Urinary biomarkers for the detection of prostate cancer in patients with high-grade prostatic intraepithelial neoplasia.

INTRODUCTION: High-grade prostatic intraepithelial neoplasia (HGPIN) is a recognized precursor stage of PCa. Men who present HGPIN in a first prostate biopsy face years of active surveillance including repeat biopsies. This study aimed to identify non-invasive prognostic biomarkers that differentiate early on between indolent HGPIN cases and those that will transform into actual PCa. METHODS: We measured the expression of 21 candidate mRNA biomarkers using quantitative PCR in urine sediment samples from a cohort of 90 patients with initial diagnosis of HGPIN and a posterior follow up of at least two years. Uni- and multivariate statistical analyses were applied to analyze the candidate biomarkers and multiplex models using combinations of these biomarkers. RESULTS: PSMA, PCA3, PSGR, GOLM, KLK3, CDH1, and SPINK1 behaved as predictors for PCa presence in repeat biopsies. Multiplex models outperformed (AUC = 0.81-0.86) the predictive power of single genes, including the FDA-approved PCA3 (AUC = 0.70). With a fixed sensitivity of 95%, the specificity of our multiplex models was of 41-58%, compared to the 30% of PCA3. The PPV of our models (30-38%) was also higher than the PPV of PCA3 (27%), suggesting that benign cases could be more accurately identified. Applying statistical models, we estimated that 33% to 47% of repeat biopsies could be prevented with a multiplex PCR model, representing an easy applicable and significant advantage over the current gold standard in urine sediment. DISCUSSION: Using multiplex RTqPCR-based models in urine sediment it is possible to improve the current diagnostic method of choice (PCA3) to differentiate between benign HGPIN and PCa cases.

Cyclooxygenase-2 inhibitor suppresses tumour progression of prostate cancer bone metastases in nude mice.

OBJECTIVE: To assess whether celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor with anti-cancer properties, has an inhibitory effect on tumour establishment and progression of prostate cancer (PCa) bone metastases. MATERIALS AND METHODS: PC-3 stable luciferase-expressing cells were injected into male nude mice by intracardiac (i.c.) and intratibial (i.t.) injections, and the effect of celecoxib on bone metastases was then recorded using bioluminescence image analysis. In cases of chemoprevention, mice received 3 mg/kg celecoxib from 1 week before cell implantation until the end of the study, and to test the therapeutic effect, mice received celecoxib 1 week after cell implantation until the end of the study. Tumour tissue samples were histologically examined and COX-2 expression was quantified at the protein level. RESULTS: Celecoxib significantly decreased cell viability and the proliferation of human PCa cells in vitro in a dose-dependent manner. Bone metastases were detected after i.c. injection in nude mice. Celecoxib (15 ppm) administered before i.c. injection did not inhibit the cellular metastatic potential, as the numbers of bone metastases were similar in both groups. However, celecoxib did decrease metastatic progression in the osseous environment when cells were injected directly into the tibia (P < 0.05). At the protein level, COX-2 expression was significantly decreased in the celecoxib treatment group (P < 0.01). CONCLUSION: In a preclinical mice model, celecoxib administered orally at the standard human dose inhibits the progression of established PCa bone metastases.

Role of serum cholesterol and statin use in the risk of prostate cancer detection and tumor aggressiveness.

The aim of this study was to analyze the relationship between statin use along with serum cholesterol levels and prostate cancer (PCa) detection and aggressiveness. Statin users of three years or more and serum cholesterol levels (SC) were assessed in 2408 men scheduled for prostate biopsy. SC was classified as normal (NSC: <200 mg/dL) or high (HSC: >200 mg/dL). High-grade PCa (HGPCa) was considered if the Gleason score was greater than 7. Statin users comprised 30.9% of those studied. The PCa detection rate was 31.2% of men on statins and 37% of non-statin users (p<0.006). The PCa detection rate was 26.3% in men with NSC and 40.6% in those with HSC (p<0.001). In the subset of NSC men, the PCa rate was 26.5% for statin users and 26.2% for non-users (p=0.939), while in men with HSC, the PCa rate was 36.4% for statin users and 42.0% for non-statin users (p=0.063). The HGPCa rate was 41.8% for statin users and 32.5% for non-users (p=0.012). NSC men had a 53.8% rate of HGPCa, while the rate was only 27.6% in HSC men (p<0.001). NSC men on statins had an HGPCa rate of 70.2%, while non-statin users had a rate of 41.2% (p<0.001). The HGPCa rate for HSC men on statins was 18.8%, while the rate was 30.0% (p=0.011) for non-users. Logistic regression analysis suggested that serum cholesterol levels could serve as an independent predictor of PCa risk, OR 1.87 (95% CI 1.56-2.24) and HGPCa risk, OR 0.31 (95% CI 0.23-0.44), while statin usage could not. Statin treatment may prevent PCa detection through serum cholesterol-mediated mechanisms. A disturbing increase in the HGPCa rate was observed in statin users who normalized their serum cholesterol.

