Publications by authors named "Paloma Ordóñez-Morán"

30 Publications

  • Page 1 of 1

High-content, targeted RNA-seq screening in organoids for drug discovery in colorectal cancer.

Cell Rep 2021 Apr;35(3):109026

Swiss Institute for Experimental Cancer Research (ISREC), École Polytechnique Fédérale de Lausanne-(EPFL-SV), 1015 Lausanne, Switzerland. Electronic address:

Organoids allow the recapitulation of intestinal homeostasis and cancerogenesis in vitro; however, RNA sequencing (RNA-seq)-based methods for drug screens are missing. We develop targeted organoid sequencing (TORNADO-seq), a high-throughput, high-content drug discovery platform that uses targeted RNA-seq to monitor the expression of large gene signatures for the detailed evaluation of cellular phenotypes in organoids. TORNADO-seq is a fast, highly reproducible time- and cost-effective ($5 per sample) method that can probe cell mixtures and their differentiation state in the intestinal system. We apply this method to isolate drugs that enrich for differentiated cell phenotypes and show that these drugs are highly efficacious against cancer compared to wild-type organoids. Furthermore, TORNADO-seq facilitates in-depth insight into the mode of action of these drugs. Our technology can easily be adapted to many other systems and will allow for more systematic, large-scale, and quantitative approaches to study the biology of complex cellular systems.
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http://dx.doi.org/10.1016/j.celrep.2021.109026DOI Listing
April 2021

Genomic Instability Profiles at the Single Cell Level in Mouse Colorectal Cancers of Defined Genotypes.

Cancers (Basel) 2021 Mar 12;13(6). Epub 2021 Mar 12.

Department of Molecular Biology, University of Geneva, 1211 Geneva, Switzerland.

The genomes of many human CRCs have been sequenced, revealing a large number of genetic alterations. However, the molecular mechanisms underlying the accumulation of these alterations are still being debated. In this study, we examined colorectal tumours that developed in mice with , , and targetable alleles. Organoids were derived from single cells and the spectrum of mutations was determined by exome sequencing. The number of single nucleotide substitutions (SNSs) correlated with the age of the tumour, but was unaffected by the number of targeted cancer-driver genes. Thus, tumours that expressed mutant , and alleles had as many SNSs as tumours that expressed only mutant . In contrast, the presence of large-scale (>10 Mb) copy number alterations (CNAs) correlated strongly with inactivation. Comparison of the SNSs and CNAs present in organoids derived from the same tumour revealed intratumoural heterogeneity consistent with genomic lesions accumulating at significantly higher rates in tumour cells compared to normal cells. The rate of acquisition of SNSs increased from the early stages of cancer development, whereas large-scale CNAs accumulated later, after inactivation. Thus, a significant fraction of the genomic instability present in cancer cells cannot be explained by aging processes occurring in normal cells before oncogenic transformation.
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http://dx.doi.org/10.3390/cancers13061267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999300PMC
March 2021

An Intrasplenic Injection Model for the Study of Cancer Stem Cell Seeding Capacity.

Methods Mol Biol 2020 ;2171:293-302

Division of Cancer & Stem Cells, School of Medicine, Biodiscovery Institute, Centre for Cancer Sciences, University of Nottingham, Nottingham, UK.

In many tumor types, only a minor pool of cancer cells-the so-called cancer stem cells-is able to colonize distant organs and give rise to secondary tumors. In humans, the liver is one of the main target organs for many metastatic tumor types, including colorectal cancer. However, mouse tumour models only rarely spontaneously metastasize to the liver. Therefore, reliable in vivo experimental metastasis assays are crucial to study cell seeding capacity and the mechanisms controlling these metastatic stem cell properties. Here, we describe an intrasplenic injection model that mimics the process of liver metastasis occurring in cancer patients.
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http://dx.doi.org/10.1007/978-1-0716-0747-3_20DOI Listing
March 2021

Specific Gene Expression in Lgr5 Stem Cells by Using Cre-Lox Recombination.

Methods Mol Biol 2020 ;2171:249-255

Division of Cancer & Stem Cells, School of Medicine, Biodiscovery Institute, Centre for Cancer Sciences, University of Nottingham, Nottingham, UK.

Intestinal stem cells are responsible for tissue renewal. The study of stem cell properties has become a major challenge in the field. We describe here a method based on Cre recombinase inducible lentivirus vectors that permits delivery of transgenes, either for overexpression or knockdown, in primary stem cells that can be cultured in an 3D intestinal organoid system. This method is an excellent approach for genetic manipulation and can complement in vivo transgenic experiments.
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http://dx.doi.org/10.1007/978-1-0716-0747-3_16DOI Listing
March 2021

Large-Scale Production of Recombinant Noggin and R-Spondin1 Proteins Required for the Maintenance of Stem Cells in Intestinal Organoid Cultures.

