Cystatin D is a candidate tumor suppressor gene induced by vitamin D in human colon cancer cells
Silvia ρlvarez-Díaz, … , Carlos López-Otín, Alberto Muñoz
Silvia ρlvarez-Díaz, … , Carlos López-Otín, Alberto Muñoz
Published July 6, 2009
Citation Information: J Clin Invest. 2009;119(8):2343-2358. https://doi.org/10.1172/JCI37205.
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Research Article Oncology

Cystatin D is a candidate tumor suppressor gene induced by vitamin D in human colon cancer cells

Abstract

The active vitamin D metabolite 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] has wide but not fully understood antitumor activity. A previous transcriptomic analysis of 1α,25(OH)2D3 action on human colon cancer cells revealed cystatin D (CST5), which encodes an inhibitor of several cysteine proteases of the cathepsin family, as a candidate target gene. Here we report that 1α,25(OH)2D3 induced vitamin D receptor (VDR) binding to, and activation of, the CST5 promoter and increased CST5 RNA and protein levels in human colon cancer cells. In cells lacking endogenous cystatin D, ectopic cystatin D expression inhibited both proliferation in vitro and xenograft tumor growth in vivo. Furthermore, cystatin D inhibited migration and anchorage-independent growth, antagonized the Wnt/β-catenin signaling pathway, and repressed c-MYC expression. Cystatin D repressed expression of the epithelial-mesenchymal transition inducers SNAI1, SNAI2, ZEB1, and ZEB2 and, conversely, induced E-cadherin and other adhesion proteins. CST5 knockdown using shRNA abrogated the antiproliferative effect of 1α,25(OH)2D3, attenuated E-cadherin expression, and increased c-MYC expression. In human colorectal tumors, expression of cystatin D correlated with expression of VDR and E-cadherin, and loss of cystatin D correlated with poor tumor differentiation. Based on these data, we propose that CST5 has tumor suppressor activity that may contribute to the antitumoral action of 1α,25(OH)2D3 in colon cancer.

Authors

Silvia ρlvarez-Díaz, Noelia Valle, José Miguel García, Cristina Peña, José M.P. Freije, Víctor Quesada, Aurora Astudillo, Félix Bonilla, Carlos López-Otín, Alberto Muñoz

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Figure 2

1α,25(OH)2D3 directly activates the CST5 promoter.

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1α,25(OH)2D3 directly activates the CST5 promoter. (A) Quantitative ...
(A) Quantitative RT-PCR analysis of CST5 RNA expression in SW480-ADH cells treated with 1α,25(OH)2D3 or vehicle that were pretreated for 30 minutes with actinomycin D (2 μg/ml) or cycloheximide (8 μg/ml) as indicated. (B) Scheme of the human CST5 gene promoter showing putative VDR binding sites grouped in regions A–D. (C) Activation of CST5 promoter constructs by 1α,25(OH)2D3 in SW480-ADH cells and in HEK293T cells cotransfected with an expression vector for wild-type VDR. Twenty-four hours after transfection, cells were treated with vehicle or 1α,25(OH)2D3 (10–7 M) for an additional 48 hours. The empty pGL3 vector was used as control. (D) The activation of the CST5 promoter by 1α,25(OH)2D3 requires a transcriptionally competent VDR. SW480-R cells were cotransfected with the promoter construct pGL3-1867 or pGL3-251 and either the wild-type VDR or the mutant ΔAF2-VDR (VDRΔAF2), or an empty vector. The cells were then treated with 1α,25(OH)2D3 (10–7 M) or vehicle for 48 hours. As control, the consensus 4xVDRE-DR3-Tk-Luc (VDRE) reporter construct was cotransfected with the wild-type or mutant VDR. Values correspond to promoter induction by 1α,25(OH)2D3 in 3 independent experiments done in triplicate. (E) 1α,25(OH)2D3 induces VDR binding to and an active chromatin conformation of the CST5 promoter in vivo. ChIP assay showing the induction by 1α,25(OH)2D3 of VDR binding, SMRT corepressor release, and increased histone H4 acetylation (AcH4) of the CST5 gene promoter in SW480-ADH cells. The CYP24 gene was used as control. The promoter regions studied are indicated.