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. 2008 Nov 1;68(15):1696-706.
doi: 10.1002/pros.20844.

Induction of FLIP expression by androgens protects prostate cancer cells from TRAIL-mediated apoptosis

Kristin A Raclaw  1 , Hannelore V Heemers, Emily M Kidd, Scott M Dehm, Donald J Tindall
Affiliations

Induction of FLIP expression by androgens protects prostate cancer cells from TRAIL-mediated apoptosis

Kristin A Raclaw et al. Prostate. .

Abstract

Background: Prostate tumors initially regress in response to androgen-ablation therapy. However, most cancers eventually relapse with an androgen-depletion-independent (ADI) phenotype that is often more aggressive than the original androgen-dependent (AD) tumor. Importantly, most relapsed tumors still rely upon androgen receptor (AR) activity for proliferation and survival. The cellular Fas/FasL-associated death domain protein-like inhibitory protein (FLIP) inhibits activation of procaspase-8 by death receptor-mediated signaling at the cell surface. In the current study, we examined the androgenic regulation of FLIP and its contribution to protecting prostate cancer cells from death receptor-mediated apoptosis.

Methods: FLIP expression in tissues from intact and castrated rats as well as androgen-treated prostate cancer cell lines (LNCaP, C4-2, LNCaP-Rf, and DU-145) was monitored via Real-Time RT-PCR and immunoblot. Induction of apoptosis by TRAIL, the death receptor ligand, was determined via microscopic observation and cell counting of fragmented nuclei following fixation and staining with Hoechst 33285.

Results: FLIP mRNA and protein expression was reduced following castration in multiple rat tissues, including dorsolateral prostate and seminal vesicles. Androgenic induction of FLIP mRNA and protein was observed in isogenic AD LNCaP and ADI LNCaP-Rf cells, but not the isogenic ADI C4-2 cell line. Protection from TRAIL-induced apoptosis by androgen was completely blocked when LNCaP-Rf cells were depleted of endogenous FLIP via siRNA transfection.

Conclusions: Androgenic protection from TRAIL-induced apoptosis is predominantly via enhanced transcription of FLIP in prostate cancer cells. Loss of androgen-sensitivity in ADI prostate cancer cells highlights this pathway as a potential target for future therapy of prostate cancer.