Methods Mol Biol 2020 ;2171:171-184

Division of Cancer and Stem Cells, School of Medicine, Centre for Cancer Sciences, Biodiscovery Institute, University of Nottingham, Nottingham, UK.

The presence of the proteins mouse R-Spondin1 (mRSpo1) and mouse Noggin (mNoggin) in a 3D-organoid culture allows for the maintenance of intestinal stem cells. Here, we describe a transient gene expression method for the production of these proteins from human embryo kidney 293 (HEK293) cells cultivated in suspension using orbitally shaken bioreactors. Plasmid DNA was delivered into cells using the cationic polymer polyethylenimine (PEI). The 7-day production cultures were performed in the presence of valproic acid (VPA), an enhancer of recombinant gene expression. Both proteins were secreted from the transfected cells. mRSpo1 was produced as a secreted Fc fusion protein (mRSpo1-Fc) and purified by protein A-based affinity chromatography. mNoggin was produced as a secreted histidine-tagged protein (mNoggin-His) and purified by immobilized metal affinity chromatography (IMAC). This transient transfection system supports a high production efficiency.
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http://dx.doi.org/10.1007/978-1-0716-0747-3_10DOI Listing
March 2021

Single-Cell Studies of Intestinal Stem Cell Heterogeneity During Homeostasis and Regeneration.

Methods Mol Biol 2020 ;2171:155-167

Division of Digestive and Liver Diseases, Department of Medicine, Columbia Center for Human Development, Columbia Stem Cell Initiative, New York, NY, USA.

Single-cell RNA-sequencing (scRNA-seq) provides a unique opportunity to study heterogeneous cell populations within tissues, including the intestinal epithelium, to gain detailed molecular insights into their biology. Many new putative markers of intestinal stem cells and their progeny have been described using single-cell transcriptomics, which has contributed to the identification of novel subpopulations of mature cell types and insight into their developmental trajectories. This approach has revealed tremendous cellular heterogeneity within the intestinal epithelium that is concordant with its diverse and multifaceted functions. We discuss the function of these subpopulations during tissue homeostasis, as well as putative subpopulations with inducible regenerative potential following tissue injury.
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http://dx.doi.org/10.1007/978-1-0716-0747-3_9DOI Listing
March 2021

Intestinal Stem Cell Niche Insights Gathered from Both and Novel Models.

Stem Cells Int 2017 7;2017:8387297. Epub 2017 Sep 7.

Swiss Institute for Experimental Cancer Research (ISREC), École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

Intestinal stem cells are located at the base of the crypts and are surrounded by a complex structure called niche. This environment is composed mainly of epithelial cells and stroma which provides signals that govern cell maintenance, proliferation, and differentiation. Understanding how the niche regulates stem cell fate by controlling developmental signaling pathways will help us to define how stem cells choose between self-renewal and differentiation and how they maintain their undifferentiated state. Tractable assay systems, which reflect the complexity of the situation but provide higher level of control, would likely be crucial in identifying new players and mechanisms controlling stem cell function. Knowledge of the intestinal stem cell niche gathered from both and novel models may help us improve therapies for tumorigenesis and intestinal damage and make autologous intestinal transplants a feasible clinical practice.
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http://dx.doi.org/10.1155/2017/8387297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5610807PMC
September 2017

Cross-Tissue Identification of Somatic Stem and Progenitor Cells Using a Single-Cell RNA-Sequencing Derived Gene Signature.

Stem Cells 2017 12 6;35(12):2390-2402. Epub 2017 Nov 6.

Laboratory of Systems Biology and Genetics, Institute of Bioengineering and Swiss Institute of Bioinformatics, CH-1015, Lausanne, Switzerland.

A long-standing question in biology is whether multipotent somatic stem and progenitor cells (SSPCs) feature molecular properties that could guide their system-independent identification. Population-based transcriptomic studies have so far not been able to provide a definite answer, given the rarity and heterogeneous nature of these cells. Here, we exploited the resolving power of single-cell RNA-sequencing to develop a computational model that is able to accurately distinguish SSPCs from differentiated cells across tissues. The resulting classifier is based on the combined expression of 23 genes including known players in multipotency, proliferation, and tumorigenesis, as well as novel ones, such as Lcp1 and Vgll4 that we functionally validate in intestinal organoids. We show how this approach enables the identification of stem-like cells in still ambiguous systems such as the pancreas and the epidermis as well as the exploration of lineage commitment hierarchies, thus facilitating the study of biological processes such as cellular differentiation, tissue regeneration, and cancer. Stem Cells 2017;35:2390-2402.
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http://dx.doi.org/10.1002/stem.2719DOI Listing
December 2017

Enhanced Rate of Acquisition of Point Mutations in Mouse Intestinal Adenomas Compared to Normal Tissue.

