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

The transcription factor SNAIL1 is a master regulator of epithelial to mesenchymal transition. SNAIL1 is a very unstable protein, and its levels are regulated by the E3 ubiquitin ligase ␤-TrCP1 that interacts with SNAIL1 upon its phosphorylation by GSK-3␤. Here we show that SNAIL1 polyubiquitylation and degradation may occur in conditions precluding SNAIL1 phosphorylation by GSK-3␤, suggesting that additional E3 ligases participate in the control of SNAIL1 protein stability. In particular, we demonstrate that the F-box E3 ubiquitin ligase FBXl14 interacts with SNAIL1 and promotes its ubiquitylation and proteasome degradation independently of phosphorylation by GSK-3␤. In vivo, inhibition of FBXl14 using short hairpin RNA stabilizes both ectopically expressed and endogenous SNAIL1. Moreover, the expression of FBXl14 is potently down-regulated during hypoxia, a condition that increases the levels of SNAIL1 protein but not SNAIL1 mRNA. FBXL14 mRNA is decreased in tumors with a high expression of two proteins up-regulated in hypoxia, carbonic anhydrase 9 and TWIST1. In addition, Twist1 small interfering RNA prevents hypoxia-induced Fbxl14 downregulation and SNAIL1 stabilization in NMuMG cells. Altogether, these results demonstrate the existence of an alternative mechanism controlling SNAIL1 protein levels relevant for the induction of SNAIL1 during hypoxia.The human SNAIL family of zinc finger transcription factors, composed of SNAIL1 and SNAIL2 (also called SNAIL and SLUG, respectively) plays a fundamental role in initiating epithelial-mesenchymal transition (EMT), 6 a key developmental program that results in the acquisition of mesenchymal characteristics by epithelial cells (1). EMT is required for essential processes, such as gastrulation and formation of neural crest cells, and is also relevant in pathological processes, such as fibrosis, cancer cell invasion, and hypoxia (1-4). Expression of SNAIL1 induces a more invasive phenotype, at least in part through its inhibition of E-cadherin gene expression (2). SNAIL1 represses transcription of E-cadherin by binding to three E-boxes present in the human E-cadherin promoter (5). Moreover, SNAIL1 has additional cellular functions independent of EMT, because it confers resistance to cell death (6 -8).SNAIL1 is a highly unstable protein and is very sensitive to proteasome inhibitors (9). SNAIL1 degradation by the proteasome requires its interaction with the E3 ubiquitin ligase ␤-TrCP1/FBXW1 and the subsequent ubiquitylation of SNAIL1 protein (9). ␤-TrCP1/FBXW1, like ␤-TrCP2/ FBXW11, recognizes the destruction motif DpSXXpS (where pS represents phosphoserine) and participates in the degradation of many substrates, including ␤-catenin (10, 11). Before its interaction with ␤-TrCP1/FBXW1, SNAIL1 degradation requires nuclear GSK-3␤ (glycogen synthase kinase-3␤) phosphorylation. This modification unmasks a nuclear export sequence (NES) and promotes SNAIL1 export from the nucleus (12). In the cytosol, SNAIL1 undergoes a second phosphorylation by GSK-3␤, which targets the prot...

SNAI1, ZEB1, E-cadherin (CDH1), and vitamin D receptor (VDR) genes regulate the epithelial-mesenchymal transition (EMT) that initiates the invasion process of many tumor cells. We hypothesized that this process could also affect the behavior of normal cells adjacent to the tumor. To verify this hypothesis, the expression level of these genes was determined by quantitative RT-PCR in tumor, normal adjacent, and normal distant tissues from 32 colorectal cancer (CC) patients. In addition, we extended the study to human HaCaT normal keratinocytes and SW480-ADH colon cancer cells co-cultured with SW480-ADH cells overexpressing the mouse Snai1 gene. Of 18 CC cases with SNAI1 expression in tumor tissue, five also had SNAI1 in normal adjacent tissue (NAT). Expression of SNAI1 in tumor tissue correlated with downregulation of CDH1 and VDR genes in both tumor (P ¼ 0.047 and P ¼ 0.014, respectively) and NAT lacking SNAI1 expression (P ¼ 0.054 and P ¼ 0.003). ZEB1 expression was directly related to VDR expression in tumor tissue (r ¼ 0.39; P ¼ 0.027) and inversely to CDH1 in NAT (r ¼ À0.46; P ¼ 0.010). CDH1 and VDR were also downregulated in SW480-ADH and MaCaT cells, respectively, when they were co-cultured with Snai1-expressing cells. Furthermore, cytokine analysis showed differences in the conditioned media obtained from the two cell types. These results indicate that histologically normal tissue adjacent to tumor tissue expressing the EMT-inducing gene SNAI1 shows alterations in the expression of epithelial differentiation genes such as CDH1 and VDR.

Purpose: Cumulative data support the role of DTAp73 variants in tumorigenic processes such as drug resistance. We evaluate the impact of TP73 isoforms and their putative target genes ABCB1, HMGB1, and CASP1 on the survival of colon cancer patients and the correlation between their expressions.Experimental Design: We determined in 77 colon cancer patients the expression of DEx2p73, DEx2/ 3p73, DNp73, TAp73, ABCB1, HMGB1, and CASP1 by quantitative real-time reverse transcriptase-PCR. Tumor characteristics, disease-free survival, and overall survival (OS) were examined in each patient. Functional experiments were carried out to check whether ectopic expression of DNp73 modifies the proliferation, drug resistance, migration, and invasion properties of colon tumor cells and the expression of ABCB1, HMGB1, and CASP1.Results: Positive correlations were observed between the expression levels of DTAp73 variants and HMGB1. Furthermore, a trend was observed for ABCB1. Overexpression of DEx2/3p73 and DNp73 isoforms was significantly associated with advanced stages (P ¼ 0.04 and P ¼ 0.03, respectively) and predicted shortened OS (P ¼ 0.04 and P ¼ 0.05, respectively). High levels of ABCB1 and HMGB1 were associated with shorter OS (P ¼ 0.04 and P ¼ 0.05, respectively). Multivariate analysis showed that, in addition to the tumor stage, ABCB1 and HMGB1 had independent relationships with OS (P ¼ 0.008). Ectopic expression of DNp73 was associated with an increase in proliferation and drug resistance.Conclusions: The positive correlation between DTAp73 variants and HMGB1 and ABCB1 expression supports them as TP73 targets. The fact that upregulation of DTAp73 isoforms was associated with shortened OS, increase in proliferation, and drug resistance confirms their oncogenic role and plausible value as prognostic markers. ABCB1 and HMGB1, putative DTAp73 target genes, strongly predict OS in an independent manner, making clear the importance of studying downstream TP73 targets that could predict the outcome of colon cancer patients better than DTAp73 variants themselves do.