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This article belongs to the Special Issue Targeting Wnt Signaling in Cancer: Opportunities Abound If We Can Avoid the Sword of Damocles., [Simple Summary]: The Wnt/β-catenin signaling pathway is aberrantly activated in most colorectal cancers and less frequently in a variety of other solid neoplasias. Many epidemiological and experimental studies and some clinical trials suggest an anticancer action of vitamin D, mainly against colorectal cancer. The aim of this review was to analyze the literature supporting the interference of Wnt/β-catenin signaling by the active vitamin D metabolite 1α,25-dihydroxyvitamin D3. We discuss the molecular mechanisms of this antagonism in colorectal cancer and other cancer types. Additionally, we summarize the available data indicating a reciprocal inhibition of vitamin D action by the activated Wnt/β-catenin pathway. Thus, a complex mutual antagonism between Wnt/β-catenin signaling and the vitamin D system seems to be at the root of many solid cancers., Abnormal activation of the Wnt/β-catenin pathway is common in many types of solid cancers. Likewise, a large proportion of cancer patients have vitamin D deficiency. In line with these observations, Wnt/β-catenin signaling and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active vitamin D metabolite, usually have opposite effects on cancer cell proliferation and phenotype. In recent years, an increasing number of studies performed in a variety of cancer types have revealed a complex crosstalk between Wnt/β-catenin signaling and 1,25(OH)2D3. Here we review the mechanisms by which 1,25(OH)2D3 inhibits Wnt/β-catenin signaling and, conversely, how the activated Wnt/β-catenin pathway may abrogate vitamin D action. The available data suggest that interaction between Wnt/β-catenin signaling and the vitamin D system is at the crossroads in solid cancers and may have therapeutic applications., The work in the authors’ laboratory is funded by the Agencia Estatal de Investigación (PID2019-104867RB-I00/AEI/10.13039/501100011033), the Agencia Estatal de Investigación—Fondo Europeo de Desarrollo Regional (SAF2016-76377-R, MINECO/AEI/FEDER, EU), the Ministerio de Economía y Competitividad (SAF2017-90604-REDT/NuRCaMeIn), and the Instituto de Salud Carlos III—Fondo Europeo de Desarrollo Regional (CIBERONC; CB16/12/00273).

Colorectal cancer results from the malignant transformation of colonic epithelial cells. Stromal fibroblasts are the main component of the tumour microenvironment, and play an important role in the progression of this and other neoplasias. Wnt/β-catenin signalling is essential for colon homeostasis, but aberrant, constitutive activation of this pathway is a hallmark of colorectal cancer. Here we present the first transcriptomic study on the effect of a Wnt factor on human colonic myofibroblasts. Wnt3A regulates the expression of 1,136 genes, of which 662 are upregulated and 474 are downregulated in CCD-18Co cells. A set of genes encoding inhibitors of the Wnt/β-catenin pathway stand out among those induced by Wnt3A, which suggests that there is a feedback inhibitory mechanism. We also show that the PKP2 gene encoding the desmosomal protein Plakophilin-2 is a novel direct transcriptional target of Wnt/β-catenin in normal and colon cancer-associated fibroblasts. PKP2 is induced by β-catenin/TCF through three binding sites in the gene promoter and one additional binding site located in an enhancer 20 kb upstream from the transcription start site. Moreover, Plakophilin-2 antagonizes Wnt/β-catenin transcriptional activity in HEK-293T cells, which suggests that it may act as an intracellular inhibitor of the Wnt/β-catenin pathway. Our results demonstrate that stromal fibroblasts respond to canonical Wnt signalling and that Plakophilin-2 plays a role in the feedback control of this effect suggesting that the response to Wnt factors in the stroma may modulate Wnt activity in the tumour cells.

The Wnt/β-catenin signalling pathway is essential for intestinal epithelium homeostasis, but its aberrant activation is a hallmark of colorectal cancer (CRC). Several studies indicate that the bioactive vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits proliferation and promotes epithelial differentiation of colon carcinoma cells in part through antagonism of the Wnt/β-catenin pathway. It is now accepted that stromal fibroblasts are crucial in healthy and pathologic intestine: pericryptal myofibroblasts are constituents of the stem cell niche and cancer-associated fibroblasts (CAFs) contribute to CRC progression. However, studies on the combined action of 1,25(OH)2D3 and Wnt factors in colon fibroblasts are lacking. Here we show by global transcriptomic studies that 1,25(OH)2D3 and Wnt3A have profound, additive, partially overlapping effects on the gene expression profile of CCD-18Co human colon myofibroblasts. Moreover, 1,25(OH)2D3 and Wnt3A inhibit CCD-18Co cell proliferation and migration, while 1,25(OH)2D3 reduces, but Wnt3A increases, their capacity to contract collagen gels (a marker of fibroblast activation). These data were largely confirmed in patient-derived primary colon normal fibroblasts and CAFs, and in fibroblasts from other origins. Our results indicate that 1,25(OH)2D3 and Wnt3A are strong regulators of colon fibroblast biology and contribute to a better knowledge of intestinal homeostasis and stromal fibroblast action in CRC., We thank IdiPAZ Biobank (PT13/0010/0003, Plataforma de Apoyo a la Investigación en Ciencias y Tecnologías de la Salud en la Red de Biobancos 2013) for providing clinical samples, Javier Pérez for the artwork, and Lucille Banham for her assistance in the preparation of the English manuscript. The work in the authors’ laboratories is supported by the Spanish Ministerio de Ciencia, Innovación y Universidades - Fondo Europeo de Desarrollo Regional (FEDER) (SAF2016-76377-R, SAF2017-90604-REDT), Consejo Superior de Investigaciones Científicas (201820I058), and Instituto de Salud Carlos III - FEDER (CIBERONC, CB16/12/00273; CIBERES, CB15/00037).

