Abstract
The Hairy/Enhancer-of-split-related with YRPW-like motif (HEY) family of proteins are transcriptional repressors and downstream effectors of Notch signaling. We previously reported that HEY1 and HEY2 selectively repress androgen receptor (AR) signaling in mammalian cell lines and have shown that in human tissue HEY1 is excluded from the nuclei in prostate cancer but not benign prostatic hyperplasia. We have now characterized a third member of this family, HEYL, which is a more potent repressor of AR activity. HEYL interacted with and repressed AR activation function-1 domain and competitively inhibited SRC1e activation of AR transcriptional activity. Using a cell line inducibly expressing exogenous HEYL, we showed that HEYL represses endogenous AR-regulated genes and reduces androgen-dependent prostate cancer cell growth. Using a trans-repression assay, we identified both trichostatin-sensitive and -insensitive domains within HEYL; however, analysis of endogenous AR target genes suggested that HEYL represses AR activity through histone deacetylase I/II-independent mechanisms. Immunohistochemical analyses of tissue indicated that, in a fashion similar to that previously reported for HEY1, HEYL is excluded from the nuclei in prostate cancer but not adjacent benign tissue. This suggests that nuclear exclusion of HEY proteins may be an important step in the progression of prostate cancer.
Highlights
C11509/A12426 and Ministerio de Ciencia e Innovacion Grants SAF200762642 and SAF2010-21013. □S The on-line version of this article contains supplemental Figs
Transient transfection experiments in COS-1 cells with an androgen receptor (AR)-responsive luciferase reporter (TAT-GRE-E1B-LUC) indicated that HEYL was a more potent repressor of AR activity than HEY1 (Fig. 1A), repressing ligand-induced activity to ϳ20% of maximum; this repression was dose-dependent (Fig. 1B). Both HEY1 and HEY2 interact with HEYL; in combination experiments, there were no additive or synergistic effects on AR activity, suggesting that Hairy/Enhancer-of-split-related with YRPW-like motif (HEY) proteins might repress AR signaling via independent mechanisms
HEYL mRNA levels were very low in the prostate cancer lines tested but were ϳ3 times higher in the AR-positive breast cancer line MCF7
Summary
C11509/A12426 and Ministerio de Ciencia e Innovacion Grants SAF200762642 and SAF2010-21013. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. HEYL Is a Potent AR Corepressor and Interacts with Activation Function-1—HEYL is 328 amino acids in length and exhibits high sequence similarity at the protein level to HEY1 (supplemental Fig. S2).
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