Androgens Stimulate Lipogenic Gene Expression in Prostate Cancer Cells by Activation of the Sterol Regulatory Element-Binding Protein Cleavage Activating Protein/Sterol Regulatory Element-Binding Protein Pathway

Hannelore Heemers, Bart Maes, Fabienne Foufelle, Walter Heyns, Guido Verhoeven and Johannes V. Swinnen

Laboratory for Experimental Medicine and Endocrinology (H.H., B.M., W.H., G.V., J.V.S.), Faculty of Medicine, Onderwijs en Navorsing, Gasthuisberg, K.U. Leuven, B-3000 Leuven, Belgium; and U465 Institut National de la Santé et de la Recherche Médicale (F.F.), Centre de Recherches Biomédicales des Cordeliers, Université Paris 6, 75270 Paris, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Using two independent prostate cancer cell lines (LNCaP and MDA-PCa-2a), we demonstrate that coordinated stimulation of lipogenic gene expression by androgens is a common phenomenon in androgen-responsive prostate tumor lines and involves activation of the sterol regulatory element-binding protein (SREBP) pathway. We show 1) that in both cell lines, androgens stimulate the expression of fatty acid synthase and hydroxymethylglutaryl-coenzyme A synthase, two key lipogenic genes representative for the fatty acid and the cholesterol synthesis pathway, respectively; 2) that treatment with androgens results in increased nuclear levels of active SREBP; 3) that the effects of androgens on promoter-reporter constructs derived from both lipogenic genes (fatty acid synthase and hydroxymethylglutaryl-coenzyme A synthase) depend on the presence of intact SREBP-binding sites; and 4) that cotransfection with dominant-negative forms of SREBPs abolishes the effects of androgens. Related to the mechanism underlying androgen activation of the SREBP pathway, we show that in addition to minor effects on SREBP precursor levels, androgens induce a major increase in the expression of sterol regulatory element-binding protein cleavage-activating protein (SCAP), an escort protein that transports SREBPs from their site of synthesis in the endoplasmic reticulum to their site of proteolytical activation in the Golgi. Both time course studies and overexpression experiments showing that increasing levels of SCAP enhance the production of mature SREBP and stimulate lipogenic gene expression support the contention that SCAP plays a pivotal role in the lipogenic effects of androgens in tumor cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FOR ALMOST 60 yr, androgens have been known to play a key role in the biology of prostate cancer (1). As illustrated by the successful results of androgen ablation therapy (at least during the initial stages of tumor development) androgens are absolutely required for survival and proliferation of prostate cancer cells. Moreover, they affect many other cellular functions, including differentiated secretory function and lipogenesis (2, 3, 4). The latter process is highly active in many cancer cells and has been associated with malignant progression and poor prognosis in several cancers, including prostate cancer (5, 6, 7, 8, 9, 10).

Examination of the mechanism by which androgens affect lipogenesis in LNCaP prostate cancer cells revealed that (at least in this cell line) androgens coordinately stimulate the expression of several genes involved in the synthesis, transport, and metabolism of both fatty acids and cholesterol (11). This finding led us to suspect that androgens do not affect the expression of all these enzymes individually but rather induce or activate a transcription factor that modulates and coordinates the expression of all these genes. In view of their central role in the coordinate control of lipogenic gene expression, sterol regulatory element-binding proteins (SREBPs) were put forward as potential mediators of the lipogenic effects of androgens in LNCaP cells (11).

SREBPs are a family of three basic helix-loop-helix leucine zipper transcription factors (SREBP-1a, -1c, and -2) that have been identified as transacting factors involved in the maintenance of intracellular cholesterol homeostasis, the control of fatty acid metabolism, and the differentiation of adipocytes (12, 13, 14). Synthesized as 125-kDa precursor proteins, SREBPs are anchored to intracellular membranes where they form a complex with SREBP cleavage-activating protein (SCAP). In sterol-deprived cells this SREBP/SCAP complex travels to the Golgi apparatus where a 68-kDa amino-terminal SREBP segment is released by a two-step mechanism of regulated intramembrane proteolysis (Rip). This transcriptionally active SREBP fragment migrates to the nucleus, where it activates the transcription of a large set of sterol-regulatory element (SRE)-containing genes encoding lipogenic enzymes belonging to the two major lipogenic pathways (fatty acid synthesis and cholesterol synthesis) (15, 16, 17).

Experiments aimed at assessing the (putative) role of SREBPs in the induction of lipogenesis by androgens revealed that (at least in LNCaP cells) androgens enhanced the expression of SREBP precursor proteins and led to increased nuclear levels of mature active SREBP. In support of the involvement of SREBPs as mediators of the lipogenic effects of androgens, the stimulatory effects of androgens on the expression of a key lipogenic enzyme (FAS) were shown to be mediated by a complex cis-acting element harboring SREBP binding sites (11). Nevertheless, as these experiments have been restricted to LNCaP cells and as this cis-acting element has also been shown to bind other transcription factors such as upstream stimulatory factor and other E box binding proteins (18), final proof for the existence of an SREBP-mediated mechanism of androgen action in prostate cancer cells demanded further studies. Moreover, the exact mechanism by which androgens would affect the SREBP pathway remained to be investigated.

