Marta Monge

Novel molecular profiles of endometrial cancer-new light through old windows.

Endometrial carcinoma (EC) is the most common gynecological malignancy in the western world. A widely accepted dualistic model, which has been established on a morphological basis, differentiates EC into two broad categories: Type I oestrogen-dependent adenocarcinoma with an endometrioid morphology and Type II non-oestrogen-dependent EC with a serous papillary or clear cell morphology. Molecular genetic evidence indicates that endometrial carcinoma, as described in other malignancies, likely develops as the result of a stepwise accumulation of alterations in cellular regulatory pathways, such as oncogene activation and tumor suppressor gene inactivation, which lead to dysfunctional cell growth. These molecular alterations appear to be specific in Type I and Type II cancers. In type I endometrioid endometrial cancer, PTEN gene silencing in conjunction with defects in DNA mismatch repair genes, as evidenced by the microsatellite instability phenotype, or mutations in the K-ras and/or beta-catenin genes, are recognized major alterations, which define the progression of the normal endometrium to hyperplasia, to endometrial intraepithelial neoplasia, and then on to carcinoma. In contrast, Type II cancers show mutations of TP53 and Her-2/neu and seem to arise from a background of atrophic endometrium. Nevertheless, despite the great effort made to establish a molecularly-based histological classification, the following issues must still be clarified: what triggers the tumor cells to invade the myometrium and what causes vascular or lymphatic dissemination, finally culminating in metastasis? RUNX1, a transcription factor, was recently identified as one of the most highly over-expressed genes in a microarray study of invasive endometrial carcinoma. Another candidate gene, which may be associated with an initial switch to myometrial infiltration, is the transcription factor ETV5/ERM. These studies, as well as those conducted for other genes possibly involved in the mitotic checkpoint as a major mechanism of carcinogenesis in non-endometrioid endometrial cancer, could help in understanding the differences in the biology and the clinical outcome among histological types.

A Differential Gene Expression Profile Reveals Overexpression of RUNX1/AML1 in Invasive Endometrioid Carcinoma

Endometrial carcinoma is the most common gynecological malignant disease in industrialized countries. Two clinicopathological types of endometrial carcinoma have been described, based on estrogen relation and grade: endometrioid carcinoma (EEC) and non-EEC (NEEC). Some of the molecular events that occur during the development of endometrial carcinoma have been characterized, showing a dualistic genetic model for EEC and NEEC. However, the molecular bases for endometrial tumorigenesis are not clearly elucidated. In the present work, we attempted to identify new genes that could trigger cell transformation in EEC. We analyzed the differential gene expression profile between tumoral and nontumoral endometrial specimens with cDNA array hybridization. Among the 53 genes for which expression was found to be altered in EEC, the acute myeloid leukemia proto-oncogene, RUNX1/AML1, was one of the most highly up-regulated. The gene expression levels of RUNX1/AML1 were quantified by real-time quantitative PCR, and protein levels were characterized by tissue array immunohistochemistry. Real-time quantitative PCR validated RUNX1/AML1 up-regulation in EEC and demonstrated a specific and significantly stronger up-regulation in those tumor stages associated with myometrial invasion. Furthermore, tissue array immunohistochemistry showed that RUNX1/AML1 up-regulation correlates to the process of tumorigenesis, from normal atrophic endometrium to simple and complex hyperplasia and then, on to carcinoma. These results demonstrate for the first time the up-regulation of RUNX1/AML1 in EEC correlating with the initial steps of myometrial infiltration.

Molecular markers of endometrial carcinoma detected in uterine aspirates.