Molecular diagnosis of endometrial cancer from uterine aspirates.

Rapid and reliable diagnosis of endometrial cancer (EC) in uterine aspirates is highly desirable. Current sensitivity and failure rate of histological diagnosis limit the success of this method and subsequent hysteroscopy is often necessary. Using quantitative reverse transcriptase-polymerase chain reaction on RNA from uterine aspirates samples, we measured the expression level of 20 previously identified genes involved in EC pathology, created five algorithms based on combinations of five genes and evaluated their ability to diagnose EC. The algorithms were tested in a prospective, double-blind, multicenter study. We enlisted 514 patients who presented with abnormal uterine bleeding. EC was diagnosed in 60 of the 514 patients (12%). Molecular analysis was performed on the remnants of aspirates and results were compared to the final histological diagnoses obtained through biopsies acquired by aspiration or guided by hysteroscopy, or from the specimens resected by hysterectomy. Algorithm 5 was the best performing molecular diagnostic classifier in the case-control and validation study. The molecular test had a sensitivity of 81%, specificity of 96%, positive predictive value (PPV) of 75% and negative predictive value (NPV) of 97%. A combination of the molecular and histological diagnosis had a sensitivity of 91%, specificity of 97%, PPV of 79% and NPV of 99% and the cases that could be diagnosed on uterine aspirate rose from 76 to 93% when combined with the molecular test. Incorporation of the molecular diagnosis increases the reliability of a negative diagnosis, reduces the need for hysteroscopies and helps to identify additional cases.

Metabolic syndrome increases the risk of aggressive prostate cancer detection.

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Metabolic syndrome can identify patients at high risk of cardiovascular disease. The prevalence of metabolic syndrome is increasing worldwide and is associated with increased age, obesity and hypogonadism. The association between metabolic syndrome and prostate cancer development has not been studied comprehensively, and published studies report divergent results. This study indicates that tumours detected in men with metabolic syndrome are more aggressive than those detected in men without this condition. OBJECTIVE: To further examine the association between metabolic syndrome (MS), prostate cancer (PC) detection risk and tumour aggressiveness. PATIENTS AND METHODS: From 2006 to 2010, 2408 men not receiving 5α-reductase inhibitors were scheduled for prostatic biopsy due to PSA above 4 ng/mL and/or abnormal digital rectal examination. MS was evaluated according to the National Cholesterol Education Program Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults, Adult Treatment Panel III definition. Tumour aggressiveness was evaluated through biopsy Gleason score, clinical stage and risk of biochemical recurrence after primary treatment. RESULTS: The rates of PC detection were 34.5% and 36.4% respectively in men with and without MS, P = 0.185. High grade PC rates (Gleason score 8-10) were 35.9% and 23.9% respectively, P < 0.001. The advanced disease rates (cT3-4 N0-1 M0-1) were 17% and 12.7% respectively, P = 0.841. The high risk PC rates (cT2c-4 or Gleason score 8-10 or PSA > 20) were 38.5% and 33.0% respectively, P = 0.581. Multivariate analysis confirmed that MS was not associated with the risk of PC detection but it was associated with an increased risk of high grade tumours (odds ratio 1.75, 95% CI 1.26-2.41), P < 0.001. CONCLUSION: MS seems not be associated with an increased risk of PC detection but it is associated with an increased risk of more aggressive tumours.

The present and future of prostate cancer urine biomarkers.

In order to successfully cure patients with prostate cancer (PCa), it is important to detect the disease at an early stage. The existing clinical biomarkers for PCa are not ideal, since they cannot specifically differentiate between those patients who should be treated immediately and those who should avoid over-treatment. Current screening techniques lack specificity, and a decisive diagnosis of PCa is based on prostate biopsy. Although PCa screening is widely utilized nowadays, two thirds of the biopsies performed are still unnecessary. Thus the discovery of non-invasive PCa biomarkers remains urgent. In recent years, the utilization of urine has emerged as an attractive option for the non-invasive detection of PCa. Moreover, a great improvement in high-throughput "omic" techniques has presented considerable opportunities for the identification of new biomarkers. Herein, we will review the most significant urine biomarkers described in recent years, as well as some future prospects in that field.