In this report, using a second independent androgen-responsive prostate cancer cell line (MDA-PCa-2a), in addition to LNCaP cells, alternative promoter-reporter constructs derived from a second androgen-regulated lipogenic gene [hydroxymethylglutaryl-coenzyme A (HMG-CoA)-synthase, belonging to the pathway of cholesterol synthesis] as well as dominant-negative forms of SREBPs, we provide this final proof. Moreover, we present evidence that induction of SCAP expression may play a pivotal role in the activation of the SREBP pathway by androgens.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Androgens Stimulate Lipogenic Gene Expression in Both LNCaP and MDA-PCa-2a Prostate Cancer Cells
In a previous study we demonstrated that, in the prostate cancer cell line LNCaP, androgens coordinately regulate the mRNA expression of several genes involved in the synthesis of fatty acids and cholesterol (11). To show that this regulation is not a peculiarity of LNCaP cells, we examined the effect of androgens on the expression of two key lipogenic genes (belonging to either the fatty acid synthesis pathway or the cholesterol synthesis pathway) in another independent androgen-responsive prostate cancer cell line, MDA-PCa-2a (19). To this end, cells were incubated for 2 d in the presence or absence of 10-8 M of the synthetic androgen R1881. Total RNA was prepared and subjected to Northern blot analysis with probes for both FAS and HMG-CoA-synthase. As demonstrated in Fig. 1Go, and rogens stimulate the expression of these genes in both LNCaP and MDA-PCa-2a cells. The absolute expression levels and the relative fold induction of these genes were different, however, in both cell lines. Both basal and androgen-induced FAS expression was higher in MDA-PCa-2a cells than in LNCaP cells. In contrast, HMG-CoA-synthase expression and induction were more pronounced in LNCaP cells.



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Figure 1. Androgen Regulation of Lipogenic Gene Expression in Two Independent Prostate Cancer Cell Lines

LNCaP cells and MDA-PCa-2a cells were cultured in the absence (-) or in the presence (+) of 10-8 M of the synthetic androgen R1881 for 2 d. Total RNA was prepared and 20 µg aliquots were subjected to Northern blot analysis with 32P-labeled probes for fatty acid synthase (FAS) or HMG-CoA-synthase (SYN) followed by autoradiography. Blots were stripped and rehybridized with 18S to demonstrate that equal amounts of RNA were present in all lanes. The data shown are representative of two independent experiments.

 
Androgen Treatment Results in Increased Nuclear Levels of Mature SREBP-1 in both LNCaP and MDA-PCa-2a Cell Lines
To test whether the observed increase in the expression of lipogenic enzymes is accompanied by activation of the SREBP pathway, nuclear extracts were prepared from cells cultured in the absence or presence of 10-8 M R1881 for 2 d. Equal amounts of protein were analyzed by immunoblot analysis using an antibody directed against the mature amino-terminal part of SREBP-1 (recognizes both SREBP-1a and SREBP-1c). As Fig. 2Go shows, androgen treatment clearly results in an increase in mature SREBP-1 levels in nuclei of both LNCaP and MDA-PCa-2a cells, indicating androgen-mediated SREBP activation in both prostate cancer cell lines. SREBP-2 levels could not be studied because of the lack of suitable antibodies recognizing mature SREBP-2 in human prostate cancer cells.



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Figure 2. Effects of Androgens on the Nuclear Content of Mature SREBP-1

After incubation for 2 d in the absence (-) or presence (+) of 10-8 M R1881, LNCaP, and MDA-PCa-2 cells were harvested and nuclear fractions were prepared. Equal amounts of protein were subjected to SDS-PAGE and Western blot analysis with IgG-2A4, a monoclonal antibody directed against the N-terminal domain of SREBP-1. Immunoreactive bands were visualized using a peroxidase-coupled goat antimouse secondary antibody and a chemiluminescence Western blot analysis system. The data shown are representative of two independent experiments.

 
Effects of Androgens on FAS and HMG-CoA-Synthase Promoter-Reporter Constructs Depend on the Presence of Intact SREBP-Binding Sites
To investigate whether this androgen-induced elevation of nuclear SREBP levels is responsible for the observed increase in lipogenic gene expression, we performed transient transfection experiments using promoter-reporter constructs derived from the two lipogenic genes studied above. In a first series of experiments, LNCaP and MDA-PCA-2a cells were transiently transfected with a 178-bp FAS promoter-reporter construct (FASwt) harboring a complex SREBP-binding site (20), or with a truncated construct that lacks this region (FASdelSRE) (Fig. 3AGo). Androgen treatment of LNCaP cells resulted in a 3-fold increase in reporter activity for the wild-type construct, and this stimulation was completely abolished when the SREBP-binding site was deleted (Fig. 3BGo). Very similar results were obtained in MDA-PCa-2a cells (Fig. 3CGo). A second set of experiments using promoter-reporter constructs derived from the HMG-CoA-synthase gene further demonstrated the importance of intact SREBP-binding sites for the androgen induction of lipogenic gene expression. As shown previously (21) and as confirmed in our studies (data not shown), the HMG-CoA-synthase gene harbors two well defined SREs (SRE-1 and SRE-2) (Fig. 4AGo), the first of which is the most critical one to confer responsiveness to the SREBP pathway. Figure 4BGo shows that androgen exposure of LNCaP cells transfected with the wild-type HMG-CoA-synthase promoter-reporter construct (SYNwt) gave rise to a 5- to 6-fold induction of reporter activity. Disruption of SRE-1 (SYNmutSRE-1) completely abolished the effects of androgens whereas mutation of SRE-2 (SYNmutSRE-2) partially decreased the response. Androgen treatment of MDA-PCa-2a cells transfected with these promoter-reporter constructs led to similar conclusions (Fig. 4CGo). The lower reporter activities obtained by transfection of HMG-CoA-synthase reporter constructs in MDA-PCa-2a cells both under basal and under androgen-stimulated conditions are consistent with the lower basal expression and lower androgen induction of the HMG-CoA-synthase gene in this cell line (see Fig. 1Go).



