Suppression of Mammary Carcinoma Growth by Retinoic Acid: Proapoptotic Genes Are Targets for Retinoic Acid Receptor and Cellular Retinoic Acid–Binding Protein II Signaling

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Abstract
Retinoic acid (RA) displays pronounced anticarcinogenic activities in several types of cancer. Whereas the mechanisms that underlie this activity remain incompletely understood, tumor suppression by RA is believed to emanate primarily from its ability to regulate transcription of multiple target genes. Here, we investigated molecular events through which RA inhibits the growth of MCF-7 mammary carcinoma cells, focusing on the involvement of the two proteins that mediate transcriptional activation by RA, the nuclear hormone receptor retinoic acid receptor (RAR) and the cellular retinoic acid-binding protein (CRABP) II, in this process. RA treatment of MCF-7 cells did not affect cell cycle distribution but triggered pronounced apoptosis. Accordingly, expression array analyses revealed that RA induces the expression of several proapoptotic genes, including caspase 7 and caspase 9. Whereas caspase 7 is an indirect responder to RA signaling, caspase 9 is a novel direct target for RAR, and it harbors a functional retinoic acid response element in its second intron. In agreement with the known role of CRABP-II in enhancing the transcriptional activity of RAR, the binding protein augmented RA-induced up-regulation of caspase 9, cooperated with RA in activating both caspase 7 and 9, and amplified the ability of RA to trigger apoptosis. Surprisingly, the data indicate that CRABP-II also displays proapoptotic activities on its own. Specifically, overexpression of CRABP-II, in the absence of RA, up-regulated the expression of Apaf1 and triggered caspase 7 and caspase 9 cleavage. These observations suggest that, in addition to its known role in direct delivery of RA to RAR, CRABP-II may have an additional, RA-independent, function.