Novel Snail1 Target Proteins in Human Colon Cancer Identified by Proteomic Analysis

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: The transcription factor Snail1 induces epithelial-to-mesenchymal transition (EMT), a process responsible for the acquisition of invasiveness during tumorigenesis. Several transcriptomic studies have reported Snail1-regulated genes in different cell types, many of them involved in cell adhesion. However, only a few studies have used proteomics as a tool for the characterization of proteins mediating EMT. [Methodology/Principal Findings]: We identified by proteomic analysis using 2D-DIGE electrophoresis combined with MALDI-TOF-TOF and ESI-linear ion trap mass spectrometry a number of proteins with variable functions whose expression is modulated by Snail1 in SW480-ADH human colon cancer cells. Validation was performed by Western blot and immunofluorescence analyses. Snail1 repressed several members of the 14-3-3 family of phosphoserine/phosphothreonine binding proteins and also the expression of the Proliferation-associated protein 2G4 (PA2G4) that was mainly localized at the nuclear Cajal bodies. In contrast, the expression of two proteins involved in RNA processing, the Cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and the Splicing factor proline/glutamine-rich (SFPQ), was higher in Snail1-expressing cells than in controls. The regulation of 14-3-3 epsilon, 14-3-3 tau, 14-3-3 zeta and PA2G4 by Snail1 was reproduced in HT29 colon cancer cells. In addition, we found an inverse correlation between 14-3-3 sigma and Snail1 expression in human colorectal tumors. [Conclusions/Significance]: We have identified a set of novel Snail1 target proteins in colon cancer that expand the cellular processes affected by Snail1 and thus its relevance for cell function and phenotype.Peer reviewe

Vitamina D y cáncer: efecto de la 1'alpha',25-dihidroxivitamina D3, sobre el fenotipo de las células de cáncer de mama y la expresión del gen DICKKOPF-4 (DKK-4) en cáncer de colon

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina. Departamento de Bioquímica. Fecha de lectura: 21 de Noviembre de 200

Vitamin D and Wnt/β-catenin pathway in colon cancer: Role and regulation of DICKKOPF genes

Colorectal cancer is a major health problem worldwide. Aberrant activation of the Wingless-type mouse mammary tumour virus integration site family (Wnt)/β-catenin signalling pathway due to mutation of adenomatous polyposis coli (APC), β-catenin (CTNNB1) or AXIN genes is the most common and initial alteration in sporadic colorectal tumours. Numerous epidemiological and experimental studies have indicated a protective action of vitamin D against colorectal cancer. Previous work has demonstrated that the most active vitamin D metabolite, 1α,25-dihydroxyvitamin D3 (125(OH) 2D3) inhibits β-catenin transcriptional activity by promoting vitamin D receptor (VDR) binding to β-catenin and the induction of E-cadherin expression. Recently, 1,25(OH)2D3 has been shown to distinctly regulate two genes encoding the extracellular Wnt inhibitors DICKKOPF-1 and DICKKOPF-4 (DKK-1, DKK-4). By an indirect transcriptional mechanism, 1,25(OH)2D3 increases the expression of DKK-1 RNA and protein, which acts as a tumour suppressor in human colon cancer cells harbouring endogenous mutations in the Wnt/β-catenin pathway. In contrast, 1,25(OH)2D3 represses DKK-4 transcription by inducing direct VDR binding to its promoter. Unexpectedly, DKK-4 is a target of the Wnt/β-catenin pathway and is up-regulated in colorectal tumours, and it has been shown to increase cell migration and invasion and to promote a proangiogenic phenotype. Together, these results show that 1,25(OH) 2D3 exerts a complex set of regulatory actions leading to the inhibition of the Wnt/β-catenin pathway in colon cancer cells that is in line with its protective effect against this neoplasia.Peer Reviewe