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Figure 3. Involvement of SREBP-Binding Sites in the Activation of FAS Promoter-Reporter Constructs by Androgens

LNCaP cells and MDA-PCa-2a cells were transiently transfected with a plasmid containing a luciferase reporter gene driven by a 178-bp FAS promoter fragment harboring a SREBP-binding site (SRE), flanked by auxiliary NF-Y and Sp-1 sites (FASwt) or with a similar construct in which the SRE was deleted (FASdelSRE) (A). The next day, cells were treated with 10-8 M R1881. After 3 d of androgen exposure, cells were lysed and luciferase acitivity was measured. Luciferase activity obtained from LNCaP cells (B) and MDA-PCa-2a cells (C) was expressed as relative luciferase units (RLU). Data shown are representative of three independent experiments. Columns, Mean of three incubations performed in triplicate; bars, SEM.

 


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Figure 4. Involvement of SREBP-Binding Sites in the Activation of HMG-CoA-Synthase Promoter-Reporter Constructs by Androgens

LNCaP cells and MDA-PCa-2a cells were transiently transfected with a plasmid containing a luciferase reporter gene driven by a HMG-CoA-synthase promoter fragment harboring two well defined SREs (SRE-1 and SRE-2) flanked by auxiliary binding sites for NF-Y and Sp-1 (SYNwt), or with similar constructs in which either SRE-1 or SRE-2 was mutated (SYNmutSRE-1 and SYNmutSRE-2) (A). The next day, cells were treated with 10-8 M R1881. After 3 d, cells were lysed and luciferase activity was measured. Luciferase activity obtained from LNCaP cells (B) and MDA-PCa-2a cells (C) was expressed as relative luciferase units (RLU). Data shown are representative of three independent experiments. Columns, Mean of three incubations performed in triplicate; bars, SEM.

 
Dominant-Negative Forms of SREBPs Abolish the Effects of Androgens on Lipogenic Gene Expression
To further corroborate the role of SREBPs in the transcriptional activation of lipogenic genes by androgens, we introduced a dominant-negative form of SREBP-1 (DN-SREBP) in LNCaP cells. This dominant-negative form was generated by deleting the amino-terminal transactivation domain of SREBP-1. The resulting protein is still able to bind to SREs but is transcriptionally inactive and blocks the access of wild-type endogenous SREBPs to SREs (22). Cotransfection of LNCaP cells with increasing amounts of an expression construct encoding DN-SREBP almost completely abolished the stimulatory effects of androgen treatment on the activity of the HMG-CoA-synthase promoter-reporter construct (Fig. 5AGo). A similar decrease in androgen responsiveness was also observed when the FAS promoter-reporter construct was used, although in this case the effects were complicated by a stimulatory effect of DN-SREBP on basal FAS expression. Cotransfection with empty expression vector was without effect (data not shown).



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Figure 5. Effects of Dominant-Negative SREBP-1 on the Induction of FAS and HMG-CoA-Synthase Promoter Activity By Androgens

A, LNCaP cells were transiently transfected with the same wild-type FAS (FAS) and HMG-CoA-synthase (SYN) promoter-reporter constructs as in Figs. 3Go and 4Go and increasing amounts of a construct encoding a dominant-negative form of SREBP-1 (DN-SREBP). The next day, cells were treated with 10-8 M R1881. After 3 d, cells were lysed, and luciferase activity was measured and expressed as relative luciferase units (RLU). Data shown are representative for two independent experiments. Columns, Mean of incubations performed in triplicate; bars, SEM. B, MDA-PCa-2a cells were cultured either without adenovirus (No virus), with adenovirus expressing a dominant-negative form of SREBP-1 (Ad-DN, 30 pfu/cell), or with adenovirus without insert (Ad-Null). The next day, 10-8 M R1881 (+) or ethanol vehicle (-) was added. Two days later, cells were harvested, followed by RNA isolation and Northern blot analysis of FAS expression.

 
To further strengthen these findings we used an alternative dominant-negative form of SREBP. This alternative DN-SREBP was generated by introduction of a point mutation in the amino-terminal fragment of SREBP-1c (amino acids 1–403), replacing tyrosine at amino acid 320 by alanine. The resulting protein no longer binds to SREs but is still able to dimerize, leading to a decreased availability of endogenous SREBP-1 (23). We used an adenoviral delivery system allowing the study of the effect of DN-SREBP on endogenous genes. As shown in Fig. 5BGo, infection with adenovirus encoding this DN-SREBP completely abolished the effects of androgens on endogenous FAS expression. Also here, the dominant-negative SREBP caused a slight elevation of basal FAS expression. Similar experiments were performed with an adenoviral vector lacking the DN insert to exclude the possibility that the observed reduction of FAS induction was due to nonspecific adverse effects related to the viral infection. Infection with the empty virus (Ad-Null) had little or no effect on the androgen induction of FAS.

