FOXO1 binds to the TAU5 motif and inhibits constitutively active androgen receptor splice variants

Abstract

Background: Aberrant activation of the androgen receptor (AR) is a major factor highly relevant to castration-resistant progression of prostate cancer (PCa). FOXO1, a key downstream effector of PTEN, inhibits androgen-independent activation of the AR. However, the underlying mechanism remains elusive.

Methods: The inhibitory effect of FOXO1 on full-length and constitutively active splice variants of the AR was examined by luciferase reporter assays and real-time reverse transcription polymerase chain reaction (RT-qPCR). In vitro protein binding assays and western blot analyses were used to determine the regions in FOXO1 and AR responsible for their interaction.

Results: We found that a putative transcription repression domain in the NH2-terminus of FOXO1 is dispensable for FOXO1 inhibition of the AR. In vitro protein binding assays showed that FOXO1 binds to the transcription activation unit 5 (TAU5) motif in the AR NH2-terminal domain (NTD), a region required for recruitment of p160 activators including SRC-1. Ectopic expression of SRC-1 augmented transcriptional activity of some, but not all AR splice variants examined. Forced expression of FOXO1 blocked the effect of SRC-1 on AR variants' transcriptional activity by decreasing the binding of SRC-1 to the AR NTD. Ectopic expression of FOXO1 inhibited expression of endogenous genes activated primarily by alternatively spliced AR variants in human castration-resistant PCa 22Rv1 cells.

Conclusions: FOXO1 binds to the TAU5 motif in the AR NTD and inhibits ligand-independent activation of AR splice variants, suggesting the PTEN/FOXO1 pathway as a potential therapeutic target for inhibition of aberrant AR activation and castration-resistant PCa growth.

Fig. 1

FOXO1 inhibition of AR transactivation independent of the putative repression domain in the NH2-terminus. A: Left, schematic diagram showing a series of COOH-terminal truncated FLAG-tagged FOXO1 mammalian expression constructs. FKH, forkhead (DNA binding) domain; NLS, nuclear localization signal; NES, nuclear exportation signal; LxxLL, nuclear receptor interaction motif; TAD, transcription activation domain. Right, inhibition of AR transcriptional activity by the NH2-terminus of FOXO1. LNCaP cells were transfected with the PSA-Luc reporter construct in combination with the empty control vector or different FOXO1 mutants. At 24 hr after transfection, cells were treated with 1 nM of R1881 (a synthetic androgen) for 12 hr and harvested for luciferase activity measurement as described in the Materials and Methods Section. Columns, mean among three individual experiments; bars, SD. Expression of FLAG-tagged FOXO1 and AR proteins were analyzed by western blot using the indicated antibodies (inset).B: Left, schematic diagram showing mutants of FOXO1 NH2-terminus (amino acids 1–267) with deletion of different mSin3a interaction domains (SIDs) identified in many transcription repressors. Right, inhibition of AR transcriptional activity by FOXO1NH2-terminus (1–267) is independent of the SID domains. LNCaP cells were transfected with the AR reporter gene 3xARE-Luc and FOXO1 mutants as indicated. Cells were treated and luciferase activities in cells were measured as in(A).

Fig. 2

FOXO1 directly interacts with the AR through a small region in the forkhead domain. A: In vitro protein binding assays with GST– FOXO1 recombination proteins and ³⁵S-labeled in vitro transcribed and translated AR. Left, schematic diagram of GST–FOXO1 recombination proteins. Right, purified GST and GST–FOXO1 fusion proteins (lower panel) were incubated with ³⁵S-labeled in vitro transcribed and translated AR proteins(middle panel)and pull-down assays were performed. Pull-down proteins were subjected to SDS–PAGE analysis and auto radiography (upper panel).B: GST pull-down assays with whole cell lysates of LNCaP cells and GST–FOXO1 recombination proteins. Left, GST–FOXO1 recombination protein diagram. Right, purified GST and GST–FOXO1 fusion proteins (lower panel) were incubated with whole cell lysate of LNCaP and pull-down assays were performed. The pull-down proteins were analyzed by western blot using anti-AR antibody (upper panel). The asterisk indicates a non specific band recognized by the anti-AR antibody.

Fig. 3

FOXO1 directly binds to AR at the TAU5 domain. A: Schematic diagram of AR NTD deletion mutants of AR fused to GST. NTD, NH2-terminal domain. TAU 5, transactivation unit 5.B: Purified GST and GST-AR fusion proteins (lower panel) were incubated with in vitro transcribed/translated ³⁵S-labeled FOXO1. Bound FOXO1 proteins were subjected to SDS–PAGE analysis and autoradiography (upper panel).

Fig. 4

FOXO1 inhibits transcriptional activity of AR variants. PC-3 (A,C) or DU145 (B) cells were transfected with the PSA-Luc reporter and the plasmid for V5-tagged wild-type FOXO1 (FOXO1-WT) or FLAG-tagged constitutively active FOXO1 (FOXO1-A3). At 48 hr after transfection, luciferase activities were measured and normalized to renilla levels. Columns, mean among three individual experiments; bars, SD. Expression of AR variants and FOXO1 in transfected PC-3 cells were analyzed by western blot (C). Immunoblotting of extracellular signal-regulated kinase 2 (ERK2) was included as a loading control.

Fig. 5

FOXO1 blocks the transcriptional activity of AR variants augmented by the transcription coactivator SRC-1. A: PC-3 cells were transfected with the PSA-luc reporter and the indicated plasmids. At 48 hr after transfection, luciferase activities were measured and normalized to renilla levels. Columns, mean among two individual experiments; bars, SD. B: Analysis of binding of AR-NTD with in vitro transcribed/translated FOXO1 and SRC-1 proteins. Purified GST-AR NTD fusion proteins were incubated with in vitro transcribed/translated ³⁵S-labeled FOXO1 and/or SRC-1 (lower panel). Bound FOXO1 and SRC-1 proteins were subjected to SDS–PAGE analysis and autoradiography (upper panel). C: PC-3 cells were transfected and luciferase activities were measured as in (A).Columns, mean among three individual experiments; bars, SD.

Fig. 6

FOXO1 regulates expression of endogenous AR target genes in 22Rv1 CRPC cells. A: Schematic diagram of AR mRNA and protein, AR siRNA targeting sites and major AR splice variants identified in 22 Rv1 cells. B,C: 22Rv1 cells were transfected with non-specific (NS) or AR siRNAs and empty vector or FLAG-tagged FOXO1. At 48 hr after transfection, cells were harvested for Western blot analysis (B) or real-time RT-PCR (C). Immunoblotting of extracellular signal-regulated kinase 2 (ERK2) was included as a loading control. Columns, mean among three individual experiments; bars, SD.