Vitamin D and cancer: an update of in vitro and in vivo data

1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D 3, Calcitriol) is a pleiotropic hormone with anti-proliferative, pro-apoptotic and pro-differentiation effects on numerous cell types, which suggest anti-cancer activity in addition to its classical regulatory action on calcium and phosphate metabolism. 1,25(OH)2D3 exerts its actions mainly via its high affinity receptor VDR through a complex network of genomic (transcriptional and post-transcriptional) and also non-genomic mechanisms, which are partially coincident in the different cells and tissues studied. Epidemiological and experimental in vitro and in vivo data support a cancer preventive role of 1,25(OH)2D3. The anti-cancer activity of 1,25(OH)2D3 and multiple analogs with reduced calcemic properties, which are thus less toxic, is under investigation in a long list of cultured cell types and in several in vivo models of wild-type and genetically-modified animals. Some vitamin D compounds have reached clinical trials, but results are still scarce.The work of the authors is supported by Grants from Fundación Científica de la Asociación Española contra el Cáncer, Fundación de Investigación Médica Mutua Madrileña, Instituto de Salud Carlos III (FIS03-C03/10) and Ministerio de Educación y Ciencia (SAF04-01015) of Spain.Peer Reviewe

Vitamin D regulates the phenotype of human breast cancer cells

1α,25-dihydroxyvitamin D3 (1,25(OH)2D 3), the most active vitamin D metabolite, regulates proliferation, survival, and differentiation in many cell types. 1,25(OH)2D 3 and several less calcemic analogs are in clinical trials against various neoplasias. We studied the effects of 1,25(OH)2D3 on a panel of human breast cancer cells, which show similar vitamin D receptor (VDR) content but variable transcriptional and anti-proliferative responsiveness. In MDA-MB-453 cells, one of the responsive lines, 1,25(OH) 2D3 increased cell and nuclear size and induced a change from a rounded to a flattened morphology. By phase contrast, laser confocal and electron microscopy, we found that 1,25(OH)2D3 changed the cytoarchitecture of actin filaments and microtubules and nuclear shape, induced filopodia and lamellipodia, and promoted cell-to-cell contacts via large cytoplasmic extensions. However, although claudin-7 and occludin content in the cells increased upon exposure to 1,25(OH)2D3, these proteins were not located at the plasma membrane probably due to the absence of E-cadherin expression. Additionally, 1,25(OH)2D3 induced the accumulation of αv-integrin, β5-integrin, focal adhesion kinase (FAK), and paxillin in focal adhesion plaques, concomitant with the increased phosphorylation of the FAK. 1,25(OH)2D 3 enhanced MDA-MB-453 and MDA-MB-468 cell adhesion to plastic but decreased adhesion to laminin. The expression of the mesenchymal marker N-cadherin and of the myoepithelial marker P-cadherin was down-regulated by 1,25(OH)2D3 in several breast cancer cell lines. Other myoepithelial proteins such as α6-integrin, β4-integrin, and smooth muscle α-actin (SMA) were also repressed by 1,25(OH)2D3 in MDA-MB-453 and MDA-MB-468 cells. Accordingly, mice lacking VDR (Vdr-/-) showed abnormally high levels of SMA and P-cadherin in their mammary gland. These findings show that 1,25(OH)2D3 profoundly affects the phenotype of breast cancer cells, and suggest that it reverts the myoepithelial features associated with more aggressive forms and poor prognosis in human breast cancer. © 2007, International Society of Differentiation.This work was supported by Grants from Ministerio de Educación y Ciencia (SAF2004-01015), Instituto de Salud Carlos III (FIS03-C03/10), Fundación de Investigación Médica Mutua Madrileña and the EU (MRTN-CT-2005-019496, NucSys).Peer Reviewe

DICKKOPF-4 is induced by TCF/β-catenin and upregulated in human colon cancer, promotes tumour cell invasion and angiogenesis and is repressed by 1α,25-dihydroxyvitamin D3