Androgens Markedly Stimulate the Expression of SCAP in Prostate Cancer Cells
Having established that the involvement of SREBPs in the coordinate regulation of lipogenic gene expression by androgens is not an event limited to LNCaP cells, we attempted to unravel the mechanism underlying the androgen activation of the SREBP pathway. We first investigated whether androgens somehow affect the expression of essential components of the SREBP pathway, such as the SREBP precursor proteins (SREBP-1a, -1c, and -2), the enzymes involved in the proteolytic cleavage of these precursor proteins [site 1 protease (S1P) and site 2 protease (S2P)], or the transporter protein SCAP. To this end, LNCaP cells were grown for 2 d in the presence or absence of R1881 (10-8 M). Total RNA was prepared and subjected to Northern blot analysis with probes for the above mentioned components. As shown by Fig. 6AGo, and rogen treatment caused a slight increase in mRNA expression for SREBP-1c and SREBP-2. SREBP-1a and S1P were not affected. Slight changes were also observed in the levels of S2P mRNA expression. Major stimulatory effects were noticed only for SCAP. This androgen-induction of SCAP expression was confirmed at the protein level (Fig. 6BGo).



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Figure 6. Effect of Androgens on the Expression of Essential Components of the SREBP Pathway

A, LNCaP cells were cultured in the presence (+) or absence (-) of 10-8 M R1881 for 2 d. Total RNA was isolated and Northern blot analysis was performed with probes for the indicated genes. Hybridization with 18S was performed to correct for loading differences (not shown). Data shown are representative of two independent experiments. B, After exposure for 2 d to 10-8 M R1881 (+) or ethanol vehicle (-), LNCaP cells were harvested and total cell extracts were prepared. Equal amounts of protein were subjected to Western blot analysis with a polyclonal antibody directed against SCAP. Data shown are representative of two independent experiments.

 
Enhanced Expression of SCAP Activates the SREBP Pathway
Given the presence of an SRE-like element in the 5'-flanking region of the human SCAP gene (24), the key question was addressed whether increased SCAP expression is actively involved in androgen-induced SREBP activation or is merely a consequence of this activation. To address this question, we cultured LNCaP cells under conditions that activate the SREBP pathway independently of androgens, i.e. sterol depletion. As shown in Fig. 7Go, sterol depletion induced a marked increase in the expression of HMG-CoA-synthase but had little effect on SCAP expression.



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Figure 7. Effect of Sterol Depletion on SCAP mRNA Expression

LNCaP cells were incubated under conditions of sterol depletion (-) or sterol repletion (+) for 1 d. Total RNA was isolated and Northern blot analysis was performed with a probe for SCAP. As a control for sterol-regulated expression, blots were stripped and reprobed with a probe directed against HMG-CoA-synthase (SYN).

 
To more directly demonstrate that SCAP has an active role in the activation of the SREBP pathway, an expression vector encoding SCAP was generated (pcDNA1.1-SCAP). COS-7 cells were transiently transfected with increasing amounts of pcDNA1.1-SCAP and an expression vector encoding a herpes simplex virus (HSV)-labeled SREBP precursor protein (pTK-HSV-BP2). Two days later, nuclear extracts were prepared and immunoblot analysis was performed using an antibody directed against the HSV-epitope. Figure 8Go shows that gradual enhancement of cellular SCAP levels does indeed result in a pronounced increase in mature nuclear SREBP.



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Figure 8. Effect of Increased Cellular SCAP Levels on Nuclear Accumulation of Mature SREBP

COS-7 cells were transiently transfected with pTK-HSV-BP2, encoding a HSV-labeled SREBP-2 precursor, and increasing amounts of an expression construct encoding human SCAP (pcDNA1.1-SCAP). Two days later, nuclear extracts were prepared. Nuclear SREBP (68 kDa) was visualized by performing Western blot analysis with an antibody directed against the HSV epitope.

 
In support of this finding, transfection of increasing amounts of pcDNA1.1-SCAP in LNCaP cells gave rise to a dose-dependent increase in FAS and HMG-CoA-synthase reporter activities only when intact SREs were present (Fig. 9Go). Moreover, addition of DN-SREBP gradually decreased the stimulatory effects of SCAP expression on reporter activities (Fig. 9CGo). Cotransfection with empty expression vectors (no SCAP or DN-SREBP insert) did not affect reporter activities (data not shown).



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Figure 9. Effects of Increased Cellular SCAP Levels on Transcription of the FAS and HMG-CoA-Synthase Gene

FAS (A) and HMG-CoA-synthase (SYN) (B) promoter-reporter constructs, described in the legends to Figs. 3Go and 4Go, were cotransfected into LNCaP cells with increasing amounts of an expression construct encoding human SCAP (pcDNA1.1-SCAP). To further examine the involvement of nSREBP in the observed effects, increasing amounts of a plasmid encoding a dominant-negative form of SREBP (DN-SREBP) were added (C). Two days later, cells were lysed. Luciferase activity was measured and was expressed in relative luciferase units (RLU). Results shown are representative of at least three independent experiments. Columns, Means of incubations performed in triplicate; bars, SEM.