Summary Aryl hydrocarbon receptor (AhR) deficiency alters tissue homeostasis. However, how AhR regulates organ maturation and differentiation remains mostly unknown. Liver differentiation entails a polyploidization process fundamental for cell growth, metabolism, and stress responses. Here, we report that AhR regulates polyploidization during the preweaning-to-adult mouse liver maturation. Preweaning AhR-null (AhR−/−) livers had smaller hepatocytes, hypercellularity, altered cell cycle regulation, and enhanced proliferation. Those phenotypes persisted in adult AhR−/− mice and correlated with compromised polyploidy, predominance of diploid hepatocytes, and enlarged centrosomes. Phosphatidylinositol-3-phosphate kinase (PI3K), extracellular signal-regulated kinase (ERK), and Wnt/β-catenin signaling remained upregulated from preweaning to adult AhR-null liver, likely increasing mammalian target of rapamycin (mTOR) activation. Metabolomics revealed the deregulation of mitochondrial oxidative phosphorylation intermediates succinate and fumarate in AhR−/− liver. Consistently, PI3K, ERK, and Wnt/β-catenin inhibition partially rescued polyploidy in AhR−/− mice. Thus, AhR may integrate survival, proliferation, and metabolism for liver polyploidization. Since tumor cells tend to be polyploid, AhR modulation could have therapeutic value in the liver., Graphical Abstract, Highlights • AhR is required for liver polyploidization during preweaning-to-adult transition • INS-R/PI3K/AKT, ERK, Wnt/β-Cat and mTOR are downregulated during liver polyploidization • Reduced polyploidy relates with enhanced mitochondrial metabolism in AhR-null liver • Understanding how AhR modulates polyploidy may provide strategies against cancer, Developmental Biology; Cancer Systems Biology; Metabolomics

Colon cancer is one of the most frequent and lethal neoplasias and the first to be associated with vitamin D deficiency. Many epidemiological studies indicate a protective effect of vitamin D on colon cancer. This is supported by preclinical studies using vitamin D, 1,25(OH)2D3 or analogues in cultured cells and experimental animals. These compounds regulate colon carcinoma cell gene expression, survival, differentiation, proliferation and invasion and inhibit angiogenesis and metastasis during colon cancer progression. Remarkably, in animal and in vitro models vitamin D compounds inhibit the Wnt/β-catenin signaling pathway, whose aberrant activation initiates and maintains colon cancer. Moreover, recent data show that 1,25(OH)2D3 protects against colon cancer acting also on tumor stromal fibroblasts. However, clinical data are scarce and inconclusive, and a definitive answer about the role of vitamin D compounds in the prevention and treatment of colon cancer requires new, large, well-designed clinical trials.

This article describes the transition from a traditional instructor-centered course, based on lectures, to a student-centered course based on active learning methodologies as part of the reform of the Spanish higher education system within the European Higher Education Area (EHEA). Specifically, we describe the use of active learning methodologies to teach metabolism to students of nutrition and dietetics during the first year of their professional training in a 4-year undergraduate degree (Bachelor of Human Nutrition and Dietetics). In the new course design, the number of didactic lectures was largely reduced and complemented with a series of activities (problems/case studies, discussion workshops, self-assessment quizzes) aimed to get students actively engaged, to encourage self-learning, and to promote sustained work throughout the length of the course. The article presents quantitative data demonstrating a clear and significant improvement in students' performance when an active approach was implemented. Importantly, the improved performance was achieved without work overload. Finally, students' responses to this new teaching methodology have been very positive and overall satisfaction high. In summary, our results strongly argue in favor of the teaching model described herein. © 2013 by The International Union of Biochemistry and Molecular Biology, 41(3):131–138, 2013

et al., SPROUTY-2 (SPRY2) is a modulator of tyrosine kinase receptor signaling with receptor- and cell type-dependent inhibitory or enhancing effects. Studies on the action of SPRY2 in major cancers are conflicting and its role remains unclear. Here we have dissected SPRY2 action in human colon cancer. Global transcriptomic analyses show that SPRY2 downregulates genes encoding tight junction proteins such as claudin-7 and occludin and other cell-to-cell and cell-to-matrix adhesion molecules in human SW480-ADH colon carcinoma cells. Moreover, SPRY2 represses LLGL2/HUGL2, PATJ1/INADL and ST14, main regulators of the polarized epithelial phenotype, and ESRP1, an epithelial-to-mesenchymal transition (EMT) inhibitor. A key action of SPRY2 is the upregulation of the major EMT inducer ZEB1, as these effects are reversed by ZEB1 knock-down by means of RNA interference. Consistently, we found an inverse correlation between the expression level of claudin-7 and those of SPRY2 and ZEB1 in human colon tumors. Mechanistically, ZEB1 upregulation by SPRY2 results from the combined induction of ETS1 transcription factor and the repression of microRNAs (miR-200 family, miR-150) that target ZEB1 RNA. Moreover, SPRY2 increased AKT activation by epidermal growth factor, whereas AKT and also Src inhibition reduced the induction of ZEB1. Altogether, these data suggest that AKT and Src are implicated in SPRY2 action. Collectively, these results show a tumorigenic role of SPRY2 in colon cancer that is based on the dysregulation of tight junction and epithelial polarity master genes via upregulation of ZEB1. The dissection of the mechanism of action of SPRY2 in colon cancer cells is important to understand the upregulation of this gene in a subset of patients with this neoplasia that have poor prognosis., This study was supported by the Ministerio de Economía y Competitividad of Spain and Fondo Europeo de Desarrollo Regional (FEDER) (grant SAF2013-43468-R to AM, SAF2011-29530 to FXR); FEDER-Instituto de Salud Carlos III (RD12/0036/0021 to AM and JMR, RD12/0036/0034 to FXR, RD12/0036/0016 to MS, RD12/0036/0012 to HGP, RD06/0020/0003, PS09/00562 and PI13/00703 to JMR); Comunidad de Madrid (S2010/BMD-2344 Colomics2 to AM); Fundación Científica de la Asociación Española contra el Cáncer (to JMR); US Department of Defense (CA093471 and CA110602 to EH); National Institutes of Health/National Cancer Institute (1R01CA155234-01 to EH); National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (1R21AR062239-01 to EH); and the Melanoma Research Alliance (to EH).

