Expression of Snail1 in epithelial cells triggers an epithelial-mesenchymal transition (EMT). Here, we demonstrate that the synthesis of Zeb2, a transcriptional repressor of E-cadherin, is up-regulated after Snail1-induced EMT. Snail1 does not affect the synthesis of Zeb2 mRNA, but prevents the processing of a large intron located in its 5-untranslated region (UTR). This intron contains an internal ribosome entry site (IRES) necessary for the expression of Zeb2. Maintenance of 5-UTR Zeb2 intron is dependent on the expression of a natural antisense transcript (NAT) that overlaps the 5 splice site in the intron. Ectopic overexpression of this NAT in epithelial cells prevents splicing of the Zeb2 5-UTR, increases the levels of Zeb2 protein, and consequently down-regulates E-cadherin mRNA and protein. The relevance of these results is demonstrated by the strong association between NAT presence and conservation of the 5-UTR intron in cells that have undergone EMT or in human tumors with low E-cadherin expression. Therefore, the results presented in this article reveal the existence of a NAT capable of activating Zeb2 expression, explain the mechanism involved in this activation, and demonstrate that this NAT regulates E-cadherin expression.

No abstract

No abstract

Tumor epithelial cells within a tumor coexist with a complex microenvironment in which a variety of interactions between its various components determine the behavior of the primary tumors. Cancer- associated fibroblasts (CAF) and M2 macrophages, characterized by high expression of different markers, including a-SMA, FSP1 and FAP, or CD163 and DCSIGN, respectively, are involved in the malignancy of different tumors. In the present study, expression of the above markers in CAF and M2 macrophages was analyzed using RT-PCR and immunohistochemistry in the normal mucosa and tumor tissue from a cohort of 289 colorectal cancer patients. Expression of CAF and M2 markers is associated with the clinical outcome of colorectal cancer patients. Moreover, the combination of CAF and M2 markers identifies three groups of patients with clear differences in the progression of the disease. This combined variable could be a decisive factor in the survival of advanced-stage patients. Taken together, these analyses demonstrate the prognostic involvement of interrelationships between DCSIGN, CD163, a-SMA, FSP1 and FAP markers in the survival of colon cancer patients.This work has been supported by Fundación Científica AECC, SAF2010-20750, S2010 ⁄BMD-2344, RTICC-RD06 ⁄ 0020 ⁄ 0020, PI12 ⁄ 02037 and Fundación Banco Santander. A.G.H. laboratory was supported by RD06 ⁄ 0020 ⁄ 0040. C.P. is the recipient of a Miguel Servet Contract from the Instituto de Salud Carlos III (CP09 ⁄ 00294) and V.G. is the recipient of a Fundación Científica AECC fellowshi

A significant proportion of extracellular nucleic acids in plasma circulate highly protected in tumor-specific exosomes, but it is unclear how the release of exosomes is modulated in carcinogenesis. We quantified by cytometry exosomes in plasma of 91 colorectal cancer patients to evaluate their potential as a tumor indicator and their repercussions on diagnosis and prognosis. We examined the involvement of TSAP6, a TP53-regulated gene involved in the regulation of vesicular secretion, in levels of circulating exosomes in plasma of colorectal patients and in HCT116 TP53-(wild-type and null) human colorectal cancer cell lines. The fraction of exosomes in cancer patients was statistically higher than in healthy controls (mean rank ¼ 53.93 vs. 24.35). High levels of exosomes in plasma of patients correlated with high levels of carcino-embryonic antigen (P ¼ 0.029) and with poorly differentiated tumors (P ¼ 0.039) and tended to have shorter overall survival than patients with low levels (P ¼ 0.056). Release of exosomes did not correlate with TSAP6 expression; and regulation of TSAP6 by TP53 was not shown either in tumor samples or in HCT116 cell lines. Although it was not suggested that the TP53/TSAP6 pathway regulates the release of exosomes into the plasma of colorectal cancer patients, the level of circulating exosomes may be used as a tumor indicator, because it correlates with poor prognosis parameters and shorter survival.