Endometrial cancer (EC) is the most frequent of the invasive tumors of the female genital tract. Although usually detected in its initial stages, a 20% of the patients present with advanced disease. To date, no characterized molecular marker has been validated for the diagnosis of EC. In addition, new methods for prognosis and classification of EC are needed to combat this deadly disease. We thus aimed to identify new molecular markers of EC and to evaluate their validity on endometrial aspirates. Gene expression screening on 52 carcinoma samples and series of real‐time quantitative PCR validation on 19 paired carcinomas and normal tissue samples and on 50 carcinoma and noncarcinoma uterine aspirates were performed to identify and validate potential biomarkers of EC. Candidate markers were further confirmed at the protein level by immunohistochemistry and Western blot. We identified ACAA1, AP1M2, CGN, DDR1, EPS8L2, FASTKD1, GMIP, IKBKE, P2RX4, P4HB, PHKG2, PPFIBP2, PPP1R16A, RASSF7, RNF183, SIRT6, TJP3, EFEMP2, SOCS2 and DCN as differentially expressed in ECs. Furthermore, the differential expression of these biomarkers in primary endometrial tumors is correlated to their expression level in corresponding uterine fluid samples. Finally, these biomarkers significantly identified EC with area under the receiver‐operating‐characteristic values ranging from 0.74 to 0.95 in uterine aspirates. Interestingly, analogous values were found among initial stages. We present the discovery of molecular biomarkers of EC and describe their utility in uterine aspirates. These findings represent the basis for the development of a highly sensitive and specific minimally invasive method for screening ECs.

Proteomic approach to ETV5 during endometrial carcinoma invasion reveals a link to oxidative stress

Endometrial cancer, the most common gynecological malignancy in western countries, is characterized by a favorable prognosis. Nonetheless, deep myometrial invasion correlates with more undifferentiated tumors, lymph-vascular invasion, node involvement and decreased global survival. We have described previously the Ets family member ERM/ETV5 specifically upregulated in endometrial endometrioid carcinoma (EEC) associated with myometrial infiltration. To understand the role of this transcription factor during myometrial infiltration, we analyzed by two-dimension differential gel electrophoresis (2D-DIGE) technology those proteins whose expression was altered in endometrial cell lines stably overexpressing ERM/ETV5. Pathway analysis pointed to actin regulation and transforming growth factor beta and progesterone signaling as processes regulated by ERM/ETV5. In addition, we characterized the specific upregulation of the nuclear dehydrogenase/reductase Hep27 as well as its ERM/ETV5-dependent mitochondrial localization. Further functional studies demonstrated a protective role of Hep 27 against apoptosis induced by oxidative stress. Overall, the ETV5-related proteomic approach performed in the Hec-1A cell line reinforces a role of this transcription factor in the regulation of the migratory and invasive tumor behavior and points to a modulated response to oxidative stress associated with the promotion of invasion in endometrial cancer. Unraveling the molecular events in EEC associated with the initiation of tumor invasion would represent an obvious improvement in the pursuit of rational targets for the onset of metastasis. This knowledge would also be a valuable tool for the molecular stratification of patients since myometrial affectation determines an increase in the rate of recurrence after a first surgical treatment and a decrease in 5 year survival.

ERM/ETV5 Up-regulation Plays a Role during Myometrial Infiltration through Matrix Metalloproteinase-2 Activation in Endometrial Cancer

We have described recently the Ets family transcription factor, ERM/ETV5, specifically up-regulated in endometrioid endometrial carcinoma (EEC) and associated with myometrial infiltration. Ets family members have been correlated to tumor progression by up-regulating the expression of matrix-degrading proteases. In the present study, we investigated the possibility that in EEC, ERM/ETV5 may act by inducing the expression of genes involved in extracellular matrix remodeling. Unraveling the molecular events associated with the initiation of tumor invasion would represent an obvious improvement for EEC patients. The overexpression of ERM/ETV5 induced scattering in the endometrial cancer cell line Hec-1A, correlating to increased matrix metalloproteinase-2 (MMP-2) gelatinase activity. Both chromatin immunoprecipitation and reversion experiments with RNA interference and specific MMP-2 inhibitor showed a functional link between ERM/ETV5 overexpression and MMP-2 activation. The increased MMP-2 activity associated with overexpressed ERM/ETV5 in a mouse model conferred invasive capacity to endometrial tumors. Orthotopically implanted overexpressing ERM/ETV5 tumors presented a more aggressive and infiltrative pattern of myometrial invasion. Finally, the specific localization of ERM/ETV5 and MMP-2 at the invasive front of myometrial infiltrating human endometrial carcinomas further reinforced the hypothesis of a role for ERM/ETV5 in the early steps of endometrial dissemination. Taken together, these results lead us to propose that in EEC, ERM/ETV5 acts through MMP-2 gelatinolytic activity to confer invasive capabilities, associated with an initial switch to myometrial infiltration. They also postulate ERM/ETV5 as a valuable marker for patient stratification and a transcription pathway that should be evaluated for therapies specifically targeting the initial steps of EEC dissemination.