 
To further evaluate whether androgen-induced elevation in SCAP levels causes the maturation of SREBP precursor proteins and leads to coordinated activation of transcription of SREBP target genes, time course studies were performed. To this end, LNCaP cells were incubated in the presence or absence of R1881 (10-8 M). After the indicated periods of time, cells were harvested and either RNA was isolated or nuclear extracts were prepared. Dot blot analysis was performed with probes for SCAP, FAS, and HMG-CoA-synthase. Expression of nuclear SREBP-1 (nSREBP-1) was evaluated by immunoblot analysis. As Fig. 10Go shows, SCAP mRNA levels were clearly enhanced within 8 h of androgen treatment. An increase in the nuclear levels of SREBP-1 was first observed after 8 h of androgen exposure. In agreement with earlier reports (3), an elevation of the mRNA levels of the SREBP-target genes FAS and HMG-CoA-synthase was not noticed until the 16-h point. These time courses support the hypothesis that androgen-induced stimulation of SCAP expression may be an important element in the activation of lipogenesis.



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Figure 10. Time Course of the Response of SCAP, FAS, and HMG-CoA-Synthase mRNA and Nuclear SREBP Protein to Androgens in LNCaP Cells

LNCaP cells were grown in the presence (hatched bars) or absence (black bars) of 10-8 M R1881. After the indicated periods of time, cells were harvested and either total RNA was isolated or nuclear extracts were prepared. Northern blot analysis was performed with probes for SCAP, FAS, or HMG-CoA-synthase (SYN). mRNA levels were quantitated using PhosphorImager screens, and hybridization signals were expressed as relative densitometric units taking the values obtained from cells at the 0 h time point as 1. nSREBP was analyzed by Western blotting with an antibody directed against SREBP-1. Immunoreactive signals were quantitated using ImageMaster 1D Elite software (Amersham Pharmacia Biotech).

 
Finally, to investigate whether androgens might directly affect transcription of the gene encoding SCAP, LNCaP cells were pretreated with actinomycin D, an inhibitor of transcription, for 30 min, followed by addition of androgens (R1881 10- 8 M) or ethanol vehicle. At the indicated time points, cells were harvested and SCAP mRNA expression was evaluated. At each time point studied, the stimulatory effects of androgens on SCAP mRNA levels were abolished after actinomycin D treatment (Fig. 11Go), indicating that SCAP induction requires transcriptional activity.



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Figure 11. Effects of Actinomycin D on the Induction of SCAP mRNA by Androgens in LNCaP Cells

LNCaP cells were pretreated with actinomycin D (10 µg/ml) or ethanol vehicle for 30 min, after which time 10-8 M R1881 or ethanol was added. After the indicated periods of time, cells were harvested. Total RNA was prepared and Northern blot analysis was carried out with a probe for SCAP. Hybridization signals were quantitated using PhosphorImager screens. mRNA values were expressed as relative densitometric units, taking the values obtained from cells at the 0 h time point as 1. Black bars, Ethanol; white bars, R1881; dotted bars, actinomycin D + ethanol; hatched bars, actinomycin D + R1881)

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present report we show that marked and coordinated stimulation of lipogenic gene expression may be a common characteristic of androgen action in androgen-responsive prostate tumor lines. Moreover, we provide conclusive evidence that both the effects of androgens on genes involved in fatty acid synthesis and their effects on genes involved in cholesterol synthesis are mediated by activation of the SREBP pathway. Finally, we demonstrate that SCAP is one of the major sites at which androgens act to stimulate the SREBP pathway.

In a previous study we demonstrated that in the human prostate cancer cell line LNCaP, androgens coordinately stimulate the expression of several genes belonging to two major lipogenic pathways: fatty acid synthesis and cholesterol synthesis (11). Here, using a second independent prostate cancer cell line (MDA-PCa-2a), we demonstrate that this regulation is not a peculiarity of LNCaP cells, but occurs also in other androgen-responsive prostate cancer cell lines. Recent findings (data not shown) confirm these observations in a third androgen-responsive tumor line, PC346c (25), suggesting that stimulation of lipogenic gene expression may be a common characteristic of androgen action in androgen-responsive tumor cell lines.

In support of the involvement of SREBPs in this regulation, we show that androgen treatment leads to increased levels of mature nuclear SREBP-1 in LNCaP and MDA-MCa-2a. That this increase in nuclear SREBP is central to the regulation of lipogenic gene expression by androgens, is unambiguously demonstrated using promoter-reporter constructs derived from two key lipogenic genes, one belonging to the FAS pathway (FAS) and one crucial for cholesterol synthesis (HMG-CoA synthase). These genes harbor different, but well characterized SREBP-binding sites. Deletion or mutation of the sites that are crucial for regulation of these genes by the SREBP pathway completely abolishes the effects of androgens. Moreover, ectopic expression of dominant negative forms of SREBP-1 counteracts the stimulatory effects of androgens both on endogenous lipogenic genes and on promoter-reporter constructs derived from these genes, further supporting the concept that SREBPs function as mediators of the lipogenic effects of androgens.