Cell Rep 2017 06;19(11):2185-2192

Department of Molecular Biology, University of Geneva, 1211 Geneva, Switzerland; Biomedical Research Foundation of the Academy of Athens, 11527 Athens, Greece. Electronic address:

The most prevalent single-nucleotide substitution (SNS) found in cancers is a C-to-T substitution in the CpG motif. It has been proposed that many of these SNSs arise during organismal aging, prior to transformation of a normal cell into a precancerous/cancer cell. Here, we isolated single intestinal crypts derived from normal tissue or from adenomas of Apc mice, expanded them minimally in vitro as organoids, and performed exome sequencing to identify point mutations that had been acquired in vivo at the single-cell level. SNSs, most of them being CpG-to-TpG substitutions, were at least ten times more frequent in adenoma than normal cells. Thus, contrary to the view that substitutions of this type are present due to normal-cell aging, the acquisition of point mutations increases upon transformation of a normal intestinal cell into a precancerous cell.
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http://dx.doi.org/10.1016/j.celrep.2017.05.051DOI Listing
June 2017

Designer matrices for intestinal stem cell and organoid culture.

Nature 2016 11 16;539(7630):560-564. Epub 2016 Nov 16.

Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences (SV) and School of Engineering (STI), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

Epithelial organoids recapitulate multiple aspects of real organs, making them promising models of organ development, function and disease. However, the full potential of organoids in research and therapy has remained unrealized, owing to the poorly defined animal-derived matrices in which they are grown. Here we used modular synthetic hydrogel networks to define the key extracellular matrix (ECM) parameters that govern intestinal stem cell (ISC) expansion and organoid formation, and show that separate stages of the process require different mechanical environments and ECM components. In particular, fibronectin-based adhesion was sufficient for ISC survival and proliferation. High matrix stiffness significantly enhanced ISC expansion through a yes-associated protein 1 (YAP)-dependent mechanism. ISC differentiation and organoid formation, on the other hand, required a soft matrix and laminin-based adhesion. We used these insights to build a fully defined culture system for the expansion of mouse and human ISCs. We also produced mechanically dynamic matrices that were initially optimal for ISC expansion and subsequently permissive to differentiation and intestinal organoid formation, thus creating well-defined alternatives to animal-derived matrices for the culture of mouse and human stem-cell-derived organoids. Our approach overcomes multiple limitations of current organoid cultures and greatly expands their applicability in basic and clinical research. The principles presented here can be extended to identify designer matrices that are optimal for long-term culture of other types of stem cells and organoids.
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http://dx.doi.org/10.1038/nature20168DOI Listing
November 2016

HOXA5 Counteracts Stem Cell Traits by Inhibiting Wnt Signaling in Colorectal Cancer.

Cancer Cell 2015 Dec;28(6):815-829

École Polytechnique Fédérale de Lausanne (EPFL), ISREC (Swiss Institute for Experimental Cancer Research), Lausanne 1015, Switzerland. Electronic address:

Hierarchical organization of tissues relies on stem cells, which either self-renew or produce committed progenitors predestined for lineage differentiation. Here we identify HOXA5 as an important repressor of intestinal stem cell fate in vivo and identify a reciprocal feedback between HOXA5 and Wnt signaling. HOXA5 is suppressed by the Wnt pathway to maintain stemness and becomes active only outside the intestinal crypt where it inhibits Wnt signaling to enforce differentiation. In colon cancer, HOXA5 is downregulated, and its re-expression induces loss of the cancer stem cell phenotype, preventing tumor progression and metastasis. Tumor regression by HOXA5 induction can be triggered by retinoids, which represent tangible means to treat colon cancer by eliminating cancer stem cells.
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http://dx.doi.org/10.1016/j.ccell.2015.11.001DOI Listing
December 2015

Polycomb Complex PRC1 Preserves Intestinal Stem Cell Identity by Sustaining Wnt/β-Catenin Transcriptional Activity.

Cell Stem Cell 2016 Jan 29;18(1):91-103. Epub 2015 Oct 29.

Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy. Electronic address:

Polycomb repressive complexes (PRCs) are among the most important gatekeepers of establishing and maintaining cell identity in metazoans. PRC1, which plays a dominant role in this context, executes its functions via multiple subcomplexes, which all contribute to H2AK119 mono-ubiquitination (H2Aubq). Despite our comprehensive knowledge of PRC1-dependent H2Aubq in embryonic stem cells and during early development, its role in adult stem cells still remains poorly characterized. Here we show that PRC1 activity is required for the integrity of the intestinal epithelium, regulating stem cell self-renewal via a cell-autonomous mechanism that is independent from Cdkn2a expression. By dissecting the PRC1-dependent transcription program in intestinal stem cells, we demonstrate that PRC1 represses a large number of non-lineage-specific transcription factors that directly affect β-catenin/Tcf transcriptional activity. Our data reveal that PRC1 preserves Wnt/β-catenin activity in adult stem cells to maintain intestinal homeostasis and supports tumor formation induced by the constitutive activation of this pathway.
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http://dx.doi.org/10.1016/j.stem.2015.09.019DOI Listing
January 2016

Complex metastatic niches: already a target for therapy?