The Wnt/β-catenin signalling pathway is activated in 90% of human colon cancers by nuclear accumulation of β-catenin protein due to its own mutation or to that of adenomatous polyposis coli. In the nucleus, β-catenin regulates gene expression promoting cell proliferation, migration and invasiveness. 1α,25-dihydroxyvitamin D3 (1,25(OH) 2D3) inhibits β-catenin signalling by inducing its binding to vitamin D receptor (VDR) and by promoting β-catenin nuclear export. The transcription factor Snail1 represses VDR expression and we demonstrate here that Snail1 also abolishes the nuclear export of β-catenin induced by 1,25(OH)2D3 in SW480-ADH cells. Accordingly, Snail1 relieves the inhibition exerted by 1,25(OH)2D3 on genes whose expression is driven by β-catenin, such as c-MYC, ectodermal-neural cortex-1 (ENC-1) or ephrin receptor B2 (EPHB2). In addition, Snail1 abrogates the inhibitory effect of 1,25(OH)2D3 on cell proliferation and migration. In xenografted mice, Snail1 impedes the nuclear export of β-catenin and the inhibition of ENC-1 expression induced by EB1089, a 1,25(OH)2D3 analogue. The elevation of endogenous SNAIL1 protein levels reproduces the effect of an ectopic Snail1 gene. Remarkably, the expression of exogenous VDR in cells with high levels of Snail1 normalizes the transcriptional responses to 1,25(OH)2D 3. However, this exogenous VDR failed to fully restore the blockage of the Wnt/β-catenin pathway by 1,25(OH)2D3. This suggests that the effects of Snail1 on this pathway are not merely due to the repression of VDR gene. We conclude that Snail1 is a positive regulator of the Wnt/β-catenin signalling pathway in part through the abrogation of the inhibitory action of 1,25(OH)2D3. © 2007 Society for Endocrinology., N Valle was supported by a contract of the Fundación Científica de la Asociación Española Contra el Cáncer. This study was supported by Grants from Ministerio de Educación y Ciencia (SAF04-01015) and Instituto de Salud Carlos III (FIS03-C03/10) of Spain.

This work is licensed under a Creative Commons Attribution 4.0 International License., The role of DICKKOPF (DKK)-1 in human cancer is controversial. DKK-1 behaves as an inhibitor of the canonical Wnt/β-catenin signaling pathway acting at the plasma membrane, although several studies have proposed effects that are independent of the inhibition of β-catenin transcriptional activity, in some cases mediated by the activation of c-Jun N-terminal kinase (JNK). Recently, a proportion of DKK-1 protein has been found within the nucleus of human intestinal epithelial cells following an apical-to-basal crypt decreasing gradient, and in that of colon carcinoma cells. Moreover, we show here that in the human mammary gland DKK-1 is also present within the nucleus of many differentiated luminal epithelial cells and in that of a small proportion of myoepithelial cells. Nuclear DKK-1 binds to actively transcribed chromatin and regulates the expression of specific genes, some of which are involved in cell proliferation, survival and stemness, and in the defense against xenobiotics. This may explain the finding that while DKK-1 is downregulated more rapidly in the nucleus than in the cytosol during colon carcinoma progression, its expression remains high in a percentage of patients who do not respond to chemotherapy. Available data suggest that the accumulation of DKK-1 in the nucleus of colon carcinoma cells depends on signals from the surrounding tumor microenvironment., Work in authors' laboratories is supported by grants from Ministerio de Economía y Competitividad of Spain-Fondo Europeo de Desarrollo Regional (FEDER) (SAF2013-43468-R), Comunidad de Madrid (S2010/BMD-2344 Colomics2), and FEDER-Instituto de Salud Carlos III (RD12/0036/0021; RD12/0036/0051, PT13/0010/0012 and PI12/01552).

et al., [Aims]: Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a master regulator of oxidant and xenobiotic metabolism, but it is unknown how it is regulated to provide basal expression of this defense system. Here, we studied the putative connection between NRF2 and the canonical WNT pathway, which modulates hepatocyte metabolism. [Results]: WNT-3A increased the levels of NRF2 and its transcriptional signature in mouse hepatocytes and HEK293T cells. The use of short interfering RNAs in hepatocytes and mouse embryonic fibroblasts which are deficient in the redox sensor Kelch-like ECH-associated protein 1 (KEAP1) indicated that WNT-3A activates NRF2 in a β-Catenin- and KEAP1-independent manner. WNT-3A stabilized NRF2 by preventing its GSK-3-dependent phosphorylation and subsequent SCF/β-TrCP-dependent ubiquitination and proteasomal degradation. Axin1 and NRF2 were physically associated in a protein complex that was regulated by WNT-3A, involving the central region of Axin1 and the Neh4/Neh5 domains of NRF2. Axin1 knockdown increased NRF2 protein levels, while Axin1 stabilization with Tankyrase inhibitors blocked WNT/NRF2 signaling. The relevance of this novel pathway was assessed in mice with a conditional deletion of Axin1 in the liver, which showed upregulation of the NRF2 signature in hepatocytes and disruption of liver zonation of antioxidant metabolism. [Innovation]: NRF2 takes part in a protein complex with Axin1 that is regulated by the canonical WNT pathway. This new WNT-NRF2 axis controls the antioxidant metabolism of hepatocytes. [Conclusion]: These results uncover the participation of NRF2 in a WNT-regulated signalosome that participates in basal maintenance of hepatic antioxidant metabolism., This work was funded by SAF2013-43271-R of the Spanish Ministry of Economy and Competitiveness.

It has been well documented that the outcome of prostate cancer treatment depends on the dose administered. Hence, techniques have been developed that allow high-dose administration without increasing the complications, e.g. external radiotherapy combined with high-dose radiation (HDR) brachytherapy. In this article we analyse the technique and protocol of real-time HDR brachytherapy together with the preliminary results that support its use. Materials and methods. Between June 1998 and December 2004, 100 patients with adenoma of the prostate were treated with 46 Gy of external irradiation to the pelvis and 2 HDR brachytherapy fractions (each of 1150 cGy) at the end of weeks 1 and 3 of a 5-week radiotherapy course. The 1997 American Joint Commission on Cancer (AJCC) system was used to establish disease stage. Patients with intermediate-risk (PSA 10-20 ng/ml or Gleason = 7 or T2c) and high-risk (two intermediate risk factors or PSA20 ng/ml or Gleason7 orT2c) without metastases were eligible for the brachytherapy. Biochemical failure was defined according to the American Society for Therapeutic Radiology and Oncology (ASTRO) consensus panel statement. SPSS statistical package was used to quantify survival (Kaplan-Meier method). Toxicity was scored according to RTOG guidelines.The mean age of patients was 67 years (range 49-78). Clinical stage was T2a in 22% of the patients, 26% T2b and 52% T3. Initial PSA was = 10 ng/ml in 22% of the patients and10 ng/ml in 78%. Median follow-up was 28 months (range: 12-79). The 5-year overall survival and actuarial biochemical control were 99% and 87% respectively. No chronic severe complications were noted.The good results of local control, disease-free survival and few complications that the external radiotherapy combined with HDR brachytherapy have shown suggest that the method should be considered as first-choice in the treatment of prostate tumours of high- and intermediate-risk.