Further studies addressing the molecular mechanism by which androgens affect the SREBP pathway revealed that androgens induce a major increase in the expression of SCAP. Since alternative routes of SREBP activation, e.g. by cholesterol depletion, do not result in a similar stimulation of SCAP expression, it is concluded that the observed increase of SCAP expression is not an indirect effect of SREBP activation, but indeed represents a more direct effect of androgens. In support of this contention is the finding that SCAP is up-regulated concordant with the accumulation of mature SREBP and well before the activation of lipogenic gene expression. The hypothesis of a direct link between the increase of SCAP expression and the stimulation of lipogenic gene expression by androgens was further corroborated by the finding that forced overexpression of SCAP led to enhanced nuclear accumulation of mature SREBP and to activation of lipogenic gene expression in an SRE- and SREBP-dependent manner. These data suggest that androgens may activate the SREBP pathway by a mechanism that differs from the one observed under conditions of sterol depletion. Low intracellular concentrations of cholesterol are sensed by SCAP and reduce its interaction with an as-yet-incompletely-characterized retention protein (26) that holds the SREBP-SCAP complex in the endoplasmic reticulum. As a result the SREBP-SCAP complex is released from the endoplasmic reticulum and moves to the Golgi complex where the SREBP is cleaved and activated. Androgens might reach the same effect by promoting SCAP production and changing the balance between SCAP and its retention protein. How androgens affect SCAP expression remains to be clarified. Preliminary evidence using the transcription inhibitor actinomycin D suggests that transcriptional activity is required to activate this gene. Whether this involves a direct or an indirect action of the AR is being investigated.

In addition to SCAP, other components of the SREBP pathway may also be affected by androgens. In this respect it worth mentioning that androgens slightly stimulate the expression of SREBP-1c and SREBP-2 precursors (Fig. 6Go). As both these genes are subject to autoregulation (27, 28), and as the increase in nuclear SREBP levels precedes the observed effects on the SREBP precursors (data not shown), it is unlikely that these genes are primary targets of androgens. The changes in SREBP precursor expression evoked by androgen treatment may, however, represent a secondary response and may provide a positive feedback mechanism to further enhance SREBP activation. Moreover, as only certain isoforms are affected, prolonged androgen exposure may cause a shift in the predominant active SREBP isoforms involved and result in a change in the set of genes that is ultimately affected.

Recent evidence from other research teams indicates that androgen regulation of lipogenic gene expression is not restricted to prostate cancer cell lines in culture, but is found also in prostate cancer xenografts in vivo (29). Castration of male mice carrying the CWR22 xenograft results in a significant decrease in the expression of FAS. Readministration of androgens restores FAS expression. The role of SREBPs has not been investigated in this system but seems likely in view of our recent finding that in clinical prostate cancer tissues lipogenic genes are also coordinately activated (9). In this respect it is noteworthy that in the breast cancer cell line MCF7, FAS expression has been shown to be affected by progestagens and that this effect seems also to be mediated by the SREBP pathway (30).

The fact that steroid hormones activate the SREBP pathway and stimulate lipogenic gene expression in prostate and breast cancer cells suggests that steroid receptor activation of the SREBP pathway may actively contribute to the overexpression of lipogenic genes in the corresponding cancers. In fact, overexpression of multiple lipogenic genes has been found to correlate with SREBP expression and with the expression of steroid receptors (31, 32, 33). Steroid receptor activation may, however, not be sufficient to explain increased lipogenic gene expression in these cancers. In this respect, recent evidence from our laboratory indicates that growth factors, which may enhance the function of steroid receptors and whose signaling is frequently dysregulated and constitutively activated in cancer, may also activate the SREBP pathway and lead to increased lipogenesis (34). The extent to which these effects are mediated by the AR remains to be examined. Another interesting question that is under further investigation is whether activation of the SREBP pathway by androgens (and other steroid hormones) is restricted to cancer cells or represents a more general physiological regulation in noncancerous tissues as well.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
The human prostate carcinoma cell line LNCaP was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and was maintained as described previously (3).

The MDA-PCa-2a cell line was kindly provided by Dr. Navone (University of Texas M.D. Anderson Cancer Center, Houston, TX) (19). Cells were maintained in a humidified atmosphere of 5% CO2 in air in BRFF-HPC1 medium (Biological Research Faculty & Facility Inc., Ijamsville, MD) supplemented with 20% FCS (Life Technologies, Inc., Paisley, Scotland, UK).

The COS-7 cell line was obtained from ATCC. Cells were maintained in a humidified atmosphere of 5% CO2 in air in DMEM medium (Life Technologies, Inc.) supplemented with 10% FCS (Life Technologies, Inc.), 3 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin.

In experiments assessing the effects of androgens, FCS was pretreated with dextran-coated charcoal (CT-FCS) (35) to reduce the background levels of steroids. To study the effects of sterols, cells were incubated in medium supplemented with 5% lipoprotein-deficient serum (LPDS) (Intracel Corp., Rockville, MD).

The synthetic androgen R1881 (methyltrienolone) was purchased from DuPont/NEN Life Science Products (Boston, MA), dissolved in absolute ethanol, and added to the cultures from a 1,000-fold concentrated stock. Cholesterol and 25-hydroxycholesterol were obtained from Sigma (St. Louis, MO), dissolved in absolute ethanol, and added to the cultures at a final concentration of 10 and 1 µg/ml, respectively. Actinomycin D was purchased from Sigma, dissolved in absolute ethanol, and added to the cultures at a final concentration of 10 µg/ml. Control cultures received similar amounts of ethanol only. Final ethanol concentrations did not exceed 0.2%.

cDNA Probes
cDNA probes for SREBP-1a, SREBP-1c, S1P, S2P, and SCAP were prepared by PCR as follows. cDNA was generated by reverse transcription of RNA from LNCaP cells by using Superscript II (Life Technologies, Inc.) and oligo(dT) as primer. This cDNA was used as template for PCR with the primer pairs listed in Table 1Go. PCR products were cloned into pGEM-T (Promega Corp., Madison, WI) and verified by nucleotide sequencing using an automated laser fluorescence automated sequencer (Pharmacia Biotech, Piscataway, NJ). Single-stranded radiolabeled PCR probes were produced as described (11). Probes for SREBP-2, FAS, HMG-CoA-synthase, and 18S rRNA probe (18S) were described previously (11).