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Figures

Figure 1
Figure 1
Androgenic manipulation regulates FLIP expression in vivo. (A) Examination of FLIP mRNA expression via real-time RT-PCR. Each experimental group contains 4 animals, with each sample analyzed in triplicate. Groups: sham-operated (Sham), castrated with vehicle injections (Cx), and castrated with testosterone/testosterone propionate injections (Cx + T). Top row graphs represent known androgen-regulated tissues: anterior prostate (AP), dorsolateral prostate (DLP), ventral prostate (VP), lacrimal gland (LG), and seminal vesicle (SV). Bottom row graphs represent androgen-independent tissues: kidney (KD), liver (LV), and spleen (SP). FLIP expression was normalized to internal r18s RNA, and data is presented as relative FLIP mRNA level, normalizing Sham to 1. Data represent the mean ± S.E. from four animals, each analyzed in triplicate. (B) Immunoblot analysis of FLIP expression in tissue lysates from seminal vesicle (top) and dorsolateral prostate (bottom). (*) in the FLIP seminal vesicle blot indicates one animal that had high FLIP expression compared to others in the Cx group, and was believed to be due to animal variation. The dorsolateral prostate FLIP blot was further analyzed by densitometric calculation (below). (C) Immunoblot analysis of AR expression in lysates of dorsolateral prostate, ventral prostate, and seminal vesicle of sham-operated rats. The blot was subjected to densitometric analysis (lower panel). Each bar represents the average pixels for the indicated group, normalized to β-actin expression. Error bars represent ± S.E.
Figure 2
Figure 2
Androgen regulation of FLIP is variable in ADI prostate cancer cells. Cells were cultured in 9% charcoal-stripped serum for 48 h prior to treatment. Immunoblot data is represented in the upper panels, real-time RT-PCR data is presented in the bar graphs below. Real-time RT-PCR data is expressed as relative mRNA expression level, and values for of one of the untreated samples are arbitrarily set at 1 unless specified otherwise. Data represent the mean ± S.E. from three independent experiments, each performed in triplicate and normalized to an internal GAPDH control. (A) Basal FLIP protein and mRNA expression in prostate cancer cell lines. Expression of FLIP in one untreated sample of LNCaP cells was arbitrarily set at 1. (B) 24h Mibolerone (MIB) dose response in AD LNCaP cells. (C) 24 h MIB dose response in ADI C4-2 cells. (D) Androgen manipulation in ADI LNCaP-Rf cells. WS, whole serum; CS, charcoal-stripped serum; MIB, 1 nM MIB treatment for 24 h.
Figure 3
Figure 3
The AR is required for androgen-induced, but not basal FLIP expression in prostate cancer cells. Cells were cultured in 9% charcoal-stripped serum. Immunoblot data is represented in the upper panels, real-time RT-PCR data is presented in the graphs below. Real-time RT-PCR data is expressed as relative mRNA expression level, and values for one untreated sample is arbitrarily set at 1 unless specified otherwise. Data represent the mean ± S.E. from three independent experiments, each performed in triplicate and normalized to an internal GAPDH control. (A) AR-null DU-145 cells do not regulate FLIP in response to androgen. Cells were treated with EtOH vehicle (−) or 1 nM MIB (+) for 24 h. (B) LNCaP cells with decreased AR expression do not display increased FLIP expression following androgen treatment. Cells were cultured in charcoal-stripped serum media, transfected with a non-targeting control siRNA (siCTRL) or siRNA specific for the AR (siAR), and treated with EtOH vehicle (−) or 1nM MIB (+) for 24h. Cells were harvested 72 h after transfection. (C) LNCaP and isogenic ADI sublines do not show changes in basal FLIP mRNA or protein following AR knock-down. Cells were cultured and transfection as in (B).
Figure 4
Figure 4
LNCaP-Rf cells are most sensitive to TRAIL-mediated apoptosis compared to other isogenic prostate cancer cells. (A) Cells were cultured in whole serum (WS), charcoal-stripped serum (CSS), or CSS and treated with MIB for 24h (CSS+MIB). FLIP mRNA levels were analyzed via real-time PCR (upper panels). Values for WS-cultured cells were arbitrarily set at 1 for each cell line. Data is normalized to an internal GAPDH control and represent the mean ± S.E. from samples analyzed in triplicate. Lower panels represent immunoblot analysis of whole cell lysates probed for AR and Erk2. (B) Cells were grown for at least 48 h in charcoal-stripped serum media before treatment with the indicated doses of TRAIL. Cells were fixed, applied to slides, and stained with Hoechst 33258. Nuclei were counted and apoptotic nuclei represented as the mean percentage ± S.E. from three independent experiments, performed in duplicate. (C) Protein lysates from cells cultured for at least 72 h in charcoal-stripped serum media were analyzed for indicated cell survival factors and apoptosis regulators via immunoblot.
Figure 5
Figure 5
Androgen-induced protection from apoptosis is largely mediated through FLIP upregulation. (A) Pre-treatment with androgen protects cells from TRAIL-mediated apoptosis. Cells were grown in charcoal-stripped serum for 48 h, and treated with EtOH vehicle (−) or 1 nM MIB for 8 h prior to TRAIL treatment (40 ng/mL) for 16 h. Cells were collected, fixed, and stained with Hoechst 33258 for nuclei counting. Data is presented as the mean apoptotic nuclei percentage ± S.E. from three independent experiments, performed in triplicate. (B) LNCaP-Rf cells were transfected with mock, non-targeting control siRNA (siCTRL), or siRNA targeted to FLIP (siFLIP) and seeded in charcoal-stripped serum media. 24 h later cells were treated with EtOH vehicle (−) or 1 nM MIB for 8 h prior to TRAIL treatment (100 ng/mL) for 16 h. Cells were collected, fixed, and stained with Hoechst 33258 for nuclei counting. Data is presented as the mean apoptotic nuclei percentage ± S.E. from three independent experiments, performed in triplicate. (C) Hoechst-stained DAPI images from cells quantified in (B). (D) Whole cell protein lysates from untreated, transfected LNCaP-Rf cells, 48 h after transfection.
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