Curr Opin Cell Biol 2014 Dec 16;31:29-38. Epub 2014 Jul 16.

École Polytechnique Fédérale de Lausanne (EPFL), ISREC (Swiss Institute for Experimental Cancer Research), Lausanne, Switzerland. Electronic address:

Metastatic spread is an inefficient process which requires generation of supportive microenvironments in which cancer cells can survive, proliferate and escape from immune attack. These niches are induced by systemic and locally produced factors and establish a tumor-supportive and immune suppressive environment which is molecularly and functionally different from the niche at the primary site. Tumor dormancy may result if the niche is not sufficiently supportive/protective. Co-evolution of cancer cells and the surrounding microenvironment creates a large number of such dynamic niches, and we are just beginning to elucidate the complexity of these interactions and their tissue-specific differences. We will discuss exciting possibilities but also challenges which are immanent when trying to target these stromal responses for diagnosis and therapy.
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http://dx.doi.org/10.1016/j.ceb.2014.06.012DOI Listing
December 2014

Autolysosomal β-catenin degradation regulates Wnt-autophagy-p62 crosstalk.

EMBO J 2013 Jul 4;32(13):1903-16. Epub 2013 Jun 4.

Cancer Research UK Colorectal Tumour Biology Group, School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK.

The Wnt/β-catenin signalling and autophagy pathways each play important roles during development, adult tissue homeostasis and tumorigenesis. Here we identify the Wnt/β-catenin signalling pathway as a negative regulator of both basal and stress-induced autophagy. Manipulation of β-catenin expression levels in vitro and in vivo revealed that β-catenin suppresses autophagosome formation and directly represses p62/SQSTM1 (encoding the autophagy adaptor p62) via TCF4. Furthermore, we show that during nutrient deprivation β-catenin is selectively degraded via the formation of a β-catenin-LC3 complex, attenuating β-catenin/TCF-driven transcription and proliferation to favour adaptation during metabolic stress. Formation of the β-catenin-LC3 complex is mediated by a W/YXXI/L motif and LC3-interacting region (LIR) in β-catenin, which is required for interaction with LC3 and non-proteasomal degradation of β-catenin. Thus, Wnt/β-catenin represses autophagy and p62 expression, while β-catenin is itself targeted for autophagic clearance in autolysosomes upon autophagy induction. These findings reveal a regulatory feedback mechanism that place β-catenin at a key cellular integration point coordinating proliferation with autophagy, with implications for targeting these pathways for cancer therapy.
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http://dx.doi.org/10.1038/emboj.2013.123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3981178PMC
July 2013

β-catenin confers resistance to PI3K and AKT inhibitors and subverts FOXO3a to promote metastasis in colon cancer.

Nat Med 2012 Jun;18(6):892-901

Vall d’Hebrón Institut d’Oncología (VHIO), Stem Cell and Cancer Laboratory, Barcelona, Spain.

The Wnt–β-catenin and PI3K-AKT-FOXO3a pathways have a central role in cancer. AKT phosporylates FOXO3a, relocating it from the cell nucleus to the cytoplasm, an effect that is reversed by PI3K and AKT inhibitors. Simultaneous hyperactivation of the Wnt–β-catenin pathway and inhibition of PI3K-AKT signaling promote nuclear accumulation of β-catenin and FOXO3a, respectively, promoting cell scattering and metastasis by regulating a defined set of target genes. Indeed, the anti-tumoral AKT inhibitor API-2 promotes nuclear FOXO3a accumulation and metastasis of cells with high nuclear β-catenin content. Nuclear β-catenin confers resistance to the FOXO3a-mediated apoptosis induced by PI3K and AKT inhibitors in patient-derived primary cultures and in corresponding xenograft tumors in mice. This resistance is reversed by XAV-939, an inhibitor of Wnt–β-catenin signaling. In the presence of high nuclear β-catenin content, activation of FOXO3a by PI3K or AKT inhibitors makes it behave as a metastasis inductor rather than a proapoptotic tumor suppressor. We show that it is possible to evaluate the β-catenin status of patients' carcinomas and the response of patient-derived cells to target-directed drugs that accumulate FOXO3a in the nucleus before deciding on a course of treatment. We propose that this evaluation could be essential to the provision of a safer and more effective personalized treatment.
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http://dx.doi.org/10.1038/nm.2772DOI Listing
June 2012

Lrig1: a new master regulator of epithelial stem cells.

EMBO J 2012 May 20;31(9):2064-6. Epub 2012 Mar 20.

Ecole Polytechnique Fédérale de Lausanne, ISREC (Swiss Institute for Experimental Cancer Research) and National Center of Competence in Research Molecular Oncology, Lausanne, Switzerland.

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http://dx.doi.org/10.1038/emboj.2012.73DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3343471PMC
May 2012

Design, synthesis, evaluation, and structure of vitamin D analogues with furan side chains.

Chemistry 2012 Jan 8;18(2):603-12. Epub 2011 Dec 8.