[Molecular biology of bone metastases]. / Biología molecular de las metástasis óseas.

Bone metastases are a frequent and devastating complication in cancer patients. Recently, significant advances in our understanding of the molecular mechanisms responsible for both osteolytic and osteoblastic bone metastases have occurred. The use of OMICS and the availability of appropriate preclinical animal models of bone metastasis have permitted the identification of factors produced by the tumor or by the host stroma in response to the tumor. These types of studies should result in a decrease of the serious skeletal morbidities associated with metastatic prostate cancer and may in the future improve overall survival. In this review the next generation of molecular targets in bone metastasis will be summarized.

A logistic model for the detection of circulating tumour cells in human metastatic colorectal cancer.

The accuracy in the diagnosis of metastatic colorectal cancer (mCRC) represents one of the challenges in the clinical management of patients. The detection of circulating tumour cells (CTC) is becoming a promising alternative to current detection techniques, as it focuses on one of the players of the metastatic disease and it should provide with more specific and sensitive detection rates. Here, we describe an improved method of detection of CTC from mCRC patients by combining immune-enrichment, optimal purification of RNA from very low cell numbers, and the selection of accurate PCR probes. As a result, we obtained a logistic model that combines GAPDH and VIL1 normalized to CD45 rendering powerful results in the detection of CTC from mCRC patients (AUROC value 0.8599). We further demonstrated the utility of this model at the clinical setting, as a reliable prognosis tool to determine progression-free survival in mCRC patients. Overall, we developed a strategy that ameliorates the specificity and sensitivity in the detection of CTC, resulting in a robust and promising logistic model for the clinical management of metastatic colorectal cancer patients.

ETV5 transcription factor is overexpressed in ovarian cancer and regulates cell adhesion in ovarian cancer cells.

Epithelial ovarian cancer is the most lethal gynecological malignancy and the fifth leading cause of cancer deaths in women in the Western world. ETS transcription factors are known to act as positive or negative regulators of the expression of genes that are involved in various biological processes, including those that control cellular proliferation, differentiation, apoptosis, tissue remodeling, angiogenesis and transformation. ETV5 belongs to the PEA3 subfamily. PEA3 subfamily members are able to activate the transcription of proteases, matrix metalloproteinases and tissue inhibitor of metalloproteases, which is central to both tumor invasion and angiogenesis. Here, we examined the role of the ETV5 transcription factor in epithelial ovarian cancer and we found ETV5 was upregulated in ovarian tumor samples compared to ovarian tissue controls. The in vitro inhibition of ETV5 decreased cell proliferation in serum-deprived conditions, induced EMT and cell migration and decreased cell adhesion to extracellular matrix components. ETV5 inhibition also decreased cell-cell adhesion and induced apoptosis in anchorage-independent conditions. Accordingly, upregulation of ETV5 induced the expression of cell adhesion molecules and enhanced cell survival in a spheroid model. Our findings suggest that the overexpression of ETV5 detected in ovarian cancer cells may contribute to ovarian tumor progression through the ability of ETV5 to enhance proliferation of ovarian cancer cells. In addition, upregulation of ETV5 would play a role in ovarian cancer cell dissemination and metastasis into the peritoneal cavity by protecting ovarian cancer cells from apoptosis and by increasing the adhesion of ovarian cancer cells to the peritoneal wall through the regulation of cell adhesion molecules.

Regional distribution of neuroendocrine cells in the urogenital duct system of the male rat.

BACKGROUND: Neuroendocrine (NE) cells are frequently present in the human prostate and urethra, whereas they are lacking in the other urogenital organs. This study was undertaken as there are only few detailed studies available on the distribution, form and function of NE cells and the structure of excretory ducts of the accessory sex organs in the male rat. METHODS: Systematic gross anatomical dissections were combined with immunohistochemical and electron microscopic studies of the excretory ducts of the urogenital glands in male rats, with particular focus on the distribution and ultrastructure of the NE cells. RESULTS: The topography and structure of the excretory ducts of the different glands were characterized in detail and analyzed for the distribution of NE cells. These are present (in falling frequencies) in the ducts of seminal vesicles and ventral and lateral prostate and are rare in ducts of coagulating gland, dorsal prostate, urethral epithelium, and excretory ducts of the (bulbo) urethral glands. They are absent in the respective glands proper, the deferent duct and ejaculatory ampulla. Approximately 40% of the NE cells of the ventral prostate ducts are of the "open" type, whereas these are less frequent (14%) in the seminal vesicle ducts, where the "closed" type prevails. CONCLUSIONS: NE cells are present in unequal quantities in the excretory ducts of the accessory sex glands, but they are absent in the glands proper and the deferent ducts. This distribution pattern points to a strictly localized function and differentiation potency of NE precursor cells.