View this table:
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Table 1. Sequences of PCR Primers for Cloning Human cDNA Probes

 
Northern Blot and Dot Blot Analysis
LNCaP cells were plated in 150-mm dishes at a density of 3 x 106 cells per dish in RPMI 1640 medium containing 5% CT-FCS. Two days later, medium was replaced, and cells were treated with 10-8 M R1881 or ethanol vehicle. For the actinomycin D experiments, LNCaP cells were pretreated with actinomycin D (10 µg/ml) for 30 min. MDA-PCa-2a cells were plated in 6-mm dishes at a density of 4 x 106 cells per dish in BRFF-HPC1 medium supplemented with 20% FCS. After 2 or 3 d, medium was changed to F12 medium supplemented with 10% CT-FCS, and cells were treated with androgens (10-8 M R1881) or ethanol vehicle. After the indicated period of androgen exposure, cells were washed with PBS, snap-frozen in liquid nitrogen, and stored at -80 C. Total RNA was prepared using a modified guanidinium/CsCl ultracentrifugation method as described (3). Northern blot and dot blot analyses were carried out as previously described (11). Blots were autoradiographed by exposure to Amersham Pharmacia Biotech hyperfilm-MP or to Kodak Biomax film (Amersham Pharmacia Biotech, Arlington Heights, IL). Hybridization signals were quantitated with PhosphorImager screens (Molecular Dynamics, Inc., Sunnyvale, CA) and corrected for differences in RNA loading as revealed by hybridization with an 18S ribosomal RNA probe.

Western Blot Analysis
To study the impact of androgen treatment on SREBP maturation, LNCaP cells were seeded in 150-mm plates at a density of 3 x 106 cells per plate in RPMI 1640 medium containing 5% CT-FCS. Two days later, medium was replaced and cells were treated with 10-8 M R1881 or ethanol for 2 d. MDA-PCa-2a cells were seeded in 60- mm plates at a density of 4 x 106 cells per plate in BRFF-HPC-1 medium supplemented with 20% FCS. Three days later, medium was replaced by F12 medium containing 10% CT-FCS, and cells were treated with 10-8 M R1881 or vehicle. After 2d of treatment, LNCaP cells and MDA-PCa-2a cells were harvested and nuclear fractions were prepared as described by Wang et al. (36) and Hua et al. (37) with modifications. Cells were scraped in 500 µl of ice-cold PBS supplemented with 2.2 µg/ml aprotinin, 10 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mM dithiothreitol (DTT), and 50 µg/ml N-acetyl-leucyl-leucyl-norleucinal (ALLN). After centrifugation at 1,000 x g at 4 C for 5 min, cells were resuspended in 10 mM HEPES-KOH (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM DTT, and 25 µg/ml ALLN. After incubation at 4 C for 10 min, the cell suspensions were passed through a 22-gauge needle 25 times (LNCaP cells) or through a 24.5-gauge needle 20 times (MDA-PCa-2a cells) and centrifuged at 1,000 x g at 4 C for 5 min. The pellets were resuspended in 10 mM HEPES-KOH (pH 7.4), 0.42 M NaCl, 2.5% (vol/vol) glycerol, 1.5 mM MgCl2, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 25 µg/ml ALLN. After incubation at 4 C for 30 min with gentle agitation, the suspension was centrifuged at top speed in a microcentrifuge at 4 C. The supernatant was collected, the protein concentration was determined, and the proteins were denatured by boiling for 3 min in SDS-gel loading buffer. Equal amounts of protein were subjected to 7.5% SDS-PAGE. Proteins were blotted onto PVDF Western blotting membranes (Roche Diagnostics, Mannheim, Germany) and incubated with an antibody against the N-terminal part of SREBP-1 (IgG2A4). The hybridoma cell line producing this monoclonal antibody against the basic helix-loop-helix leucine zipper domain of human SREBP-1 (IgG-2A4) was obtained from the American Type Culture Collection. Immunoreactive proteins were visualized with horseradish peroxidase-conjugated goat antimouse IgG (Amersham Pharmacia Biotech) by using an enhanced chemilumi-nescence detection kit (Renaissance, NEN Life Science Products, Dreiech, Germany).