Departamento de Química Orgánica, Universidad de Santiago y Unidad Asociada al CSIC, Avda de las Ciencias s/n, 15782 Santiago de Compostela, Spain.

Based on the crystal structures of human vitamin D receptor (hVDR) bound to 1α,25-dihydroxy-vitamin D(3) (1,25 D) and superagonist ligands, we previously designed new superagonist ligands with a tetrahydrofuran ring at the side chain that optimize the aliphatic side-chain conformation through an entropy benefit. Following a similar strategy, four novel vitamin D analogues with aromatic furan side chains (3a, 3b, 4a, 4b) have now been developed. The triene system has been constructed by an efficient stereoselective intramolecular cyclization of an enol triflate (A-ring precursor) followed by a Suzuki-Miyaura coupling of the resulting intermediate with an alkenyl boronic ester (CD-side chain, upper fragment). The furan side chains have been constructed by gold chemistry. These analogues exhibit significant pro-differentiation effects and transactivation potency. The crystal structure of 3a in a complex with the ligand-binding domain of hVDR revealed that the side-chain furanic ring adopts two conformations.
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http://dx.doi.org/10.1002/chem.201102695DOI Listing
January 2012

β-catenin represses expression of the tumour suppressor 15-prostaglandin dehydrogenase in the normal intestinal epithelium and colorectal tumour cells.

Gut 2012 Sep 14;61(9):1306-14. Epub 2011 Nov 14.

Cancer Research UK Colorectal Tumour Biology Research Group, School of Cellular and Molecular Medicine, University Walk, University of Bristol, Bristol, UK.

Background: Cyclooxygenase-2 (COX-2) overexpression in colorectal cancer increases levels of its pro-tumorigenic product prostaglandin E2 (PGE(2)). The recently identified colorectal tumour suppressor 15-prostaglandin dehydrogenase (15-PGDH) catalyses prostaglandin turnover and is downregulated at a very early stage in colorectal tumorigenesis; however, the mechanism responsible remains unclear. As Wnt/β-catenin signalling is also deregulated early in colorectal neoplasia, a study was undertaken to determine whether β-catenin represses 15-PGDH expression.

Methods: The effect of modulating Wnt/β-catenin signalling (using β-catenin siRNA, mutant TCF4, Wnt3A or GSK3 inhibition) on 15-PGDH mRNA, protein expression and promoter activity was determined in colorectal cell lines by immunoblotting, qRT-PCR and reporter assays. The effect of β-catenin deletion in vivo was addressed by 15-PGDH immunostaining of β-catenin(-/lox)-villin-creERT2 mouse tissue. 15-PGDH promoter occupancy was determined using chromatin immunoprecipitation and PGE(2) levels by ELISA.

Results: The study shows for the first time that β-catenin knockdown upregulates 15-PGDH in colorectal adenoma and carcinoma cells without affecting COX-2 protein levels. A dominant negative mutant form of TCF4 (dnTCF4), unable to bind β-catenin, also upregulated 15-PGDH; conversely, increasing β-catenin activity using Wnt3A or GSK3 inhibition downregulated 15-PGDH. Importantly, inducible β-catenin deletion in vivo also upregulated intestinal epithelial 15-PGDH. 15-PGDH regulation occurred at the protein, mRNA and promoter activity levels and chromatin immunoprecipitation indicated β-catenin/TCF4 binding to the 15-PGDH promoter. β-catenin knockdown decreased PGE(2) levels, and this was significantly rescued by 15-PGDH siRNA.

Conclusion: These data suggest a novel role for β-catenin in promoting colorectal tumorigenesis through very early 15-PGDH suppression leading to increased PGE(2) levels, possibly even before COX-2 upregulation.
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http://dx.doi.org/10.1136/gutjnl-2011-300817DOI Listing
September 2012

Vitamin D receptor deficiency enhances Wnt/β-catenin signaling and tumor burden in colon cancer.

PLoS One 2011 15;6(8):e23524. Epub 2011 Aug 15.

Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain.

Aberrant activation of the Wnt/β-catenin pathway is critical for the initiation and progression of most colon cancers. This activation provokes the accumulation of nuclear β-catenin and the induction of its target genes. Apc(min/+) mice are the most commonly used model for colon cancer. They harbor a mutated Apc allele and develop intestinal adenomas and carcinomas during the first months of life. This phenotype is caused by the mutation of the second Apc allele and the consequent accumulation of nuclear β-catenin in the affected cells. Here we describe that vitamin D receptor (VDR) is a crucial modulator of nuclear β-catenin levels in colon cancer in vivo. By appropriate breeding of Apc(min/+) mice and Vdr(+/-) mice we have generated animals expressing a mutated Apc allele and two, one, or none Vdr wild type alleles. Lack of Vdr increased the number of colonic Aberrant Crypt Foci (ACF) but not that of adenomas or carcinomas in either small intestine or colon. Importantly, colon ACF and tumors of Apc(min/+)Vdr(-/-) mice had increased nuclear β-catenin and the tumors reached a larger size than those of Apc(min/+)Vdr(+/+). Both ACF and carcinomas in Apc(min/+)Vdr(-/-) mice showed higher expression of β-catenin/TCF target genes. In line with this, VDR knock-down in cultured human colon cancer cells enhanced β-catenin nuclear content and target gene expression. Consistently, VDR depletion abrogated the capacity of 1,25(OH)(2)D(3) to promote the relocation of β-catenin from the nucleus to the plasma membrane and to inhibit β-catenin/TCF target genes. In conclusion, VDR controls the level of nuclear β-catenin in colon cancer cells and can therefore attenuate the impact of oncogenic mutations that activate the Wnt/β-catenin pathway.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0023524PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3156234PMC
February 2012