An orthotopic endometrial cancer mouse model demonstrates a role for RUNX1 in distant metastasis

Endometrial carcinoma is the most common malignancy of the female genital tract in industrialized countries. Metastasis is the major cause of endometrial cancer deaths. Therefore, there is a vital need for clinically relevant in vivo models allowing the elucidation of the molecular and cellular mechanisms underlying metastatic behavior. In this study, we describe an innovative experimental orthotopic model of human endometrial carcinoma. Implantation in the bifurcation of the uterine horns resulted in tumors integrated into the myometrial compartment, which can be used and further exploited for the study of in vivo angiogenesis, myometrial invasion, and the metastatic capacity of endometrial cancer cells. This orthotopic model also represents a suitable tool to analyze how tumorigenesis and distant metastasis of endometrial cancer might be influenced by gene alteration, by modulating its expression in the original cancer cell line. One of the candidate genes implicated in endometrial cancer is the transcription factor RUNX1. The over‐expression of RUNX1 in the endometrial cancer cell line HEC1A and the transplantation of these cells to the uterus of nude mice were associated specifically with distant metastasis in the lung. RUNX1 plays a role in the establishment of metastases in endometrial cancer. Translated to the clinics, these models would be equivalent to an advanced undifferentiated carcinoma with node affectation (stage IIIC) and distant metastasis (stage IVB). These patients would be candidates for adjuvant therapy, not efficient until today, and therefore, our models are actually suitable for the design and evaluation of experimental therapies.

Up-regulation of ERM/ETV5 correlates with the degree of myometrial infiltration in endometrioid endometrial carcinoma.

To elucidate alterations in gene expression in endometrioid endometrial carcinoma (EEC), differential gene expression profiling was previously described in both tumour and non‐tumour contexts, and the up‐regulation of the RUNX1/AML1 proto‐oncogene in EEC was characterized. Among the set of genes found to be up‐regulated significantly in EEC, the most relevant, ERM/ETV5, corresponds to the PEA3 subfamily and is a member of the Ets family of transcription factors that contain the Ets DNA‐binding domain and are involved in matrix remodelling. In the present work, an attempt was made to characterize the expression of ERM/ETV5 in EEC throughout the process of tumourigenesis. Gene expression levels of ERM/ETV5 were quantified by real‐time quantitative PCR (RT‐Q‐PCR) using a large panel of samples ranging from non‐invasive IA to metastatic IIIA stages, and protein expression was characterized by tissue array immunohistochemistry (TMA). RT‐Q‐PCR validated ERM/ETV5 up‐regulation in EEC and demonstrated a specific and significant increase restricted to those tumour stages associated with myometrial invasion. TMA showed that ERM/ETV5 up‐regulation correlated mainly with the transition from atrophic endometrium to hyperplasia and carcinoma during tumour progression. Furthermore, ERM/ETV5 gene and protein expression levels were associated with low tumour grade. Finally, ERM/ETV5 up‐regulation correlated with that of RUNX1/AML1. All of these results lead to the proposal of a co‐operative role between ERM/ETV5 and RUNX1/AML1 during the early events of endometrial tumourigenesis, which may be associated with a switch to myometrial infiltration. Copyright