This work is licensed under a Creative Commons Attribution 3.0 License.-- et al., Sporadic colorectal cancer (CRC) insurgence and progression depend on the activation of Wnt/β-catenin signaling. Dickkopf (DKK)-1 is an extracellular inhibitor of Wnt/β-catenin signaling that also has undefined β-catenin-independent actions. Here we report for the first time that a proportion of DKK-1 locates within the nucleus of healthy small intestine and colon mucosa, and of CRC cells at specific chromatin sites of active transcription. Moreover, we show that DKK-1 regulates several cancerrelated genes including the cancer stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) and Ral-binding protein 1-associated Eps domain-containing 2 (REPS2), which are involved in detoxification of chemotherapeutic agents. Nuclear DKK-1 expression is lost along CRC progression; however, it remains high in a subset (15%) of CRC patients (n = 699) and associates with decreased progression-free survival (PFS) after chemotherapy administration and overall survival (OS) [adjusted HR, 1.65; 95% confidence interval (CI), 1.23-2.21; P = 0.002)]. Overexpression of ALDH1A1 antiand REPS2 associates with nuclear DKK-1 expression in tumors and correlates with decreased OS (P = 0.001 and 0.014) and PFS. In summary, our findings demonstrate a novel location of DKK-1 within the cell nucleus and support a role of nuclear DKK-1 as a predictive biomarker of chemoresistance in colorectal cancer., This work was supported by grants from the Ministerio de Economía y Competitividad of Spain and Fondo Europeo de Desarrollo Regional (FEDER) (SAF2013-43468-R, A.M.; BFU2010-19659, J.M.G-S.; SAF2010-19256, B.J.; SAF2011-23089, C.L-O.), Comunidad de Madrid (S2010/BMD-2344 Colomics2, A.M., F.R. and J.M.G-S.) and FEDER-Instituto de Salud Carlos III (RD12/0036/0021, A.M.; RD12/0036/0051, PT13/0010/0012 and PI12/01552, F.R.; ProteoRed Networked Proteomics Platform, F.C.).

The relation between thyroid status and diseases and cancer is unclear. No detailed analysis of thyroid hormone receptor (TR) expression in human breast cancer has been reported. We have analysed the expression and mutational status of the TRalpha1, encoded by the c-erbA proto-oncogene, TRbeta1 and TRbeta2 isoforms in 70 sporadic breast cancers. Alterations in the RNA level of TRbeta1, TRalpha1, or both were found in a number of patients. No expression of TRbeta2 RNA was detected. Western blotting analysis confirmed the differences in expression at the protein level in those cases where sufficient tumor sample was available. Additionally, tumor-specific truncated TRbeta1 RNA was found in six patients. Strikingly, three transcripts shared the same breakpoint. Only one tumor carried the corresponding deletion at the genomic DNA level, suggesting that the remaining abnormal TRbeta1 transcripts are aberrant splicing products. Though no significant correlation was found between TRbeta1 alteration and any clinical parameter, it showed a tendency to associate with early age of onset (50 years). Our results reveal specific alterations in the expression of TRbeta and TRalpha genes in a subset of breast cancer patients, suggesting that deregulation of thyroid hormone target genes may be involved in the generation of this neoplasia.

This work was supported by grants 08.1/0069/2000 1 from the Comunidad Autonoma de Madrid and SAF2001-2291 from Ministerio de Ciencia y Tecnologia of Spain.

This work was supported by Grants SAF95-0738 and SAF98-0060 from Comision Interministerial de Ciencia y Tecnologia, Spain. JMG-S was supported by a predoctoral fellowship from Comunidad Autonoma de Madrid.

Wnt/β-catenin signalling pathway is crucial for the formation of many tissues and organs during development. In recent years, this pathway has also been found to regulate the biology of stem cells in the intestine and probably in other organs in adult life. Abnormal activation of Wnt/β-catenin signalling, which controls the expression of a high number of genes, is critical for the initiation and progression of most colorectal cancers. In line with this, the gene expression signature induced by activation of the Wnt/β-catenin pathway defines the intestinal stem cells present at the bottom of the crypts and also colon cancer stem cells. This supports the importance of inhibitors of the Wnt/β-catenin pathway as potential agents in colorectal cancer therapy. However, the complexity, wide activity in the organism modulating the biology of several cell types, and characteristics of this pathway have delayed the identification of suitable targets and so, the development of such inhibitors that are only now reaching the clinic.

This is an open-access article distributed under the terms of the Creative Commons Attribution License., Many studies in different biological systems have revealed that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) modulates signaling pathways triggered at the plasma membrane by agents such as Wnt, transforming growth factor (TGF)-β, epidermal growth factor (EGF), and others. In addition, 1α,25(OH)2D3 may affect gene expression by paracrine mechanisms that involve the regulation of cytokine or growth factor secretion by neighboring cells. Moreover, post-transcriptional and post-translational effects of 1α,25(OH)2D3 add to or overlap with its classical modulation of gene transcription rate. Together, these findings show that vitamin D receptor (VDR) cannot be considered only as a nuclear-acting, ligand-modulated transcription factor that binds to and controls the transcription of target genes. Instead, available data support the view that much of the complex biological activity of 1α,25(OH)2D3 resides in its capacity to interact with membrane-based signaling pathways and to modulate the expression and secretion of paracrine factors. Therefore, we propose that future research in the vitamin D field should focus on the interplay between 1α,25(OH)2D3 and agents that act at the plasma membrane, and on the analysis of intercellular communication. Global analyses such as RNA-Seq, transcriptomic arrays, and genome-wide ChIP are expected to dissect the interactions at the gene and molecular levels., The work in the authors’ laboratory is supported by Ministerio de Economía y Competitividad of Spain (SAF2010-18302,BFU2010-19659), Fondo Europeo de Desarrollo Regional-Instituto de Salud Carlos III(RD12/0036/0021,RD12/0036/0041), and Comunidad de Madrid (S2010/BMD-2344, Colomics2).

