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 4

CST5 silencing affects SW480-ADH cell proliferation and gene expression.

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CST5 silencing affects SW480-ADH cell proliferation and gene expression...
(A) Phase-contrast images of control shRNA and CST5 shRNA cells that were treated with 10–7 M 1α,25(OH)2D3 or vehicle for 48 hours. Western blot analysis showing decreased level of cystatin D protein in CST5 shRNA cells. Scale bar: 20 μm. (B) CST5 knockdown abrogates the inhibition of cell proliferation by 1α,25(OH)2D3. Control shRNA and CST5 shRNA cells were treated with vehicle or 1α,25(OH)2D3 for the indicated times. Proliferation of cells in 1α,25(OH)2D3- versus vehicle-treated cultures is shown. (C) Quantitative RT-PCR analysis showing decreased CDH1 and CYP24 and increased LEF1 RNA expression in CST5 shRNA cells. (D) Western blot analysis showing changes in E-cadherin, c-MYC, and LEF-1 proteins in CST5 shRNA cells. (E) CST5 knockdown increases the expression of SNAI1 RNA (left) and protein (right). Numbers between the blots in A, D, and E correspond to mean of the fold increase values obtained in 2 experiments.*P < 0.05, **P < 0.01, ***P < 0.001. The Western blot in E shows noncontiguous lanes run on the same gel.