Aberrant activation of the Wnt/β-catenin signaling pathway is a hallmark of colon cancer. We show that the Wnt antagonist DICKKOPF-4 (DKK-4) gene is repressed by 1α,25-dihydroxyvitamin D3 (1,25(OH) 2D3) in human colon cancer cells. This effect correlated with the inhibition of the DKK-4 promoter. Chromatin immunoprecipitation assays revealed that 1,25(OH)2D3 induces early and transient binding of the vitamin D receptor (VDR) and the SMRT corepressor to the region adjacent to the transcription start site of DKK-4. Additionally, we demonstrate that the DKK-4 gene is a new downstream target of TCF/β-catenin. Remarkably, expression of DKK-4 messenger RNA (mRNA) was not detected in a series of 29 human normal (N) colon biopsies but expression was upregulated in all the matched tumour (T) tissues. An inverse correlation existed between the expression of DKK-4 and VDR RNA in the Ts. Ectopic DKK-4 expression increased the migration and invasion properties of colon cancer cells and conditioned media (CM) from DKK-4-expressing cells enhanced the capacity to migrate and form capillary-like tubules of human primary microvascular endothelial cells. In conclusion, DKK-4 is upregulated in colon cancer and is associated with the acquisition of malignant properties by neoplastic cells. DKK-4 downregulation is a novel effect of 1,25(OH)2D3 that may contribute to its anticancer action. © 2008 Macmillan Publishers Limited All rights reserved.This study was supported by grants from Ministerio de Educación y Ciencia (SAF2004-01015, SAF2007-60341 and SAF2004-04152), Ministerio de Sanidad y Consumo ISCIII-RETIC (RD06/0020/0009), Fundación de Investigación Médica Mutua Madrileña, Comunidad de Madrid (S-GEN-0266-2006) and the European Union (MRTN-CT-2005-019496, NucSys).Peer Reviewe

The Wnt antagonist DICKKOPF-1 gene is a downstream target of β-catenin/TCF and is downregulated in human colon cancer

Wnt glycoproteins regulate homeostasis and development by binding to membrane Frizzled-LRP5/6 receptor complexes. Wnt signaling includes a canonical pathway involving cytosolic β-catenin stabilization, nuclear translocation and gene regulation, acting as a co-activator of T-cell factor (TCF) proteins, and noncanonical pathways that activate Rho, Rac, JNK and PKC, or modulate Ca2+ levels. DICKKOPF-1 (DKK-1) encodes a secreted Wnt antagonist that binds to LRP5/6 and induces its endocytosis, leading to inhibition of the canonical pathway. We show that activation of canonical signaling by Wnt1 or ectopic expression of active β-catenin, TCF4 or LRP6 mutants induces transcription of the human DKK-1 gene. Multiple β-catenin/TCF4 sites in the DKK-1 gene promoter contribute to this activation. In contrast, Wnt5a, which signals through noncanonical pathways, does not activate DKK-1. Northern and Western blot studies show that activation of the Wnt/β-catenin pathway by treatment with lithium or Wnt3a-conditioned medium, or by stable expression of either Wnt1 or β-catenin, increases DKK-1 RNA and protein, thus initiating a negative feedback loop. However, we found that DKK-1 expression decreases in human colon tumors, which suggests that DKK-1 acts as a tumor suppressor gene in this neoplasia. Our data indicate that the Wnt/β-catenin pathway is downregulated by the induction of DKK-1 expression, a mechanism that is lost in colon cancer. © 2005 Nature Publishing Group All rights reserved.This study was supported by Grants from Fundación de Investigación Médica Mutua Madrileña, Fundación Científica de la Asociación Española contra el Cáncer, Instituto de Salud Carlos III (FIS03-C03/10) and Ministerio de Educación y Ciencia (SAF01-2291) of Spain.Peer Reviewe

Immunofluorescence analysis of 14-3-3 proteins in Snail1 and Mock cells.

<p>Representative confocal microscopy images showing the expression and localization of 14-3-3 β, ε and σ (red) in SW480-ADH Mock and Snail1 cells. GFP expression (green) was unaltered by Snail1 and was used as a control. Scale bar, 10 µm.</p