MicroRNAs (miRNAs) are noncoding RNAs that regulate expression of target mRNAs and are controlled by tumor suppressors and oncogenes. Altered expression of specific miRNAs in several tumor types and its association with poor prognosis parameters have been reported. Fewer data are available on its impact on patients' survival. We studied the impact of the expression of miR-17-5p, miR-106a, and miR-126 on survival and its correlation with the levels of their target mRNAs and host gene and TP53 alterations. We assessed in 110 colon cancer patients the levels of miR-17-5p, miR-106a, miR-126, E2F1, and EGFL7 by quantitative real-time RT-PCR and loss of heterozygosity (LOH) in the TP53 region. Tumor characteristics, disease-free survival (DFS), and overall survival (OS) were examined in each patient. Altered expression of miR-17-5p, miR-106a, and EGFL7 was associated with pathological tumor features of poor prognosis. Downregulation of miR-106a predicted shortened DFS (P = 0.03) and OS (P = 0.04). miR-17-5p correlated with DFS only at early stages (P = 0.07). Inverse correlations were found between miR-17-5p and miR-106a levels and their target expression, E2F1 (P = 0.04 and P = 0.03, respectively). No correlation was found between miR-126 expression and its host gene levels, EGFL7. miR-106a deregulation was revealed as a marker of DFS and OS independent of tumor stage. The lack of association between expression of miR-126 and its host gene EGFL7 suggests their regulation by independent stimuli. Inverse correlation between miR-17-5p and miR-106a and E2F1 levels supports E2F1 as a target mRNA for the two miRNAs.

Purpose: Cancer-associated fibroblasts (CAF) are essential components of the stroma that play a critical role in cancer progression. This study aimed to identify novel CAFs markers that might contribute to the invasion and the prognosis of colorectal cancer.Experimental Design: The azoxymethane/dextran sodium sulfate mouse model of sporadic colon cancer represents an adequate source for the isolation of CAFs and normal fibroblasts. By using the explants technique, we purified CAFs and normal fibroblasts from colon tissues. Whole-cell extracts and supernatants were subjected to in-depth quantitative proteomic analysis by tandem mass spectrometry. Further validations of upregulated proteins in CAFs were carried out by chemokine microarray and immunohistochemical analyses of mouse and human tissues.Results: Using a fold-change of 1.4 or more, we found 132 and 125 differentially expressed proteins in whole-cell extracts and supernatants, respectively. We found CAFs-associated proinflammatory and desmoplastic signatures. The proinflammatory signature was composed of several cytokines. Among them, CCL2 and CCL8 caused an increase in migration and invasion of colorectal cancer KM12 cells. The desmoplastic signature was composed of 30 secreted proteins. In mouse and human samples, expression of LTBP2, CDH11, OLFML3, and, particularly, FSTL1 was significantly increased in the tumoral stroma, without significant expression in the cancer epithelial cells. The combination of CALU and CDH11 stromal expression showed a significant association with disease-free survival and poor prognosis.Conclusion: We have identified LTBP2, CDH11, OLFML3, and FSTL1 as selective biomarkers of cancer stroma, and CALU and CDH11 as candidate stromal biomarkers of prognostic significance in colon cancer.

The identification of tumour biomarkers that detect the presence of disease using noninvasive diagnostic procedures is a key part of cancer research. We determined in plasma the vesicle-related microRNA (miRNA) expression profile of nonsmall cell lung cancer (NSCLC) and evaluate whether plasma miRNAs can be both discriminating (between patients and healthy controls) and prognostic markers.365 human miRNAs were analysed by Taqman1 low-density arrays (Applied Biosystems, Foster City, CA, USA) in the plasma from 28 NSCLC patients and 20 controls. Five selected miRNAs (let7f, miR-20b, miR-30e-3p, miR-223 and miR-301) were validated independently by real-time PCR in plasma from 78 NSCLC and 48 controls and correlated with pathologic parameters and survival.Levels of let-7f, miR-20b and miR-30e-3p were decreased in plasma vesicles of NSCLC patients. Moreover, levels of let-7f and miR-30e-3p distinguished between two groups of patients for stage of disease and therefore possibility of surgery. Plasma levels of miR-30e-3p and let-7f were associated with short disease-free survival and overall survival, respectively. NSCLC patients and healthy controls differ in vesicle-related miRNAs in plasma. Levels of let-7f and miR-30e-3p in NSCLC patients are associated with poor outcome. Thus, plasma vesiclerelated miRNAs obtained by noninvasive methods could serve as circulating tumour biomarkers of discriminating and prognostic value.