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.

A three-gene panel on urine increases PSA specificity in the detection of prostate cancer.

BACKGROUND: Several studies have demonstrated the usefulness of monitoring an RNA transcript, such as PCA3, in post-prostate massage (PM) urine for increasing the specificity of prostate-specific antigen (PSA) in the detection of prostate cancer (PCa). However, a single marker may not necessarily reflect the multifactorial nature of PCa. METHODS: We analyzed post-PM urine samples from 154 consecutive patients, who presented for prostate biopsies because of elevated serum PSA (>4 ng/ml) and/or abnormal digital rectal exam. We tested whether the putative PCa biomarkers PSMA, PSGR, and PCA3 could be detected by quantitative real-time PCR in post-PM urine sediment. We combined these findings to test if a combination of these biomarkers could improve the specificity of actual diagnosis. Afterwards, we specifically tested our model for clinical usefulness in the PSA diagnostic "gray zone" (4-10 ng/ml) on a target subset of 82 men with no prior biopsy. RESULTS: By univariate analysis, we found that the PSMA, PSGR, and PCA3 scores were significant predictors of PCa. Using a multiplex model, the area under the multi receiver-operating characteristic curve was 0.74 versus 0.82 in the diagnostic "gray zone." Fixing the sensitivity at 96%, we obtained a specificity of 34% and 50% in the gray zone. CONCLUSIONS: Taken together, these results provide a strategy for the development of a more accurate model for PCa diagnosis. In the future, a multiplexed, urine-based diagnostic test for PCa with a higher specificity, but the same sensitivity as the serum-PSA test, could be used to determine better which patients should undergo biopsy.

33% radius evaluation to assess bone mineral density in prostate cancer patients.

PURPOSE: Dual-energy X-ray absorptiometry (DXA) is the standard method to assess bone mineral density (BMD). The International Society for Clinical Densitometry recommends the measurement of BMD at lumbar spine, total hip and femoral neck, but in certain circumstances the 33% radius may be the recommended area to measure BMD. The aim of this study has been to analyze whether 33% radius should be considered the recommended area to assess BMD in prostate cancer patients. METHODS: This is a retrospective study where BMD was assessed by DXA at 33% radius, lumbar spine, total hip, and femoral neck (cDXA) in 141 prostate cancer patients. Twenty-eight patients were hormone naïve while 113 were subjected to androgen suppression (AS) during the mean period of 29 months. Osteoporosis was diagnosed when T-score was lower than -2.5 and osteopenia when it ranged between -1 and -2.5. RESULTS: The osteoporosis rate was 29.8% at 33% radius, 23.4% at femoral neck, 19.9% at lumbar spine, and 12.8% at total hip. The overall osteoporosis rate at cDXA was 29.1%. Osteoporosis was detected in 52.2% at 33% radius and 36.2% at cDXA. Normal BMD was found in 17.7% at 33% radius and 34.8% at cDXA. The 33% radius was the only site where a significant increase in the osteoporosis rate was detected in patients subjected to AS compared to those hormone naïve (33 and 13.8%). CONCLUSIONS: The 33% radius seems more sensible than the central skeleton areas to detect bone mass loss in patients with prostate cancer.

Acute inflammation promotes early cellular stimulation of the epithelial and stromal compartments of the rat prostate.

