BACKGROUND Telomere maintenance has been proposed as an essential step for tumor cell immortalization. The objectives of the current study were to investigate the mechanisms implicated in telomere length in colorectal carcinoma (CRC) and to evaluate the prognostic impact of telomere status. METHODS Ninety‐one colorectal carcinoma samples that were obtained from patients who underwent surgery were analyzed to investigate the factors related to telomere function. The authors studied telomerase activity, terminal restriction fragment (TRF) length, and telomeric‐repeat binding factor (TRF1) expression and analyzed the prognostic implications of those factors. RESULTS Most tumors (81.3%) displayed telomerase activity. Overall, telomeres in CRC specimens were significantly shorter compared with telomeres in normal, adjacent specimens (P = 0.02). Moreover, tumors that demonstrated shortened telomeres displayed higher TRF1 levels than tumors without telomere shortening. In relation to patient prognosis, a significantly poor clinical course was observed in the group of patients who had tumors with longer telomeres (P = 0.02), and this finding emerged as an independent prognostic factor in a Cox proportional hazards model (P = 0.04; relative risk, 6.48). Among patients with tumors classified as telomerase‐positive, telomere length ratios (the ratio of tumor tissues to normal tissues) ≤ 0.66 or TRF1 over‐expression conferred a favorable outcome (P = 0.03 and P = 0.05, respectively). CONCLUSIONS The majority of CRC specimens in the current study displayed telomerase reactivation. However, only those tumors that displayed telomere elongation conferred a poor prognosis. Conversely, colorectal tumors that over‐expressed TRF1 demonstrated telomere shortening, and patients with those tumors had a better clinical course. Cancer 2006. © 2005 American Cancer Society.

BackgroundAbout 20% of patients diagnosed with endometrial cancer (EC) are considered high-risk with unfavorable prognosis. In the framework of the European Network for Individualized Treatment in EC (ENITEC), we investigated the presence and phenotypic features of Circulating Tumor Cells (CTC) in high-risk EC patients.MethodsCTC isolation was carried out in peripheral blood samples from 34 patients, ranging from Grade 3 Stage IB to Stage IV carcinomas and recurrences, and 27 healthy controls using two methodologies. Samples were subjected to EpCAM-based immunoisolation using the CELLection™ Epithelial Enrich kit (Invitrogen, Dynal) followed by RTqPCR analysis. The phenotypic determinants of endometrial CTC in terms of pathogenesis, hormone receptor pathways, stem cell markers and epithelial to mesenchymal transition (EMT) drivers were asked. Kruskal-Wallis analysis followed by Dunn’s post-test was used for comparisons between groups. Statistical significance was set at p < 0.05.ResultsEpCAM-based immunoisolation positively detected CTC in high-risk endometrial cancer patients. CTC characterization indicated a remarkable plasticity phenotype defined by the expression of the EMT markers ETV5, NOTCH1, SNAI1, TGFB1, ZEB1 and ZEB2. In addition, the expression of ALDH and CD44 pointed to an association with stemness, while the expression of CTNNB1, STS, GDF15, RELA, RUNX1, BRAF and PIK3CA suggested potential therapeutic targets. We further recapitulated the EMT phenotype found in endometrial CTC through the up-regulation of ETV5 in an EC cell line, and validated in an animal model of systemic dissemination the propensity of these CTC in the accomplishment of metastasis.ConclusionsOur results associate the presence of CTC with high-risk EC. Gene-expression profiling characterized a CTC-plasticity phenotype with stemness and EMT features. We finally recapitulated this CTC-phenotype by over-expressing ETV5 in the EC cell line Hec1A and demonstrated an advantage in the promotion of metastasis in an in vivo mouse model of CTC dissemination and homing.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-223) contains supplementary material, which is available to authorized users.

The microRNAs (miRNAs) are a class of small, 20–22 nucleotides in length, endogenously expressed noncoding RNAs that regulate multiple targets posttranscriptionally. Interestingly, miRNAs have emerged as regulators of most physiological and pathological processes, including metastatic tumor progression, in part by controlling a reversible process called epithelial-to-mesenchymal transition (EMT). The activation of EMT increases the migratory and invasive properties fundamental for tumor cell spread while activation of the reverse mesenchymal-to-epithelial transition is required for metastasis outgrowth. The EMT triggering leads to the activation of a core of transcription factors (EMT-TFs) – SNAIL1/SNAIL2, bHLH (E47, E2-2, and TWIST1/TWIST2), and ZEB1/ZEB2 – that act as E-cadherin repressors and, ultimately, coordinate EMT. Recent evidence indicates that several miRNAs regulate the expression of EMT-TFs or EMT-activating signaling pathways. Interestingly, some miRNAs and EMT-TFs form tightly interconnected negative feedback loops that control epithelial cell plasticity, providing self-reinforcing signals and robustness to maintain the epithelial or mesenchymal cell status. Among the most significant feedback loops, we focus on the ZEB/miR-200 and the SNAIL1/miR-34 networks that hold a clear impact in the regulation of the epithelial-mesenchymal state. Recent insights into the p53 modulation of the EMT-TF/miRNA loops and epigenetic regulatory mechanisms in the context of metastasis dissemination will also be discussed. Understanding the regulation of EMT by miRNAs opens new avenues for the diagnosis and prognosis of tumors and identifies potential therapeutic targets that might help to negatively impact on metastasis dissemination and increasing patient survival.

Although it is becoming clear that certain miRNAs fulfil a fundamental role in the regulation of the epithelial-to-mesenchymal transition (EMT), a comprehensive study of the miRNAs associated with this process has yet to be performed. Here, we profiled the signature of miRNA expression in an in vitro model of EMT, ectopically expressing in MDCK cells one of seven EMT transcription factors (SNAI1, SNAI2, ZEB1, ZEB2, TWIST1, TWIST2 or E47) or the EMT inducer LOXL2. In this way, we identified a core subset of deregulated miRNAs that were further validated in vivo, studying endometrial carcinosarcoma (ECS), a tumour entity that represents an extreme example of phenotypic plasticity. Moreover, epigenetic silencing through DNA methylation of miRNA genes of the miR-200 family and miR-205 that are down-regulated during EMT was evident in both the in vitro (MDCK transfectants) and in vivo (ECS) models of EMT. The strong correlation between expression and DNA methylation suggests a major role for this epigenetic mark in the regulation of the miR-141-200c locus.