intermediate mesenchymal (IM; SKOV-3), intermediate epithelial (IE; OAW-42) and epithelial (E; OV-90). M- and IM-cells depicted the highest migration capacity when grown in monolayers, and aggregates derived from M- and IM-cell lines showed lower cell death, higher adhesion to extracellular matrices and higher invasion capacity than E- and IE-aggregates. The analysis of E-cadherin, N-cadherin, cytokeratin 19 and vimentin mRNA levels in 20 advanced-stage high-grade serous human OC ascites showed an IM phenotype in all cases, characterized by higher proportions of N- to E-cadherin and vimentin to cytokeratin 19. In particular, higher E-cadherin mRNA levels were associated with cancer antigen 125 levels more than 500 U/mL and platinum-free intervals less than 6 months. Altogether, E-cadherin expression levels were found relevant for the assessment of OC progression and aggressiveness.

Development of a sequential workflow based on LC-PRM for the verification of endometrial cancer protein biomarkers in uterine aspirate samples.

About 30% of endometrial cancer (EC) patients are diagnosed at an advanced stage of the disease, which is associated with a drastic decrease in the 5-year survival rate. The identification of biomarkers in uterine aspirate samples, which are collected by a minimally invasive procedure, would improve early diagnosis of EC. We present a sequential workflow to select from a list of potential EC biomarkers, those which are the most promising to enter a validation study. After the elimination of confounding contributions by residual blood proteins, 52 potential biomarkers were analyzed in uterine aspirates from 20 EC patients and 18 non-EC controls by a high-resolution accurate mass spectrometer operated in parallel reaction monitoring mode. The differential abundance of 26 biomarkers was observed, and among them ten proteins showed a high sensitivity and specificity (AUC > 0.9). The study demonstrates that uterine aspirates are valuable samples for EC protein biomarkers screening. It also illustrates the importance of a biomarker verification phase to fill the gap between discovery and validation studies and highlights the benefits of high resolution mass spectrometry for this purpose. The proteins verified in this study have an increased likelihood to become a clinical assay after a subsequent validation phase.

Nidogen 1 and Nuclear Protein 1: novel targets of ETV5 transcription factor involved in endometrial cancer invasion.

Endometrial cancer is the most frequent malignancy of the female genital tract in western countries. Our group has previously characterized the upregulation of the transcription factor ETV5 in endometrial cancer with a specific and significant increase in those tumor stages associated with myometrial invasion. We have shown that ETV5 overexpression in Hec1A endometrial cancer cells induces epithelial to mesenchymal transition resulting in the acquisition of migratory and invasive capabilities. In the present work, we have identified Nidogen 1 (NID1) and Nuclear Protein 1 (NUPR1) as direct transcriptional targets of ETV5 in endometrial cancer cells. Inhibition of NID1 and NUPR1 in ETV5 overexpressing cells reduced cell migration and invasion in vitro and reduced tumor growth and dissemination in an orthotopic endometrial cancer model. Importantly, we confirmed a significant increase of NUPR1 and NID1 protein expression in the invasion front of the tumor compared to their paired superficial zone, concomitant to ETV5 overexpression. Altogether, we conclude that NID1 and NUPR1 are novel targets of ETV5 and are actively cooperating with ETV5 at the invasion front of the tumor in the acquisition of an invasive phenotype to jointly drive endometrial cancer invasion.

Molecular bases of endometrial cancer: new roles for new actors in the diagnosis and the therapy of the disease.

Endometrial carcinoma (EC) is the most commonly diagnosed gynecologic malignancy in the western world. The majority of these cancers are curable, but a subset about 15-20% of endometrial tumors exhibits an aggressive phenotype. Based on clinic-pathological and molecular characteristics, EC has been classified into two groups: Type I estrogen-dependent adenocarcinomas, which have a good prognosis and an endometrioid histology, and Type II or non-estrogen-dependent EC associated with poor prognosis and non-endometrioid histology. EC develops as a result of a stepwise accumulation of alterations that seem to be specific of each histological type. However, more knowledge is needed to better understand the differences in the biology and the clinical outcome of EC. We would like to highlight the need to explore new potential biomarkers of EC as a tool for the detection and monitoring of aggressive endometrial tumors that, at the same time, will allow us to develop novel and more selective molecular targeted therapies against EC.

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, TGFß, ETV5 and microRNAs are deeply related to the EMT process in EC.