1473

CST5

-;cystatin D;CST5;cystatin D

cystatin 5|cystatin-5|cystatin-D|cysteine-proteinase inhibitor

20p11.21

protein-coding

The active vitamin D metabolite 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] has wide but not fully understood antitumor activity. A previous transcriptomic analysis of 1alpha,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 1alpha,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/beta-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 1alpha,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 1alpha,25(OH)2D3 in colon cancer.

Many studies support a protective action of vitamin D against colon cancer. 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) exerts wide gene regulatory effects in human colon cancer cells. We previously reported that 1,25(OH)2D3 increases cytosolic Ca2+ concentration and transiently activates RhoA and its effector the Rho-associated coiled-kinase (ROCK), and later p38MAPK-MSK. We found that the inhibition of ROCK signaling by Y27632 or that of MSK by Ro318220 prevent the formation of epithelioid islands of SW480-ADH cells by 1,25(OH)2D3 and disrupts the adhesive phenotype of HT29 cells. ROCK and MSK inhibition also abrogates the induction of 1,25(OH)2D3 24-hydroxylase (CYP24), E-cadherin, and vinculin and the repression of cyclin D1 by 1,25(OH)2D3. Moreover, 1,25(OH)2D3 does not promote the localization of the tight junction protein occludin at the plasma membrane in cells expressing a dominant negative RhoA (N19-RhoA). In addition, 1,25(OH)2D3 specifically increases the level of the cysteine protease-inhibitor cystatin D, whereas that of cystatin SN is unaffected. The increase of cystatin D protein caused by 1,25(OH)2D3 is abrogated in N19-RhoA cells. Thus, activation of the RhoA-ROCK-p38MAPK-MSK signaling pathway is essential for the regulation of the phenotype and of the CST5/cystatin D candidate tumor suppressor and other target genes by 1,25(OH)2D3 in colon cancer cells.