Synthesis and biological evaluation of 1α,25-dihydroxyvitamin D(3) analogues hydroxymethylated at C-26.

J Med Chem 2011 Jun 10;54(11):3950-62. Epub 2011 May 10.

Departamento de Química Orgánica y Unidad Asociada al CSIC, Universidad de Santiago de Compostela, Santiago de Compostela, Spain.

We designed by docking and synthesized two novel analogues of 1α,25-dihydroxyvitamin D(3) hydroxymethylated at C-26 (2 and 3). The syntheses were carried out by the convergent Wittig-Horner approach via epoxide 12a as a common key intermediate. The antiproliferative and transactivation potency of the compounds was evaluated in colon and breast cancer cell lines. The analogues showed a similar but reduced activity compared to 1,25(OH)(2)D(3). Analogue 3 was more potent than analogue 2, and in some assays it exhibited potency similar to that of the natural ligand.
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http://dx.doi.org/10.1021/jm200276yDOI Listing
June 2011

Synthesis, structure, and biological activity of des-side chain analogues of 1α,25-dihydroxyvitamin D3 with substituents at C18.

ChemMedChem 2011 May 29;6(5):788-93. Epub 2011 Mar 29.

Laboratorium voor Experimentele Geneeskunde en Endocrinologie, Katholieke Universiteit Leuven, Leuven, Belgium.

An improved synthetic route to 1α,25-dihydroxyvitamin D(3) des-side chain analogues 2 a and 2 b with substituents at C18 is reported, along with their biological activity. These analogues display significant antiproliferative effects toward MCF-7 breast cancer cells and prodifferentiation activity toward SW480-ADH colon cancer cells; they are also characterized by a greatly decreased calcemic profile. The crystal structure of the human vitamin D receptor (hVDR) complexed to one of these analogues, 20(17→18)-abeo-1α,25-dihydroxy-22-homo-21-norvitamin D(3) (2 a) reveals that the side chain introduced at position C18 adopts the same orientation in the ligand binding pocket as the side chain of 1α,25-dihydroxyvitamin D(3).
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http://dx.doi.org/10.1002/cmdc.201100021DOI Listing
May 2011

The effects of 1,25-dihydroxyvitamin D3 on colon cancer cells depend on RhoA-ROCK-p38MAPK-MSK signaling.

J Steroid Biochem Mol Biol 2010 Jul 17;121(1-2):355-61. Epub 2010 Mar 17.

Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, and Hospital Universitario Puerta de Hierro, Arturo Duperier 4, 28029 Madrid, Spain.

Many studies support a protective action of vitamin D against colon cancer. 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) exerts wide gene regulatory effects in human colon cancer cells. We previously reported that 1,25(OH)2D3 increases cytosolic Ca2+ concentration and transiently activates RhoA and its effector the Rho-associated coiled-kinase (ROCK), and later p38MAPK-MSK. We found that the inhibition of ROCK signaling by Y27632 or that of MSK by Ro318220 prevent the formation of epithelioid islands of SW480-ADH cells by 1,25(OH)2D3 and disrupts the adhesive phenotype of HT29 cells. ROCK and MSK inhibition also abrogates the induction of 1,25(OH)2D3 24-hydroxylase (CYP24), E-cadherin, and vinculin and the repression of cyclin D1 by 1,25(OH)2D3. Moreover, 1,25(OH)2D3 does not promote the localization of the tight junction protein occludin at the plasma membrane in cells expressing a dominant negative RhoA (N19-RhoA). In addition, 1,25(OH)2D3 specifically increases the level of the cysteine protease-inhibitor cystatin D, whereas that of cystatin SN is unaffected. The increase of cystatin D protein caused by 1,25(OH)2D3 is abrogated in N19-RhoA cells. Thus, activation of the RhoA-ROCK-p38MAPK-MSK signaling pathway is essential for the regulation of the phenotype and of the CST5/cystatin D candidate tumor suppressor and other target genes by 1,25(OH)2D3 in colon cancer cells.
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http://dx.doi.org/10.1016/j.jsbmb.2010.02.031DOI Listing
July 2010

Nuclear receptors: genomic and non-genomic effects converge.

