| Laboratory of Alberto Muñoz Terol |
Identification and study of genes regulated by vitamin D in human colon cancer cells
Following previous studies and on the basis of results from transcriptomic analyses, we have studied the regulation of KDM6B/JMJD3 gene by 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), the most active vitamin D derivative in the organism. KDM6B/JMJD3 is an epigenetic regulator that encodes a histone demethylase that prevents the repressive activity of the Polycomb complex on gene transcription. KDM6B/JMJD3 methylase removes methyl residues of histone H3 Lysine 27. Our data show that 1,25(OH)2D3 induces the expression of KDM6B/JMJD3 and that this demethylase mediates partially the effects of 1,25(OH)2D3 on the expression of several target genes. Moreover, the repression of KDM6B/JMJD3 by means of gene knockdown increases intercellular adhesion and abolishes the induction of beta-catenin nuclear export by 1,25(OH)2D3 (Pereira et al., Hum Mol Genet, 2011). In collaboration with Dr. Fthe existance of ed a dikrect relation berween the level of expression of and VDRFf several target genes. MOreover, the repressélix Bonilla's group (Hosp. Univ. Puerta de Hierro, Majadahonda, Madrid) we have confirmed that the expression levels of KDM6B/JMJD3 and VDR are directly related in human colon tumors. An in-depth study has shown that 1,25(OH)2D3 modulates variably the level of expression of several members of the Jumonji (JmjC) and LSD families of demethylases (Pereira et al., Cell Cycle, 2012). In addition, a comparative proteomics analysis led to the identification of novel proteins regulated by 1,25(OH)2D3 in colon cancer cells; among them, several are involved in splicing and microRNA synthesis (SFPQ, SMARCE, KHSRP, TARDBP, PARP1...) (Cristobo et al., J Proteomics, 2011). By using microRNA microarrays we have been able to characterize several microRNA species as putative targets of 1,25(OH)2D3, and we selected microRNA-22 (miR-22) for an in-depth study. We confirmed that miR-22 is induced by 1,25(OH)2D3 and that it contributes to its effects on cell proliferation and migration and on gene regulation (Álvarez-Díaz et al., Hum Mol Genet, 2012). Our data and also all others existing in the literature on the action of vitamin D and its derivatives in colon cancer have recently been reviewed by us (Pereira et al., Endocr-Rel Cancer, 2012). Finally, we continue studying the biological activity of a few genes that we previously identified as targets of 1,25(OH)2D3 in colon cancer cells such as DICKKOPF-1, SPROUTY-2 and CST5/Cystatin D.
Vitamin D effects on Wnt/beta-catenin pathway and on bladder cancer
Based on our own previous data, which have been confirmed by other researchers in diverse biological systems, indicating that 1,25(OH)2D3 antagonizes the Wnt/beta-catenin signaling pathway in colon cancer cells, we have now performed studies in animals to analyze whether such antagonism takes place also in vivo. To this end, we generated animals carrying one mutated allele of the Apc tumor suppressor gene and either two, one, or none alleles of Vdr gene coding for the vitamin D receptor by means of appropriated crosses between Apcmin/+ and Vdr+/- mice. Results obtained show that Vdr deficiency does not alter the number of tumors developed in mice but in contrast increases tumor growth and the number of aberrant crypt foci. In addition, Vdr deficiency enhances the level of nuclear beta-catenin and the expression of Wnt/beta-catenin target genes. Accordingly, Vdr deficiency abolishes the capacity of 1,25(OH)2D3 to promote the relocation of beta-catenin from the cell nucleus towards the plasma membrane in cultured colon cancer cells. In summary, we demonstrate that Vdr controls also in vivo the amount of nuclear beta-catenin and that it may attenuate the impact of oncogenic mutations such as Apc loss that activate the Wnt/beta-catenin pathway (Larriba et al., PLos ONE, 2011).
Regarding Wnt/beta-catenin pathway, which is cfucial in the initiation and progression of colon cancer, in collaboration with Drs. Héctor G. Pálmer and others at the Vall'd Hebron Institute of Oncologia (VHIO, Barcelona) we have reported how the combined presence of beta-catenin and FOXO3a within the cell nucleus confers resistance to the apoptosis induced by the latter and causes a profound alteration of the gene expression profile promoting metastasis in an animal model while it is linked to metastasis and lower survival in colorectal cancer patients (Tenbaum et al., Nat Med, 2012). Notably, given the action of 1,25(OH)2D3 decreasing beta-catenin nuclear level, these results support the proposed protective effect of this agent against colorectal cancer.
In collaboration with Drs. Nuria Malats and Francisco X. Real at Centro Nacional de Investigaciones Oncológicas (CNIO, Madrid) we have extended our studies on vitamin D to bladder cancer. In a population-based study combined with genetic/molecular analyses we have found an association of low circulating levels of 25(OH)D3 with increased risk of urothelial bladder cancer, particularly in patients with muscle-invasive tumors that express low level of FGFR3. Moreover, in cultured bladder cells, 1,25(OH)2D3 increases FGFR3 expression, whose mutation and over-expression is a marker of good prognostic in this type of cancer. Altogether, these data suggest that vitamin D deficient patients may have an increased risk of developing aggressive bladder tumors (Amaral et al., J Natl Cancer Inst, 2012).
Biological activity of novel vitamin D3 derivatives
In the frame of a long lasting collaboration with Drs. Antonio Mouriño and Miguel Maestro (Univ. Santiago de Compostela and La Coruña, respectively), we have studied the biological activity of novel vitamin D derivatives synthesized in their laboratories. Our contribution has been the analysis of the effects of these compounds on the survival, proliferation and phenotype of human colon cancer cells, and on the expression of selected genes that play crucial roles in this neoplasia such as CDH1/E-cadherin and c-MYC. Also, we have studied some genes identified by us as having probable tumor suppressive action such as CST5/cystatin D. In addition, we checked the ability of the vitamin D compounds to induce the expression of VDR. Several of the compounds studied can be considered as highly interesting due to their high activity/toxicity (calcemic effect) ratio (Verlinden et al., ChemMedChem, 2011; Regueira et al., J Med Chem 2011; Fraga et al., Chemistry 2012; Carballa et al., J Med Chem, 2012).