15 pages, 9 figures., The activity of c-Jun, the major component of the transcription factor AP-1, is potentiated by amino-terminal phosphorylation on serines 63 and 73 (Ser-63/73). This phosphorylation is mediated by the Jun amino-terminal kinase (JNK) and required to recruit the transcriptional coactivator CREB-binding protein (CBP). AP-1 function is antagonized by activated members of the steroid/thyroid hormone receptor superfamily. Recently, a competition for CBP has been proposed as a mechanism for this antagonism. Here we present evidence that hormone-activated nuclear receptors prevent c-Jun phosphorylation on Ser-63/73 and, consequently, AP-1 activation, by blocking the induction of the JNK signaling cascade. Consistently, nuclear receptors also antagonize other JNK-activated transcription factors such as Elk-1 and ATF-2. Interference with the JNK signaling pathway represents a novel mechanism by which nuclear hormone receptors antagonize AP-1. This mechanism is based on the blockade of the AP-1 activation step, which is a requisite to interact with CBP. In addition to acting directly on gene transcription, regulation of the JNK cascade activity constitutes an alternative mode whereby steroids and retinoids may control cell fate and conduct their pharmacological actions as immunosupressive, anti-inflammatory, and antineoplastic agents., C.C. and J.M.G.-S. were supported by a postdoctoral contract from Ministerio de Educación y Ciencia of Spain and a predoctoral fellowship from the Comunidad Autónoma de Madrid, Spain. This work was supported by a grant (SAF95-0738) from Comisión Interministerial de Ciencia y Tecnología, Plan Nacional de Investigación y Desarrollo of Spain. The publication costs of this article were defrayed in part by payment of page charges.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license., The Wnt/β-catenin signaling pathway is abnormally activated in most colorectal cancers and in a proportion of other neoplasias. This activation initiates or contributes to carcinogenesis by regulating the expression of a large number of genes in tumor cells. The active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits Wnt/β-catenin signaling by several mechanisms at different points along the pathway. Additionally, paracrine actions of 1,25(OH)2D3 on stromal cells may also repress this pathway in neighbouring tumor cells. Here we review the molecular basis for the various mechanisms by which 1,25(OH)2D3 antagonizes Wnt/β-catenin signaling, preferentially in human colon carcinoma cells, and the consequences of this inhibition for the phenotype and proliferation rate. The effect of the vitamin D system on Wnt/beta-catenin signaling and tumor growth in animal models will also be commented in detail. Finally, we revise existing data on the relation between vitamin D receptor expression and vitamin D status and the expression of Wnt/β-catenin pathway genes and targets in cancer patients., The work in the authors’ laboratory is supported by Ministerio de Economía y Competitividad of Spain (SAF2010-18302, BFU2010-19659), Fondo Europeo de Desarrollo Regional-Instituto de Salud Carlos III (RD12/0036/0021) and Comunidad de Madrid (S2010/BMD-2344, Colomics2).

et al., Dishevelled (Dvl) proteins are intracellular effectors of Wnt signaling that have essential roles in both canonical and noncanonical Wnt pathways. It has long been known that Wnts stimulate Dvl phosphorylation, but relatively little is known about its functional significance. We have previously reported that both Wnt3a and Wnt5a induce Dvl2 phosphorylation that is associated with an electrophoretic mobility shift and loss of recognition by monoclonal antibody 10B5. In the present study, we mapped the 10B5 epitope to a 16-amino acid segment of human Dvl2 (residues 594-609) that contains four Ser/Thr residues. Alanine substitution of these residues (P4m) eliminated the mobility shift induced by either Wnt3a or Wnt5a. The Dvl2P4m mutant showed a modest increase in canonical Wnt/β-catenin signaling activity relative to wild type. Consistent with this finding, Dvl2 4Pm preferentially localized to cytoplasmic puncta. In contrast to wild-type Dvl2, however, the P4m mutant was unable to rescue Wnt3a-dependent neurite outgrowth in TC-32 cells following suppression of endogenous Dvl2/3. Earlier work has implicated casein kinase 1δ/ε as responsible for the Dvl mobility shift, and a CK1δ in vitro kinase assay confirmed that Ser594, Thr595, and Ser597 of Dvl2 are CK1 targets. Alanine substitution of these three residues was sufficient to abrogate the Wnt-dependent mobility shift. Thus, we have identified a cluster of Ser/Thr residues in the C-terminal domain of Dvl2 that are Wnt-induced phosphorylation (WIP) sites. Our results indicate that phosphorylation at theWIPsites reduces Dvl accumulation in puncta and attenuates β-catenin signaling, whereas it enables noncanonical signaling that is required for neurite outgrowth. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc., This work was supported, in whole or in part, by National Institutes of Health Grant R01 CA123238 (to A. M. C. B.) and Postdoctoral Fellowship F32 CA117662 (to C. L. A.). This work was also supported by a fellowship from the Ministerio de Educación, Cultura, y Deportes, of Spain (to J. M. G.-S.), by New York State Department of Health postdoctoral Fellowship NYS C021339 (to Y. T.), and by charitable donations to Strang Cancer Prevention Center. This research also was supported in part by the Intramural Research Program of the National Institutes of Health, National Cancer Institute.

This article describes the transition from a traditional instructor-centered course, based on lectures, to a student-centered course based on active learning methodologies as part of the reform of the Spanish higher education system within the European Higher Education Area (EHEA). Specifically, we describe the use of active learning methodologies to teach metabolism to students of nutrition and dietetics during the first year of their professional training in a 4-year undergraduate degree (Bachelor of Human Nutrition and Dietetics). In the new course design, the number of didactic lectures was largely reduced and complemented with a series of activities (problems/case studies, discussion workshops, self-assessment quizzes) aimed to get students actively engaged, to encourage self-learning, and to promote sustained work throughout the length of the course. The article presents quantitative data demonstrating a clear and significant improvement in students' performance when an active approach was implemented. Importantly, the improved performance was achieved without work overload. Finally, students' responses to this new teaching methodology have been very positive and overall satisfaction high. In summary, our results strongly argue in favor of the teaching model described herein.

3ª Edición, Capítulo 13, Sección II: Mechanisms of action.-- El pdf es la versión pre-print., Wnt factors regulate tissue formation during development and tissue homeostasis in adult life. Upon binding to plasma membrane receptors, Wnt factors trigger Wnt/β-catenin (canonical) signaling pathway or several other β-catenin-independent (non-canonical) pathways. Activation of Wnt/β-catenin signaling leads to nuclear accumulation of β-catenin which acts as a transcriptional coactivator of T-cell factor/lymphoid enhancer factor (TCF/LEF) target genes involved in numerous cellular processes. Aberrant activation of Wnt/β-catenin signaling occurs in several human cancers and other diseases. The most active vitamin D3 metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its receptor (VDR) regulate Wnt/β-catenin signaling in a tissue-specific manner. 1,25(OH)2D3/VDR inhibits β-catenin/TCF transcriptional activity in colon cancer cells, while up-regulation of the Wnt/β-catenin pathway by either ligand-activated or unliganded VDR promotes bone formation or hair follicle differentiation, respectively. This crosstalk with Wnt/β-catenin signaling may contribute to the protective action of 1,25(OH)2D3 against colon cancer and to its effects on tissue homeostasis.