Cell Cycle 2009 Jun;8(11):1675-80

Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain

The nuclear receptor superfamily mediates the regulatory activities of many hormones, nutrients and metabolites on the homeostasis and physiology of cells and tissues. Classically, ligand binding induced the ability of nuclear receptors to modulate the transcription rate of target genes (genomic effects), which led to consider them as ligand-activated transcription factors. Later, rapid actions of nuclear receptor ligands were reported that did not involve changes in gene expression. These (non-genomic) effects have been attributed in some cases to receptors different to those mediating gene transcription but most evidences indicate that they result from the activity of a population of nuclear receptor molecules acting outside the cell nucleus. Recent studies on estrogen and vitamin D, and their receptors (ERalpha/beta, VDR) support now the idea that non-genomic and genomic effects may integrate in a unique mode of action of nuclear receptor ligands, in which the non-genomic effects constitute signaling pathways required for the effects at the genome level. Here, we will discuss these novel findings and also those indicating transcriptional regulation through ligand-dependent and -independent crosstalk of nuclear receptors with beta-catenin or VDR-interacting repressor (VDIR).
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http://dx.doi.org/10.4161/cc.8.11.8579DOI Listing
June 2009

RhoA-ROCK and p38MAPK-MSK1 mediate vitamin D effects on gene expression, phenotype, and Wnt pathway in colon cancer cells.

J Cell Biol 2008 Nov;183(4):697-710

Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain.

The active vitamin D metabolite 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits proliferation and promotes differentiation of colon cancer cells through the activation of vitamin D receptor (VDR), a transcription factor of the nuclear receptor superfamily. Additionally, 1,25(OH)(2)D(3) has several nongenomic effects of uncertain relevance. We show that 1,25(OH)(2)D(3) induces a transcription-independent Ca(2+) influx and activation of RhoA-Rho-associated coiled kinase (ROCK). This requires VDR and is followed by activation of the p38 mitogen-activated protein kinase (p38MAPK) and mitogen- and stress-activated kinase 1 (MSK1). As shown by the use of chemical inhibitors, dominant-negative mutants and small interfering RNA, RhoA-ROCK, and p38MAPK-MSK1 activation is necessary for the induction of CDH1/E-cadherin, CYP24, and other genes and of an adhesive phenotype by 1,25(OH)(2)D(3). RhoA-ROCK and MSK1 are also required for the inhibition of Wnt-beta-catenin pathway and cell proliferation. Thus, the action of 1,25(OH)(2)D(3) on colon carcinoma cells depends on the dual action of VDR as a transcription factor and a nongenomic activator of RhoA-ROCK and p38MAPK-MSK1.
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http://dx.doi.org/10.1083/jcb.200803020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2582889PMC
November 2008

The Wnt antagonist DICKKOPF-1 gene is induced by 1alpha,25-dihydroxyvitamin D3 associated to the differentiation of human colon cancer cells.

Carcinogenesis 2007 Sep 21;28(9):1877-84. Epub 2007 Apr 21.

Instituto de Investigaciones Biomédicas Alberto Sols, Facultad de Medicina, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, 4, 28029 Madrid, Spain.

The Wnt-beta-catenin pathway is aberrantly activated in most colon cancers. DICKKOPF-1 (DKK-1) gene encodes an extracellular Wnt inhibitor that blocks the formation of signalling receptor complexes at the plasma membrane. We report that 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3], the most active vitamin D metabolite, increases the level of DKK-1 RNA and protein in human SW480-ADH colon cancer cells. This effect is dose dependent, slow and depends on the presence of a transcription-competent nuclear vitamin D receptor (VDR). Accordingly, 1,25(OH)2D3 activates a 2300 bp fragment of the human DKK-1 gene promoter. Chromatin immunoprecipitation assays revealed that 1,25(OH)2D3 treatment induced a pattern of histone modifications which is compatible with transcriptionally active chromatin. DKK-1 is expressed at high level in colon cancer cell lines with a differentiated phenotype such as Caco-2 or HT-29. Exogenous expression of E-cadherin into SW480-ADH cells results in a strong adhesive phenotype and a 17-fold increase in DKK-1 RNA. In contrast, an E-cadherin blocking antibody inhibits 1,25(OH)2D3-induced differentiation of SW480-ADH cells and DKK-1 gene expression. Remarkably, in vivo treatment with the vitamin D analogue EB1089 induced DKK-1 protein expression in SW480-ADH cells xenografted in immunodeficient mice, and a correlation was observed in the expression of VDR and DKK-1 RNA in a series of 32 human colorectal tumours. These data indicate that 1,25(OH)2D3 activates the transcription of the DKK-1 gene, probably in an indirect way that is associated to the promotion of a differentiated phenotype. DKK-1 gene induction constitutes a novel mechanism of inhibition of Wnt signalling and antitumour action by 1,25(OH)2D3.
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http://dx.doi.org/10.1093/carcin/bgm094DOI Listing
September 2007

The inhibition of Wnt/beta-catenin signalling by 1alpha,25-dihydroxyvitamin D3 is abrogated by Snail1 in human colon cancer cells.