ETV5 cooperates with LPP as a sensor of extracellular signals and promotes EMT in endometrial carcinomas

Endometrial carcinoma (EC) is the most frequent among infiltrating tumors of the female genital tract, with myometrial invasion representing an increase in the rate of recurrences and a decrease in survival. We have previously described ETV5 transcription factor associated with myometrial infiltration in human ECs. In this work, we further investigated ETV5 orchestrating downstream effects to confer the tumor the invasive capabilities needed to disseminate in the early stages of EC dissemination. Molecular profiling evidenced ETV5 having a direct role on epithelial-to-mesenchymal transition (EMT). In particular, ETV5 modulated Zeb1 expression and E-Cadherin repression leading to a complete reorganization of cell–cell and cell–substrate contacts. ETV5-promoted EMT resulted in the acquisition of migratory and invasive capabilities in endometrial cell lines. Furthermore, we identified the lipoma-preferred partner protein as a regulatory partner of ETV5, acting as a sensor for extracellular signals promoting tumor invasion. All together, we propose ETV5-transcriptional regulation of the EMT process through a crosstalk with the tumor surrounding microenvironment, as a principal event initiating EC invasion.

Subtractive proteomic approach to the endometrial carcinoma invasion front.

Tumor invasion defines the transition between tissue-restricted carcinomas, related to good outcome as optimal surgery becomes possible, and metastatic tumors associated with poor prognosis and a dramatic decrease in survival. In endometrial cancer, myometrial infiltration represents a determinant parameter highly valuable in prognosis. To date, the identification of proteins involved in endometrial carcinoma invasion has been essentially conducted by immunohistochemical methods, without a global perception on the invasive front. Laser microdissection presents nowadays limitations to the profound spatiotemporal regulation from both the tumor and the surrounding stroma occurring at the invasive front. In this work, we attempted an alternative proteomic approach to characterize specific components of the tumor invasive front or its reactive stroma, by comparing the invasive area of an endometrial carcinoma with the noninvasive superficial tumor area and normal tissue from the same patients. This strategy led us to identify proteins involved in cellular morphology, assembly and movement, differentially expressed at the invasive front, as well as pathways like cell-to-cell signaling and interaction and a modulated response to oxidative stress as events related to endometrial carcinoma invasion. In conclusion, we could identify new players of myometrial infiltration by applying a subtractive proteomic approach to the endometrial carcinoma invasion front.

Annexin-A2 as predictor biomarker of recurrent disease in endometrial cancer

Endometrial carcinomas, the most common malignant tumour of the female genital tract, are usually diagnosed at an early stage with uterine‐confined disease and an overall favourable prognosis. However, up to 20% of endometrial carcinomas will end up in recurrent disease, associated with a drop in survival and representing the major clinical challenge. Management of this group of risk patients relies on robust biomarkers that may predict which endometrial carcinomas will relapse. For this, we performed a proteomic analysis comparing primary lesions with recurrences and identified ANXA2 as a potential biomarker associated with recurrent disease that we further validated in an independent series of samples by immunohistochemistry. We demonstrated in vitro a role for ANXA2 in the promotion of metastasis rather than interfering with sensitivity to radio/chemotherapy. In addition, ANXA2 silencing resulted in a reduced metastatic pattern in a mice model of endometrial cancer dissemination, with a limited presence of circulating tumor cells. Finally, a retrospective study in a cohort of 93 patients showed that ANXA2 effectively predicted those endometrioid endometrial carcinomas that finally recurred. Importantly, ANXA2 demonstrated a predictive value also among low risk Stage I endometrioid endometrial carcinomas, highlighting the clinical utility of ANXA2 biomarker as predictor of recurrent disease in endometrial cancer. Retrospective and prospective studies are ongoing to validate ANXA2 as a potential tool for optimal stratification of patients susceptible to receive radical surgery and radio/chemotherapy.