9 páginas, 4 figuras.-- et al., [Background]: Wnt factors control cell differentiation through semi-independent molecular cascades known as the β-catenin-dependent (canonical) and -independent (non-canonical) Wnt signalling pathways. Genetic and epigenetic alteration of components of the canonical Wnt signalling pathway is one of the primary mechanisms underlying colon cancer. Despite increasing evidence of the role of the non-canonical pathways in tumourigenesis, however, the underlying molecular mechanisms are poorly understood. [Results]: Here we report that the receptor tyrosine kinase-like orphan receptor 2 (ROR2), a transmembrane receptor for Wnt factors that activates non-canonical pathways, is frequently repressed by aberrant promoter hypermethylation in human colon cancer cell lines and primary tumours. By restoring ROR2 activity in colon cancer cells harbouring ROR2 promoter hypermethylation, we show that the role of ROR2 in colon cancer cells is mediated, at least in part, by canonical Wnt and that its epigenetic-dependent loss can be pro-tumourigenic. [Conclusions]: Our data show the importance of epigenetic alterations of ROR2 in colon cancer, highlighting the close interconnection between canonical and non-canonical Wnt signalling pathways in this type of tumour., AM is funded by the Spanish Ministerio de Ciencia e Innovación (MICINN; SAF2007-60341, ISCIII-RETIC RD06/0020/0009), VC receives a fellowship from the Spanish FPU Research Programme, EL and CH are recipients of fellowships from the Spanish FIS Research Programme. The Instituto Universitario de Oncología is supported by Obra Social Cajastur, Spain. This work was supported by the MICINN (PI061267; PS09/02454; Ref. 200820I172).

We evaluate whether 1,25(OH)2D3 downregulates TP73 variants in colon and breast carcinomas, the role of survivin in this context, and the significance of this network in the clinic. Tumor cells were treated/untreated with 1,25(OH)2D3 and transiently transfected with survivin. Levels of survivin and TP73 variants were evaluated by quantitative RT-PCR and Western blotting. In 75 colon and 60 breast cancer patients, the expressions of survivin and TP73 isoforms were determined. Tumor characteristics were examined in each patient. Survivin protein levels were also evaluated in a subgroup of patients and cell lines. Decrease in survivin and TAp73 transcripts and protein and ΔNp73 mRNA was detected after 1,25(OH)2D3 treatment. Ectopic survivin expression led to an increase in the TAp73, ΔNp73, ΔEx2p73, and ΔEx2-3p73 transcripts. In cancer patients, direct correlations were observed between TP73 variants and survivin levels. 1,25(OH)2D3 negatively regulate survivin and TP73 variants in colon and breast cancer cells. Positive regulation of TP73 isoforms by survivin may exist, which reinforces the possibility that the downregulation of TP73 forms by 1,25(OH)2D3 is survivin-dependent., Supported by: Fundación MMA, Grant numbers: FIS PI08/0605, ISCIII-RTICC-RD06/0020/0020, ISCIII-ATC and SAF2007-60214.

Tumours of the Ewing family, which comprise Ewing's sarcoma and peripheral primitive neuroectodermal tumours, are highly aggressive and mostly affect children and adolescents. They are characterized by chromosomal translocations leading to the generation of fusion proteins between EWS (or very rarely FUS) and members of the E-twenty-six (ETS) family of transcription factors that are capable of transforming cells. EWS/FLI1, the most frequent fusion, is thought to cause transformation through activation or repression of specific target genes. We present evidence demonstrating that the Wnt inhibitor and β-catenin/T-cell factor (TCF)-responsive gene DICKKOPF-1 (DKK-1) is a transcriptional target of EWS/FLI1, which can inhibit both basal and β-catenininduced transactivation of the DKK-1 promoter. Moreover, our data indicate that EWS/FLI1 has a more general effect on β-catenin/TCF-mediated transcription since it can block transactivation of a consensus β-catenin/TCF reporter construct. Consistently, Ewing tumour cells expressing different EWS/ETS translocations cannot engage β-catenin/TCF-dependent transcription, whereas silencing of EWS/FLI1 restores β-catenin responsiveness in A673 and RD-ES Ewing tumour cells. Accordingly, gene set enrichment analysis shows that β-catenin/TCF target genes are significantly enriched among genes downregulated by EWS/FLI1 in the Ewing cell line A673. Mechanistically, the inhibitory effect of EWS/FLI1 can be overcome by a constitutively active TCF4 protein (TCF4-VP16). Moreover, EWS/FLI1 binds lymphoid enhancer factor 1, a TCF family member, and interferes with its binding to β-catenin, which could explain its negative effect on β-catenin/TCF-mediated transcription. Our results show that EWS/FLI1 inhibits both DKK-1 expression as well as β-catenin/TCF-dependent transcription, which could contribute to progression of tumours of the Ewing family. © The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org., Ministerio de Educación y Ciencia (BFU2007-63038/BMC to J.M.G-S., SAF2007-62101 to J.A.); Fundación Científica de la Asociación Española Contra el Cáncer and Fundación Roviralta (to J.A.).

Colorectal cancer is a major health problem worldwide. Aberrant activation of the Wingless-type mouse mammary tumour virus integration site family (Wnt)/beta-catenin signalling pathway due to mutation of adenomatous polyposis coli (APC), beta-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, 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits beta-catenin transcriptional activity by promoting vitamin D receptor (VDR) binding to beta-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/beta-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/beta-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/beta-catenin pathway in colon cancer cells that is in line with its protective effect against this neoplasia.

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).

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).

