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

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

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

SPROUTY2 is a β-catenin and FOXO3a target gene indicative of poor prognosis in colon cancer

et al.SPROUTY2 (SPRY2) is an intracellular regulator of receptor tyrosine kinase signaling involved in cell growth, differentiation and tumorigenesis. Here, we show that SPRY2 is a target gene of the Wnt/β-catenin pathway that is abnormally activated in more than 90% of colon carcinomas. In human colon cancer cells, SPRY2 expression is induced by β-catenin in co-operation with the transcription factor FOXO3a instead of lymphoid enhancer factor/T-cell factor proteins. We found binding of β-catenin to the SPRY2 promoter at FOXO3a response elements. In vivo, cells marked by nuclear β-catenin and FOXO3a express SPRY2 in proliferative epithelial tissues, such as intestinal mucosa and epidermis. Consistently, inducible β-catenin deletion in mice reduced Spry2 expression in the small intestine. Moreover, SPRY2 protein expression correlated with nuclear β-catenin and FOXO3a colocalization in human colon carcinomas. Importantly, the amount of SPRY2 protein correlated with shorter overall survival of colon cancer patients. Our data reveal SPRY2 as a novel Wnt/β-catenin and FOXO3a target gene indicative of poor prognosis in colon cancer.Experiments were supported by grants from Fondo Europeo de Desarrollo Regional-Instituto de Salud Carlos III and Spanish Cooperative Research Network on Cancer (RTICC) (FIS-PI081356, RD12/0036/0001, RD12/0036/0021 and RD12/0036/0012), Plan Nacional de Biomedicina, Ministerio de Economía y Competitividad (SAF-18302) and Comunidad de Madrid (S2010/BMD-2344 Colomics2). HGP was supported by the Miguel Servet Program, Instituto de Salud Carlos III (ISCIII) and PO-M by an EMBO research fellowship.Peer reviewe

p120 Catenin-associated Fer and Fyn tyrosine kinases regulate β-catenin Tyr-142 phosphorylation and β-catenin-α-catenin interaction

β-Catenin has a key role in the formation of adherens junction through its interactions with E-cadherin and α-catenin. We show here that interaction of β-catenin with α-catenin is regulated by the phosphorylation of β-catenin Tyr-142. This residue can be phosphorylated in vitro by Fer or Fyn tyrosine kinases. Transfection of these kinases to epithelial cells disrupted the association between both catenins. We have also examined whether these kinases are involved in the regulation of this interaction by K-ras. Stable transfectants of the K-ras oncogene in intestinal epithelial IEC18 cells were generated which show little α-catenin-β-catenin association with respect to control clones; this effect is accompanied by increased Tyr-142 phosphorylation and activation of Fer and Fyn kinases. As reported for Fer, Fyn kinase is constitutively bound to p120 catenin; expression of K-ras induces the phosphorylation of p120 catenin on tyrosine residues increasing its affinity for E-cadherin and, consequently, promotes the association of Fyn with the adherens junction complex. Yes tyrosine kinase also binds to p120 catenin but only upon activation, and stimulates Fer and Fyn tyrosine kinases. These results indicate that p120 catenin acts as a docking protein facilitating the activation of Fer/Fyn tyrosine kinases by Yes and demonstrate the role of these p120 catenin-associated kinases in the regulation of β-catenin-α-catenin interaction.This work was supported by grants 01/045-00 (from Fundació La Caixa) to A.G.H.; PM-99-0132 and PM99-0064 (from Ministerio de Ciencia y Tecnología) to A.G.H. and M.D., respectively; and 2001SGR00410 and 2001SGR00197 (from Direcció General de Recerca). J.C. and S.M. were recipients of predoctoral fellowships from the Ministerio de Educación y Ciencia and UAB, respectively.Peer Reviewe

Genetic signatures of differentiation Induced by 1α ,25-Dihydroxyvitamin D3 in human colon cancer cells

Epidemiological and preclinical data indicate that vitamin D and its most active metabolite 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] have anticancer activity. Accordingly, clinical trials are under way using several nonhypercalcemic 1α,25(OH)2D3 analogues against various neoplasms including colon cancer. 1α,25(OH)2D3 induces proliferation arrest and epithelial differentiation of human SW480-ADH colon cancer cells. We examined the gene expression profiles associated with 1α,25(OH)2D3 exposure using oligonucleotide microarrays. 1α,25(OH)2D3 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 1α,25(OH)2D3 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, G0S2, 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 1α,25(OH)2D3 in SW480-ADH cells differed from those in LS-174T cells, which lack E-cadherin and do not differentiate in response to 1α,25(OH)2D3. Data from this study reveal that 1α,25(OH)2D3 causes a profound change in gene expression profiles and provide a mechanistic basis to the ongoing clinical studies using nonhypercalcemic vitamin D3 derivatives for colon cancer prevention and treatment.Grant support: Fundación Científica de la Asociación Española contra el Cáncer and SAF2001-2291 from Ministerio de Ciencia y Tecnología, Spain.Peer Reviewe