Endocr Relat Cancer 2007 Mar;14(1):141-51

Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain.

The Wnt/beta-catenin signalling pathway is activated in 90% of human colon cancers by nuclear accumulation of beta-catenin protein due to its own mutation or to that of adenomatous polyposis coli. In the nucleus, beta-catenin regulates gene expression promoting cell proliferation, migration and invasiveness. 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits beta-catenin signalling by inducing its binding to vitamin D receptor (VDR) and by promoting beta-catenin nuclear export. The transcription factor Snail1 represses VDR expression and we demonstrate here that Snail1 also abolishes the nuclear export of beta-catenin induced by 1,25(OH)(2)D(3) in SW480-ADH cells. Accordingly, Snail1 relieves the inhibition exerted by 1,25(OH)(2)D(3) on genes whose expression is driven by beta-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)(2)D(3) on cell proliferation and migration. In xenografted mice, Snail1 impedes the nuclear export of beta-catenin and the inhibition of ENC-1 expression induced by EB1089, a 1,25(OH)(2)D(3) 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)(2)D(3). However, this exogenous VDR failed to fully restore the blockage of the Wnt/beta-catenin pathway by 1,25(OH)(2)D(3). 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/beta-catenin signalling pathway in part through the abrogation of the inhibitory action of 1,25(OH)(2)D(3).
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http://dx.doi.org/10.1677/ERC-06-0028DOI Listing
March 2007

Effects of 1alpha,25-dihydroxyvitamin D3 in human colon cancer cells.

Anticancer Res 2006 Jul-Aug;26(4A):2669-81

Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid, E-28029 Madrid, Spain.

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.
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August 2006

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

Front Biosci 2005 Sep 1;10:2723-49. Epub 2005 Sep 1.

Instituto de Investigaciones Biomedicas, Consejo Superior de Investigaciones Cientificas, Universidad Autonoma de Madrid, Madrid, Spain.

1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3, 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.
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http://dx.doi.org/10.2741/1731DOI Listing
September 2005

The transcription factor SNAIL represses vitamin D receptor expression and responsiveness in human colon cancer.

Nat Med 2004 Sep 22;10(9):917-9. Epub 2004 Aug 22.

Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, E-28029 Madrid, Spain.

Several non-hypercalcemic analogs of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) show antitumor activity in a subset of cancer patients. High vitamin D receptor (VDR) expression, which is associated with good prognosis but is lost during tumor progression. We show that the SNAIL transcription factor represses VDR gene expression in human colon cancer cells and blocks the antitumor action of EB1089, a 1,25(OH)(2)D(3) analog, in xenografted mice. In human colon cancers, elevated SNAIL expression correlates with downregulation of VDR.
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http://dx.doi.org/10.1038/nm1095DOI Listing
September 2004

Genetic signatures of differentiation induced by 1alpha,25-dihydroxyvitamin D3 in human colon cancer cells.

Cancer Res 2003 Nov;63(22):7799-806

Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier 4, 28029 Madrid, Spain.

Epidemiological and preclinical data indicate that vitamin D and its most active metabolite 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] have anticancer activity. Accordingly, clinical trials are under way using several nonhypercalcemic 1alpha,25(OH)(2)D(3) analogues against various neoplasms including colon cancer. 1alpha,25(OH)(2)D(3) induces proliferation arrest and epithelial differentiation of human SW480-ADH colon cancer cells. We examined the gene expression profiles associated with 1alpha,25(OH)(2)D(3) exposure using oligonucleotide microarrays. 1alpha,25(OH)(2)D(3) changed the expression levels of numerous previously unreported genes, including many involved in transcription, cell adhesion, DNA synthesis, apoptosis, redox status, and intracellular signaling. Most genes were up-regulated, and only a small fraction were down-regulated. Fourteen of 17 candidate genes studied were validated as 1alpha,25(OH)(2)D(3) target genes by Northern and Western blotting or immunocytochemistry. They included c-JUN, JUNB, JUND, FREAC-1/FoxF1, ZNF-44/KOX7, plectin, filamin, keratin-13, G(0)S2, and the putative tumor suppressors NES-1 and protease M. There was little overlap between genes regulated after short (4 h) or long (48 h) exposure. Gene regulatory effects of 1alpha,25(OH)(2)D(3) in SW480-ADH cells differed from those in LS-174T cells, which lack E-cadherin and do not differentiate in response to 1alpha,25(OH)(2)D(3). Data from this study reveal that 1alpha,25(OH)(2)D(3) causes a profound change in gene expression profiles and provide a mechanistic basis to the ongoing clinical studies using nonhypercalcemic vitamin D(3) derivatives for colon cancer prevention and treatment.
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November 2003