Colorectal cancer is a major health problem worldwide. Epidemiological studies and work on experimental animals strongly suggest a protective effect of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) against colon neoplasia. 1,25(OH)2D3 is a pleiotropic hormone that has multiple actions in the organism. By binding to the widely expressed high affinity vitamin D receptor (VDR) it regulates the transcription rate of many genes. Other non-genomic effects of 1,25(OH)2D3 also appear to modulate the physiology of numerous cell types. Human normal and cancer colon epithelial cells express VDR and the key enzymes involved in 1,25(OH)2D3 synthesis and degradation and are, thus, responsive to the hormone. 1,25(OH)2D3 inhibits proliferation, induces differentiation and sometimes the apoptosis of human colon cancer cells. A great variety of mechanisms and signaling pathways are involved. Since VDR mediates most, if not all, 1,25(OH)2D3 actions, the control of VDR expression is a crucial aspect of 1,25(OH)2D3 biology. Here, the molecular mechanisms underlying the actions of 1,25(OH)2D3 are reviewed and the repression of the VDR gene by the transcription factor SNAIL in human colon cancer cells is discussed. Understanding these mechanisms may provide the basis for the potential use of this hormone and its non-hypercalcemic derivatives in the prevention and treatment of 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.

Secreted signaling proteins of the Wnt family are known to regulate a diverse range of developmental processes, and their signaling pathway through beta-catenin is frequently activated in cancer. The identification of both Frizzled and LRP5/6 (LRP: low-density lipoprotein receptor-related protein) proteins as components of cell-surface receptors for Wnt proteins has raised questions about their individual functions. We have investigated this issue through a structure-function analysis of Frizzled and LRP proteins that have been implicated in Wnt1 signaling. Consistent with other reports, we find that LRP6/Arrow proteins deleted for their extracellular domain are able to activate the Wnt/beta-catenin signaling pathway. Importantly, our results demonstrate that this signaling from LRP6/Arrow derivatives can occur in a Frizzled- and ligand-independent manner. Furthermore, we show that the PPSP motifs within the intracellular domain of LRP6 are required for signaling. In contrast to results with LRP6, overexpression of Frizzled proteins did not activate the pathway. Based on evidence of ligand binding to both Frizzled and LRP6, current models suggest that both proteins are components of a Wnt receptor complex that signals to beta-catenin. In light of these models, our data imply that LRP5/6/Arrow proteins constitute the distal signal-initiating component of these receptors. The results also support the notion that LRP5/6 are candidate oncogenes.

Thyroid hormone (triiodothyronine, T3) is a pleiotropic regulator of growth, differentiation and tissue homeostasis in higher organisms that acts through the control of target gene expression. Most, if not all, major T3 actions are mediated by specific high affinity nuclear receptors (TR) which are encoded by two genes, THRA and THRB. Several TRα and TRβ receptor isoforms are expressed. Abundant and contradictory literature exists on the relationship between circulating thyroid hormone levels, thyroid diseases and human cancer. In 1986, a connection between TR and cancer became evident when the chicken TRα1 was characterized as the c-erbA proto-oncogene, the cellular counterpart of the retroviral v-erbA oncogene. V-erbA causes erythroleukemias and sarcomas in birds, and hepatocellular carcinomas in transgenic mice. In recent years, many studies have analyzed the presence of quantitative (abnormal levels) or qualitative (mutations) alterations in the expression of THR genes in different types of human neoplasias. While their role in tumor generation or progression is currently unclear, both gross chromosomal and minor mutations (deletions, aberrant splicing, point mutations) and changes in the level of expression of THRA and THRB genes have been found. Together with other in vitro data indicating connections between TR and p53, Rb, cyclin D and other cell cycle regulators and oncogenes, these results suggest that THRA and THRB may be involved in human cancer. © 2002 Elsevier Science Ireland Ltd. All rights reserved., The work of the authors is supported by Grants 08.1/0069/2000 from the Comunidad Autónoma de Madrid and SAF2001-2291 from Ministerio de Ciencia y Tecnología of Spain.

Glucocorticoid hormones have multiple important physiological and therapeutic activities. They function by controlling the expression of target genes through the binding to specific glucocorticoid receptors (GR) which belong to the nuclear receptor superfamily of ligand-modulated transcription factors (Beato et al. 1995; Mangelsdorf et al. 1995). Glucocorticoids regulate their target genes mainly at the transcriptional level, though examples of post-transcriptional regulation have also been reported. Transcriptionally regulated genes can be directly modulated through the binding of ligand-bound GR homodimers to regulatory sequences termed glucocorticoid response elements (GREs). This mechanism is known as trans-activation. In addition, the transcription of target genes can be regulated via interactions (“cross-talk”) of hormone-activated GR with other transcription factors. These interactions are often mutually antagonistic, but in other cases are unidirectionally antagonistic or even synergistic, depending on the factors involved and also the specific gene and cell type (Gottlicher et al. 1998). The inhibition of the gene regulatory effects of other factors by GR is commonly termed as trans-repression (Vayssiere et al. 1997; Resche-Rigon and Gronemeyer 1998).

et al., The β-catenin signaling pathway is deregulated in nearly all colon cancers. Nonhypercalcemic vitamin D3 (1α,25-dehydroxyvitamin D3) analogues are candidate drugs to treat this neoplasia. We show that these compounds promote the differentiation of human colon carcinoma SW480 cells expressing vitamin D receptors (VDRs) (SW480-ADH) but not that of a malignant subline (SW480-R) or metastasic derivative (SW620) cells lacking VDR. 1α,25(OH)2D3 induced the expression of E-cadherin and other adhesion proteins (occludin, Zonula occludens [ZO]-1, ZO-2, vinculin) and promoted the translocation of β-catenin, plakoglobin, and ZO-1 from the nucleus to the plasma membrane. Ligand-activated VDR competed with T cell transcription factor (TCF)-4 for β-catenin binding. Accordingly, 1α,25(OH)2D3 repressed β-catenin–TCF-4 transcriptional activity. Moreover, VDR activity was enhanced by ectopic β-catenin and reduced by TCF-4. Also, 1α,25(OH)2D3 inhibited expression of β-catenin–TCF-4-responsive genes, c-myc, peroxisome proliferator-activated receptor δ, Tcf-1, and CD44, whereas it induced expression of ZO-1. Our results show that 1α,25(OH)2D3 induces E-cadherin and modulates β-catenin–TCF-4 target genes in a manner opposite to that of β-catenin, promoting the differentiation of colon carcinoma cells., H.G. Pálmer and J.M. González-Sancho were recipients of fellowships from the Comunidad Autónoma de Madrid. This work was supported by a grant from the Plan Nacional de Investigación y Desarrollo (SAF98-0060), Ministerio de Ciencia y Tecnología of Spain.

