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 1

1α,25(OH)2D3 induces cystatin D expression.

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1α,25(OH)2D3 induces cystatin D expression. (A) Kinetics of CST5 RNA...
(A) Kinetics of CST5 RNA (top) and protein (bottom) induction by 1α,25(OH)2D3. SW480-ADH cells were incubated with 1α,25(OH)2D3 (10–7 M) or vehicle for the indicated times, and the levels of CST5 RNA and protein were measured by quantitative RT-PCR (top) or Western blotting (bottom) as explained in Methods. Normalized mean values and SD obtained in 3 independent experiments are shown. (B) Dose-curve induction of CST5 RNA (top) and protein (bottom) by 1α,25(OH)2D3. Numbers between the blots in A and B correspond to mean of the fold increase values obtained in 2 experiments. (C) Immunofluorescence analysis of cystatin D induction. Images of SW480-ADH and SW480-R cells treated with 1α,25(OH)2D3 (10–7 M) or vehicle for 48 hours. The relocalization of β-catenin was analyzed as control of 1α,25(OH)2D3 action. Scale bars: 20 μm. (D) The 1α,25(OH)2D3 analog EB1089 induces cystatin D expression in vivo. Left: Immunohistochemical analysis of cystatin D expression in tumors generated by SW480-ADH cells in immunodeficient mice that were treated with EB1089 (10–7 M) or placebo. Scale bar: 200 μm. Right: Quantification of cystatin D expression by estimation of staining intensity as described in Methods. The number of samples analyzed per group and the percentage corresponding to each level of cystatin D staining are shown.