Vitamin D Receptor Deficiency Enhances Wnt/β-Catenin Signaling and Tumor Burden in Colon Cancer

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. Apcmin/+ 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 Apcmin/+ 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 Apcmin/+Vdr-/- mice had increased nuclear β-catenin and the tumors reached a larger size than those of Apcmin/+Vdr+/+. Both ACF and carcinomas in Apcmin/+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)2D3 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

SNAIL vs vitamin D receptor expression in colon cancer: therapeutics implications

Vitamin D analogues with reduced hypercalcemic activity are under clinical investigation for use against colon cancer and other neoplasias. However, only a subset of patients responds to this therapy, most probably due to loss of vitamin D receptor (VDR) expression during tumour progression. Recent data show that SNAIL transcription factor represses VDR expression, and thus abolishes the antiproliferative and prodifferentiation effects of VDR ligands in cultured cancer cells and their antitumour action in xenografted mice. Accordingly, upregulation of SNAIL in human colon tumours associates with downregulation of VDR. These findings suggest that SNAIL may be associated with loss of responsiveness to vitamin D analogues and may thus be used as an indicator of patients who are unlikely to respond to this therapy

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

The active vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) 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)2D3 has several nongenomic effects of uncertain relevance. We show that 1,25(OH)2D3 induces a transcription-independent Ca2+ 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)2D3. RhoA–ROCK and MSK1 are also required for the inhibition of Wnt–β-catenin pathway and cell proliferation. Thus, the action of 1,25(OH)2D3 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

PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex

PTEN controls three-dimensional (3D) glandular morphogenesis by coupling juxtamembrane signalling to mitotic spindle machinery. While molecular mechanisms remain unclear, PTEN interacts through its C2 membrane-binding domain with the scaffold protein β-Arrestin1. Because β-Arrestin1 binds and suppresses the Cdc42 GTPase-activating protein ARHGAP21, we hypothesize that PTEN controls Cdc42-dependent morphogenic processes through a β-Arrestin1-ARHGAP21 complex. Here we show that PTEN knockdown (KD) impairs β-Arrestin1 membrane localization, β-Arrestin1-ARHGAP21 interactions, Cdc42 activation, mitotic spindle orientation and 3D glandular morphogenesis. Effects of PTEN-deficiency were phenocopied by β-Arrestin1 KD or inhibition of β-Arrestin1-ARHGAP21 interactions. Conversely, silencing of ARHGAP21 enhanced Cdc42 activation and rescued aberrant morphogenic processes of PTEN-deficient cultures. Expression of the PTEN C2 domain mimicked effects of full-length PTEN but a membrane-binding defective mutant of the C2 domain abrogated these properties. Our results show that PTEN controls multicellular assembly through a membrane-associated regulatory protein complex composed of β-Arrestin1, ARHGAP21 and Cdc42

SPROUTY-2 represses the epithelial phenotype of colon carcinoma cells via upregulation of ZEB1 mediated by ETS1 and miR-200/miR-150

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 LLGLL2/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 (EGF) 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 A.M., SAF2011-29530 to F.X.R.); FEDERInstituto de Salud Carlos III (RD12/0036/0021 to A.M. and J.M.R., RD12/0036/0034 to F.X.R., RD12/0036/0016 to M.S., RD12/0036/0012 to H.G.P., RD06/0020/0003, PS09/00562 and PI13/00703 to J.M.R.); Comunidad de Madrid (S2010/BMD-2344 Colomics2 to A.M.); Fundación Científica de la Asociación Española contra el Cáncer (to J.M.R.); U.S. Department of Defense (CA093471 and CA110602 to E.H.); National Institutes of Health/National Cancer Institute (1R01CA155234-01 to E.H.); National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (1R21AR062239-01 to E.H.); and the Melanoma Research Alliance (to E. H.)

The Vitamin D Receptor Is a Wnt Effector that Controls Hair Follicle Differentiation and Specifies Tumor Type in Adult Epidermis

We have investigated how Wnt and vitamin D receptor signals regulate epidermal differentiation. Many epidermal genes induced by β-catenin, including the stem cell marker keratin 15, contain vitamin D response elements (VDREs) and several are induced independently of TCF/Lef. The VDR is required for β-catenin induced hair follicle formation in adult epidermis, and the vitamin D analog EB1089 synergises with β-catenin to stimulate hair differentiation. Human trichofolliculomas (hair follicle tumours) are characterized by high nuclear β-catenin and VDR, whereas infiltrative basal cell carcinomas (BCCs) have high β-catenin and low VDR levels. In mice, EB1089 prevents β-catenin induced trichofolliculomas, while in the absence of VDR β-catenin induces tumours resembling BCCs. We conclude that VDR is a TCF/Lef-independent transcriptional effector of the Wnt pathway and that vitamin D analogues have therapeutic potential in tumors with inappropriate activation of Wnt signalling

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

El pdf del artículo es la versión pre-print.-- et al.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 b-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. © 2012 Nature America, Inc. All rights reserved.Experiments were supported by a VHIO starting grant and grants from Fondo de Investigaciones Sanitarias–Instituto de Salud Carlos III (ISCIII) (FIS-PI081356, RETICC-RD06/0020/0075 and RETICC-RD06/0020/0009), and Plan Nacional de Biomedicina, Ministerio de Ciencia e Innovación (SAF-18302). S.P.T. was supported by a Fundació Olga Torres Fellowship, I.P. was funded by the Fundación Científica de la Asociación Española Contra el Cancer (AECC), and H.G.P. was supported by the Miguel Servet Program, ISCIII.Peer Reviewe

