Abstract
The ability of androgens to inhibit apoptosis in both normal and malignant prostatic cells has been well documented. However, the underlying mechanisms are understood poorly. Here we demonstrated that forkhead transcription factor FKHR (FOXO1)-induced death of LNCaP cells was blocked by a synthetic androgen R1881. Androgen treatment also resulted in a reduction in transcriptional activity of FKHR in these cells. Moreover, treatment of LNCaP cells with R1881 led to a decrease in the intact FKHR protein (70 kDa) and an increase in a faster migrating protein band (60 kDa). Androgen-enhanced appearance of the 60-kDa protein was diminished specifically by lysosomal acidic cysteine protease inhibitors. Mass spectrometry analyses of the purified FLAG-tagged 70- and 60-kDa proteins demonstrated that the 60-kDa species is a FKHR protein product that lacks about 120 amino acid residues of the C-terminal end. Mutagenesis of the basic amino acid Arg(537) in the protease cleavage region, as suggested by mass spectrometry, abrogated both the androgen-induced accumulation of the 60-kDa product and decrease in cell death induced by FKHR, suggesting that the residue Arg(537) is a potential protease cleavage site. Finally, ectopic expression of the first 537 amino acids of FKHR produced an inhibitory effect on transcriptional activity of the intact protein. Together, these results suggest that androgens induce increased activity of an acidic cysteine protease, which in turn cleaves FKHR. This provides a mechanism by which androgens protect prostate cancer cells from the killing effect of FKHR.
Highlights
The tumor suppressor gene PTEN is mutated frequently in a variety of tumors including prostate, brain, and endometrium [7,8,9]
We demonstrated that forkhead transcription factor FKHR (FOXO1)-induced death of LNCaP cells was blocked by a synthetic androgen R1881
Androgens Protect LNCaP Cells from Apoptosis Induced by FKHR—Ectopic expression of PTEN in LNCaP cells induces apoptosis, which can be inhibited by androgen treatment [40, 41]
Summary
Materials—N-terminal FLAG-tagged pcDNA3 expression plasmids for wild-type FKHR, FKHR(WT), a constitutively active form, FKHR(AAA), and the luciferase reporter construct 3ϫIRS-Luc were kindly provided by Drs K. Two truncation mutants WT(⌬) and AAA(⌬) were constructed from FKHR(WT) and FKHR(AAA), respectively, by deleting a C-terminal fragment after the residue Arg537. A polyclonal antibody against FKHR was purchased from Cell Signaling Technology (Beverly, MA). A monoclonal antibody against PTEN (6H2.1) was purchased from. Erk (D-2) monoclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. Mouse monoclonal antibodies against ␣-FLAG were purchased from Sigma. Cell Lines and Cell Culture—The prostate cancer cell line LNCaP (purchased from the American Type Culture Collection, Manassas, VA) was cultured in RPMI 1640 medium containing 10% fetal bovine serum. Cells were mixed with DNA in 400 l of RPMI 1640 medium. Western Blot Analysis—Protein samples were prepared by lysing cells in radioimmune precipitation assay buffer
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