Dissecting tumor anatomy: Intratumoral cell heterogeneity defines response to target-directed therapies

Resumen del trabajo presentado al XXXIV Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Barcelona del 5 al 8 de septiembre de 2011.-- et al.Accumulated evidences indicate that most solid tumors are not homogeneous but built of cancer cell populations with divers biological properties. They follow a hierarchical organization in which self-renewing cancer stem cells (CSC) are in the apex of a differentiation process within the cancerous tissue. CSC can also compose the small reservoir of drug-resistant cells responsible for tumor relapse or can give rise to metastasis. Our laboratory is exploring such heterogeneity and describing novel populations of cancer cells within colon carcinomas responsible for drugresistance, relapse or metastasis, all clinical determinants of patients' survival. Blocking signaling pathways that drive CSC distinctive properties is a new strategy being recently explored in clinical oncology by the use of novel targetdirected drugs. Wnt/β-catenin and PI3K/AKT are two of these pathways playing a central role in CSC homeostasis. We have described the function of their corresponding effectors - β-catenin and FOXO3a - cooperating in colon cancer. Their activation promotes cell scatteringand metastasis regulating a set of common target genes. Unexpectedly, the anti-tumoral AKT inhibitor API-2 promotes nuclear FOXO3a accumulation and metastasis from cells with high nuclear β-catenin. β-catenin confers resistance to FOXO3a-induced apoptosis promoted by PI3K and AKT inhibitors in patient-derived cells enriched in CSC, that is reverted by Wnt/β-catenin inhibitor XAV-939. Our findings could have a serious impact on therapy since we demonstrate that nuclear β-catenin heterogeneity compromises the response of different cancer cell populations to anti-tumoral drugs currently in clinical trials and directed against PI3K/AKT oncogenic signal.Peer Reviewe

SPROUTY-2 and E-cadherin regulate reciprocally and dictate colon cancer cell tumourigenicity

El pdf del artículo es la versión post-print.-- et al.SPROUTY-2 (SPRY2) regulates receptor tyrosine kinase signalling and therefore cell growth and differentiation. In this study, we show that SPRY2 expression in colon cancer cells is inhibited by the active vitamin D metabolite 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3) through E-cadherin-dependent and-independent mechanisms. In turn, SPRY2 represses both basal and 1,25(OH) 2 D 3-induced E-cadherin expression. In line with this, SPRY2 induces ZEB1 RNA and protein, but not that of other epithelial-to-mesenchymal transition inducers that repress the CDH1/E-cadherin promoter. Consistently, SPRY2 and E-cadherin protein levels inversely correlate in colon cancer cell lines and xenografted tumours. Moreover, SPRY2 knockdown by small hairpin RNA increases CDH1/E-cadherin expression and, reciprocally, CDH1/E-cadherin knockdown increases that of SPRY2. In colon cancer patients, SPRY2 is upregulated in undifferentiated high-grade tumours and at the invasive front of low-grade carcinomas. Quantification of protein expression in 34 tumours confirmed an inverse correlation between SPRY2 and E-cadherin. Our data demonstrate a tumourigenic action of SPRY2 that is based on the repression of E-cadherin, probably by the induction of ZEB1, and a reciprocal regulation of SPRY2 and E-cadherin that dictates cell phenotype. We propose SPRY2 as a candidate novel marker for high-grade tumours and a target of therapeutic intervention in colon cancer. © 2010 Macmillan Publishers Limited All rights reserved.This study was supported by the Ministerio de Ciencia e Innovación of Spain and Fondo Europeo de Desarrollo Regional (SAF2007-60341 and RD06/0020/0009 to AM, RD06/0020/0020 to FB, and SAF2006-04247, RD06/0020/0003 and FIS-Intrasalud (PS09/00562) to JMR), Comunidad de Madrid (S-GEN-0266/2006 to AM and FB), Spanish Association Against Cancer (to JMR), and European Union (MRTN-CT-2005-019496, NucSys, to AM).Peer Reviewe

Vitamin D3 promotes the differentiation of colon carcinoma cells by the induction of E-cadherin and the inhibition of β-catenin signaling

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.Peer reviewe