The immunosuppressive and antiinflammatory actions of glucocorticoid hormones are mediated by their transrepression of activating protein-1 (AP-1) and nuclear factor-kappa B (NFκB) transcription factors. Inhibition of the c-Jun NH2-terminal kinase (JNK) signaling pathway, the main mediator of AP-1 activation, has been described in extracts of hormone-treated cells. Here, we show by confocal laser microscopy, enzymatic assays, and immunoblotting that the synthetic glucocorticoid dexamethasone inhibited tumor necrosis factor α (TNF-α)–induced phosphorylation and activation of JNK in the cytoplasm and nucleus of intact HeLa cells. As a result, c-Jun NH2-terminal domain phosphorylation and induction were impaired. Dexamethasone did not block the TNF-α–induced JNK nuclear translocation, but rather induced, per se, nuclear accumulation of the enzyme. Consistently with previous findings, a glucocorticoid receptor mutant (GRdim), which is deficient in dimerization, DNA binding, and transactivation, but retains AP-1 transrepressing activity, was as efficient as wild-type GR in mediating the same effects of dexamethasone on JNK in transfected Cos-7 cells. Our results show that glucocorticoids antagonize the TNF-α–induced activation of AP-1 by causing the accumulation of inactive JNK without affecting its subcellular distribution., This work was supported by grants from the Plan Nacional de Investigación y Desarrollo (SAF98-0060, 1FD97-0281-CO2-01), Comisión Interministerial de Ciencia y Tecnología, and Plan General de Conocimiento (PM96-0035), Ministerio de Educación y Cultura of Spain.

Tenascin-C (Tn-C) is an extracellular matrix protein with growth-, invasive-, and angiogenesis-promoting activities. Tn-C is upregulated during wound healing, tumorigenesis, and other pathological conditions. Highly malignant gliomas with poor prognosis exhibit high levels of Tn-C expression. Here we demonstrate that Tn-C RNA expression in glioma C6 cells is inhibited in a dose-dependent manner by retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (1,25-D3). No additive or synergistic effects were found. Inhibition is maximum 24 hr after RA or 1,25-D3 treatment, prior to a delayed cytotoxic effect starting at day 4-5 of treatment, and correlates with a reduction in the synthesis of Tn-C protein. Tn-C expression is also inhibited, but to a lesser extend by prostaglandin D2 (PGD2). Furthermore, both RA and 1,25-D3, but not PGD2 abolish the induction of Tn-C by the tumor promoter 12-O- tetradecanoyl phorbol 13-acetate. The inhibition of Tn-C expression might be relevant for the anti-cancer activity of RA and 1,25-D3., J.M.G-S. and L.F.G-F. were supported by post-doctoral contracts from Comunidad Autónoma de Madrid, and C.N-Y. by a BEFI fellowship (98/9100) from Fondo de Investigaciones Sanitarias (FIS) del Ministerio de Sanidad y Consumo, respectively.

Tenascin-C is an extracellular matrix protein with growth-, invasion- and angiogenesis-promoting activities. Tenasein-C is upregulated in breast carcinoma and stromal cells, and in many other cell types during tumorigenesis. We demonstrate that tenascin-C RNA expression is inhibited by 1,25-dihydroxyvitamin D3 (1,25-D3) in a variety of mouse and human mammary epithelial cell lines exhibiting normal or malignant phenotype. In EpH4 cells, the inhibition is maximum 24 h after 1,25-D3 treatment and correlates with a dose-dependent reduction in the synthesis of tenascin-C protein. Furthermore, 1,25-D3 also abolishes the induction of tenascin-C by serum or the tumor promoter 12-O-tetradecanoyl phorbol 13-acetate. The inhibition of tenascin-C expression may be relevant for the anticancer activity of 1,25- D3., This research was supported by Grants SAF95-0738 and SAF98-0060 from the Comisión Interministerial de Ciencia y Tecnología, Spain. J.M.G-S. was supported by a pre-doctoral fellowship from Comunidad Autónoma de Madrid.

Tenascin-C is an extracellular matrix glycoprotein involved in cell adhesion and migration, and neurite outgrowth. Since these processes have been found to be under thyroid control in the developing rat brain, we have investigated the effect of congenital hypothyroidism on tenascin-C expression. At birth, in situ hybridization studies in hypothyroid rats show an abnormal up-regulation of tenascin-C in some areas (caudate-putamen, geniculate nuclei, ependymal epithelium of the lateral ventricles, hippocampus) and down-regulation in others (occipital and retrosplenial cortex, subiculum). With subsequent development, hypothyroid animals show higher tenascin-C expression also in the upper layers of the cerebral cortex and subplate, and the Bergmann glia of the cerebellum. Significantly, thyroxine treatment of hypothyroid rats led to normalization of tenascin-C levels in most areas. In agreement with the messenger RNA data, hypothyroid rats contain an uniformly higher level of immunoreactive tenascin-C protein throughout the brain, particularly in the cerebellum. Suggesting a direct cellular effect, thyroid hormone also decreases tenascin-C expression in two glial cell lines (C6, B3.1) expressing thyroid receptors. Our results show that congenital hypothyroidism causes specific alterations in the pattern of tenascin-C expression in the rat brain which may at least partially be responsible for some of the developmental disturbances observed in this syndrome., This work was supported by research grants SAF95-0738 from the Comisión Interministerial de Ciencia y Tecnología, Plan Nacional de I+D of the Ministerio de Educación y Ciencia and 94/0273 from the Fondo de Investigaciones Sanitarias of the Ministerio de Sanidad of Spain.

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.

Stromelysins are a group of proteases which degrade the extracellular matrix and activate other secreted proteases. Stromelysin (ST)-1 and ST-2 genes are induced by tumor promoters, oncogenes and growth factors, and have been involved in acquisition of the malignant phenotype. We show here that the thyroid hormone (T3) increases ST-1 and ST-2 expression in a non-transformed mouse mammary epithelial cell line (EpH4) in a way that is dependent on the level of thyroid receptor/c-erbA (TR alpha-1) expression. In agreement with this, T3 increases the secreted stromelysin activity and enhances the gelatinolytic activity of type IV collagenase. We have also demonstrated that T3 affects the epithelial polarity of EpH4 cells, diminishing the transepithelial electrical resistance of monolayers cultured on permeable filters, causing an abnormal distribution of polarization markers and the disruption of the organized 3-D structures formed by these cells in type I collagen gels. These results indicate that the ligand-activated TR alpha-1 plays an important role in regulating the morphogenetic and invasive capacities of mammary epithelial cells. Because the c-erbA locus is altered in several types of carcinoma, an altered or deregulated TR alpha-1 expression may also be important for breast cancer development and metastasis.