Efectos de la 1'alfa', 25-dihidroxivitamina D3 en células de cáncer de cólon humano

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 21-09-2001La sefialización por la ruta de la fi-catenina se encuentra alterada en prácticamente todos los cánceres de colon. Los análogos no hipercalcémicos de la vitamina D, (la,25(OH)zD1) son drogas con posible acción sobre este tipo de neoplasia. Nosotros mostramos que estos compuestos promueven la diferenciación de células de carcinoma de colon humano, SW480, que expresan el receptor de la vitamina D (VDR) (SW480- ADH), mientras que no tienen efecto sobre la sublínea SW480-R o la linea metastásica SW620, que han perdido la expresión de VDR. la,25(OH)zD, induce la expresión de E-cadherina y otras proteínas de adhesión intercelular (ocludina, ZO-1, 20-2, vinculina). Este tratamiento también provocó el aumento progresivo de la resistencia transepitelial (TER) en las células SW480-ADH, indicando la adquisición de uniones ocluyentes funcionales. La actinomicina D bloqiieo completamente la inducción de E-cadherina, mientras la cicloheximida mostró una fuerte inhibición. Por otro lado, el tratamiento con la,25(OH)?D, indujo 1.8 veces la transcripción de al construcción con 1.1 kb del promotor del gen humano de la E-cadherina. Sin embargo, la vida media del mRNA de la E-cadherina no se vió afectada por la,25(OH)zD1. en la células SW480-ADH. Todo ello indica que probablemente la la,25(OH)~D3 induce la expresión de E-cadherina por un mecanismo transcripcional que requiere la síntesis de proteinas tic t~ovo. La la,25(OH).& promueve la translocación de p-catenina, placoglobina y 20-1 desde el núcleo y el citoplasma a la membrana citoplasmatica, inhibiendo probablemente así su actividad transcripcional. El VDR activado por su ligando es capaz de competir con el TCF-4 por su unión a fi-catenina. De acuerdo con ello, observamos que el TCF-4 es capaz de bloquear la actividad transcripcional del VDR, y que por el contrario, la B-catenina es capaz de potenciar la inducción por la,25(OH)zD3 de un VDRE consenso. En consecuencia, observamos que la,25(OH)rD, reprime los genes diana de fi-catenianlTCF-4: C-myc, PPARG, TcJl y CD44, mientras que induce ZO-1. Tres análogos de la vitamina D con bajo efecto hipercalcémico, MC903, KH1060 y EB1089, actualmente utilizados en varios ensayos clínicos contra diversas neoplasisas, fueron capaces de inducir los mismos efectos que la la,25(OHhD~s obre la células SW480-ADH. Los tres compuestos indujeron Ecadherina, inhibieron la actividad p-cateninaRCF-4, y promovieron la diferenciación morfológica y la relocalización de ptatenina en estas c6lulas. mientras que no tuvieron ningún efecto sobre las lineas SW480-R y SW620 defectivas en VDR.The p-catenin signaling pathway is deregulated in nearly al1 colon cancers. Nonhipercalcemic vitamin 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 (VDR) (SW480-ADH), but not of a malignant subline (SW480-R) or metastasic denvative (SW620) cells lacking VDR. la,25(OH)~Djin duced the expression of E-cadhenn and other adhesion proteins (occludin, 20-1, 20-2, vinculin). la,25(OH)~D,) progressively increased the transepithelial resistance (TER) of SW480-ADH cells, indicating the formation of functional tight junctions. Actinomycin D completely blocked la,25(OH)zD3) induction of E-cadhenn, whereas cycloheximide strongly inhibited it. 1.8-fold induction of a 1.1 kb promoter construct of the humrin E-cadhenn gene, was observed. In contrast, 1a,7S(OH)2Dj did not modulate the half-life of E-cadhenn mRNA in SW480-ADH cells. All of this indicates that probably, la,2S(OH)zD3 induces the E-cadhenn expresion by a transcriptional mechanism that requires protein synthesis de novo. la,Z5(OH):D3 promoted the translocation of 0-catenin, plakoglobin, and 20-1 from the nucleus and citoplasm to the plasma membrane, probably inhibiting their transcriptional activity. Ligand-activated VDR competed wvith TCF-4 for p-catenin binding, and inhibited the transcriptional activity of PtateniniTCF-4 complexes. In line with this, TCF-4 was able to hinder VDR tnnscnptional activaty, and in contrast, B-catenin fostered the induction by l a,25(OH)zD3 of a consensus VDRE Accordinyly, l a,25(OH)zD3 repressed p-catenin-TCF-4-responsive genes c-myc, PPARG, Tcf-1 and CD44 bvhile induced ZO-1 Three synthetic vitamin D analogues with low calcemic propierties, MC903, KH1060, and EB1089, currently used in clinical trials to treat several neoplasias, induced the same effects that la,2S(OH)-D3, in SW80-ADH cells. All three compounds induced Ecadhenn, inhibited the psateniniTCF-4 activity, and promoted de morphological differentiation and nuclear export of P-catenin in SW480-ADH cells, but had no effect in VDRdefective SW480-R or SW620 cells