BACKGROUND: It has been proposed that prostatic inflammation plays a pivotal role in the pathophysiology of benign hyperplasia and prostate cancer. However, little information is available about the prostatic reaction to bacterial compounds in vivo. Our aim was therefore to evaluate the early effects of bacterial infection on rat ventral prostate compartments. METHODS: Using a rat model of acute bacterial prostatitis by Escherichia coli, we analyzed the histological and ultrastructural changes in the prostate at 24, 48, and 72 hr postinfection. Prostatic tissues were immunostained for prostatic binding protein (PBP), ACTA2, ErbB1, and ErbB2 receptors, TUNEL, and markers of cell proliferation. Dot and Western blots for PBP, ACTA2, ErbB1, ErbB2, and TGFbeta1 were also performed. RESULTS: The prostatic epithelium became hypertrophied, with increases in PBP and ErbB1 expression at 24 hr postinfection. Moreover, inflammation induced the expression of ErbB2, a receptor strongly involved in carcinogenesis. These alterations were more pronounced at 48 hr, but the epithelium also showed apoptosis and finally atrophy at 72 hr postinfection, with a decrease in PBP and ErbB receptors. Interestingly, the epithelial cells exhibited a high level of proliferation in response to the bacteria. The stromal reaction to acute inflammation was initially characterized by smooth muscle hypertrophy. Afterwards, muscle cells acquired a secretory phenotype, with a reduction in ACTA2 at 72 hr postinfection. CONCLUSIONS: Prostatic inflammation, even at the early stages, promotes atrophic and proliferative changes, and the upregulation of ErbB receptors together with dedifferentiation of smooth muscle cells. These data suggest that repetitive reinfections could lead to uncontrolled growth in the prostate gland.

PSGR and PCA3 as biomarkers for the detection of prostate cancer in urine.

BACKGROUND: Several studies have demonstrated the usefulness of monitoring an RNA transcript in urine, such as PCA3, for prostate cancer (PCa) diagnosis. PCa screening would benefit from additional biomarkers of higher specificity and could be used in conjunction with prostate-specific antigen (PSA) testing, in order to better determine biopsy candidates. METHODS: We used urine sediments after prostate massage (PM) from 215 consecutive patients, who presented for prostate biopsy. We tested whether prostate-specific G-protein coupled receptor (PSGR), a biomarker previously described to be over-expressed in PCa tissue, could also be detected by quantitative real-time PCR in post-PM urine sediment. We combined these findings with prostate cancer gene 3 (PCA3), the current gold standard for PCa diagnosis in urine, to test if a combination of both biomarkers could improve the sensitivity of PCA3 alone. RESULTS: By univariate analysis we found that PSGR and PCA3 were significant predictors of PCa. Receiver operator characteristic curve analysis and its multivariate extension, multivariate ROC (MultiROC), were used to assess the outcome predictive values of the individual and the paired biomarkers. We obtained the following area under the curve values: PSA (0.602), PSGR (0.681), PCA3 (0.656), and PSGRvPCA3 (0.729). Then, we tested whether a combination of PSGR and PCA3 could improve specificity by fixing the sensitivity at 95%. We obtained specificities of 15% (PSGR), 17% (PCA3), and 34% (PSGRvPCA3). CONCLUSIONS: A multiplexed model including PSGR and PCA3 improves the specificity for the detection of PCa, especially in the area of high sensitivity. This could be clinically useful for determining which patients should undergo biopsy.

Behavior of the PCA3 gene in the urine of men with high grade prostatic intraepithelial neoplasia.

OBJECTIVE: An ideal marker for the early detection of prostate cancer (PCa) should also differentiate between men with isolated high grade prostatic intraepithelial neoplasia (HGPIN) and those with PCa. Prostate Cancer Gene 3 (PCA3) is a highly specific PCa gene and its score, in relation to the PSA gene in post-prostate massage urine (PMU-PCA3), seems to be useful in ruling out PCa, especially after a negative prostate biopsy. Because PCA3 is also expressed in the HGPIN lesion, the aim of this study was to determine the efficacy of PMU-PCA3 scores for ruling out PCa in men with previous HGPIN. PATIENTS AND METHODS: The PMU-PCA3 score was assessed by quantitative PCR (multiplex research assay) in 244 men subjected to prostate biopsy: 64 men with an isolated HGPIN (no cancer detected after two or more repeated biopsies), 83 men with PCa and 97 men with benign pathology findings (BP: no PCa, HGPIN or ASAP). RESULTS: The median PMU-PCA3 score was 1.56 in men with BP, 2.01 in men with HGPIN (p = 0.128) and 9.06 in men with PCa (p = 0.008). The AUC in the ROC analysis was 0.705 in the subset of men with BP and PCa, while it decreased to 0.629 when only men with isolated HGPIN and PCa were included in the analysis. Fixing the sensitivity of the PMU-PCA3 score at 90%, its specificity was 79% in men with BP and 69% in men with isolated HGPIN. CONCLUSIONS: The efficacy of the PMU-PCA3 score to rule out PCa in men with HGPIN is lower than in men with BP.

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.

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.

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.

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.