AKT-Independent Protection of Prostate Cancer Cells from Apoptosis Mediated through Complex Formation between the Androgen Receptor and FKHR

Abstract

Recent studies suggested that the protection of cell apoptosis by AKT involves phosphorylation and inhibition of FKHR and related FOXO forkhead transcription factors and that androgens provide an AKT-independent cell survival signal in prostate cancer cells. Here, we report receptor-dependent repression of FKHR function by androgens in prostate cancer cells. Transcriptional analysis demonstrated that activation of the androgen receptor caused an inhibition of both wild-type FKHR and a mutant in which all three known AKT sites were mutated to alanines, showing that the repression is AKT independent. In vivo and in vitro coprecipitation studies demonstrated that the repression is mediated through protein-protein interaction between FKHR and the androgen receptor. Mapping analysis localized the interacting domains to the carboxyl terminus between amino acids 350 and 655 of FKHR and to the amino-terminal A/B region and the ligand binding domain of the receptor. Further analysis demonstrated that the activated androgen receptor blocked FKHR's DNA binding activity and impaired its ability to induce Fas ligand expression and prostate cancer cell apoptosis and cell cycle arrest. These studies identify a new mechanism for androgen-mediated prostate cancer cell survival that appears to be independent of the activity of the receptor on androgen response element-mediated transcription and establish FKHR and related FOXO forkhead proteins as important nuclear targets for both AKT-dependent and -independent survival signals in prostate cancer cells.

ACKNOWLEDGMENTS

We thank Betty A. Bing for technical help, F. G. Barr for permission to use FKHR in the studies, K. L. Guan for the Flag-tagged wild-type and mutant FKHR plasmids, M. E. Greenberg for the FKHRL1 and FHRE-Luc plasmids, B. W. O'Malley and P. S. Meltzer for receptor coactivator plasmids, S. Mosselman for plasmid pKCRE.ERβ, and Paul Byvoet for reading the manuscript. Fas ligand expression and cell cycle were analyzed by the Flow Cytometry Core Facility at the H. Lee Moffitt Cancer Center and Research Institute.

The work was supported by Public Health Service grants R29 CA79530 (W.B.) and R01 CA93666 (W.B.) and DOD Prostate Cancer Idea Grant DAMD17-02-1-0140 (W.B.).