To detect SCAP protein in total cell extracts, LNCaP cells were seeded in RPMI 1640 medium supplemented with 5% CT-FCS in 60-mm dishes at a density of 5 x 105 cells per dish. Two days later medium was replaced and cells were treated with 10-8 M R1881 or ethanol vehicle. After the indicated period of treatment, cells were collected in SDS lysis buffer and boiled for 3 min. Equal amounts of protein were analyzed by 7.5% SDS-PAGE as described above, using a polyclonal antibody against SCAP (K-19, Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Transient Transfection
The promoter-reporter constructs, FASwt and FASdelSRE, and the HMG-CoA-synthase promoter-reporter constructs, SYNwt, SYNmutSRE-1, and SYNmutSRE-2, were described previously (11, 34). The construct DN-SREBP, encoding amino acids 90–460 of SREBP-1, was generated by cloning the corresponding PCR fragment—using pSREBP-1a (ATCC79810) as a template—into the pIRES vector (CLONTECH Laboratories, Inc., Palo Alto, CA). The construct pTK-HSV-BP2 was obtained from ATCC (no. 99530). pcDNA1.1-SCAP was constructed by cloning the entire coding sequence for SCAP into the pcDNA1.1 vector (Invitrogen, San Diego, CA).

In experiments assessing the effects of androgens or sterols, LNCaP cells were seeded in 60-mm dishes at a density of 1 x 106 cells per dish in RPMI 1640 medium containing 5% CT-FCS. The next day, transfection mixtures were prepared. For each dish, 2 ml of serum-free DMEM medium (Life Technologies, Inc.) were supplemented with 5 µg of promoter-reporter construct. In the case of cotransfection experiments, the indicated amounts of the DN-SREBP construct were added. After addition of 15 µl Transfast (Promega Corp.) the transfection mixture was incubated for 15 min at room temperature. Cells were washed with serum-free medium, the transfection mixture was added, and cell cultures were incubated for 30 min at 37 C. Four milliliters of RPMI medium with 5% CT-FCS (androgen effects) or 5% LPDS (sterol effects) were added. The next day, medium was replaced (RPMI with either 5% CT-FCS or 5% LPDS), and cells were treated with either 10-8 M R1881 for 3 d (androgen effects) or cholesterol (10 µg/ml) + 25-hydroxycholesterol (1 µg/ml) or mevastatin (10 µM) (Sigma) for 1 d. After the indicated time of treatment, cells were washed with PBS and lysed in 500 µl passive lysis buffer (Promega Corp.). Aliquots of 10 µl of cleared lysate were assayed for luciferase activity by using a luciferase reporter assay kit from Promega Corp. and a Berthold Microlumat LB96P luminometer.

In experiments determining the effect of SCAP (over)expression on lipogenic gene transcription, LNCaP cells were seeded in 60 mm dishes at a density of 1 x 106 cells in RPMI 1640 medium supplemented with 10% FCS. The next day, transfection was performed as described using 3 µg promoter-reporter construct, the indicated amounts of pcDNA1.1 and/or DN-SREBP, and 9 µl Transfast per dish. The following day, medium was replaced. One day later, cells were lysed and luciferase activity was assayed as described.

In experiments assessing the impact of SCAP (over)expression on SREBP maturation, COS-7 cells were seeded in 60-mm dishes at a density of 4 x 105 cells per dish in DMEM medium containing 10% FCS. The next day, cells were transfected—2.5 µg pTK-HSV-BP2 and the indicated amounts of pcDNA1.1-SCAP per dish—using FuGene 6 Transfection Reagent (Roche Molecular Biochemicals) in accordance with the manufacturer’s instructions. The following day, medium was replaced. Nuclear extracts were prepared 24 h later as described above. Equal amounts of protein were subjected to 7.5% SDS-PAGE and analyzed for recombinant SREBP expression, using an antibody directed against the HSV epitope (Novagen, Madison, WI).

Treatment with Recombinant Adenovirus
The construction and propagation of recombinant adenoviruses expressing a dominant-negative form of SREBP-1 (Ad-DN) and of adenoviruses without insert (Ad-Null) were described previously (38, 39). MDA-PCa-2a cells were seeded in 60-mm dishes at a density of 4 x 106 cells per dish in BRFF-HPC1 medium supplemented with 20% FCS. Three days later, medium was removed and replaced by 2 ml F12 medium supplemented with 10% CT-FCS. Virus (30 pfu/cell) was added as indicated. Cultures were incubated for 4 h with frequent agitation. Fresh medium was added to a final volume of 5 ml. The next day, cells were treated with 10-8 M R1881 or ethanol vehicle. After 2 d, cells were washed and snap-frozen for RNA isolation as described previously (11).


    ACKNOWLEDGMENTS
 
We acknowledge the excellent technical assistance of Frank Vanderhoydonc.


    FOOTNOTES
 
Address requests for reprints to: Dr. J.V. Swinnen, LEGENDO, Gasthuisberg, K. U. Leuven, Onderwijs en Navorsing 9, Herestraat 49, B-3000 Leuven, Belgium. E-mail: Johan.Swinnen{at}med.kuleuven.ac.be

This work was supported by a grant "Geconcerteerde Onderzoeksactie van de Vlaamse Gemeenschap," by research grants from the Fund for Scientific Research-Flanders (Belgium) (F.W.O.), by a Cancer Research Grant from FB Insurance and VIVA, by a grant from the Belgische Federatie tegen Kanker, by a specialization grant from the Flemish Institute for the Enhancement of Scientific-Technological Research in the Industry (IWT) (to H.H.), and by a grant Interuniversity Poles of Attraction Programme-Belgian State, Prime Minister’s Office, Federal Office for Scientific, Technical and Cultural Affairs.

Abbreviations: ALLN, N-acetyl-leucyl-leucyl-norleucinal; CT-FCS, Charcoal-treated FCS; DTT, dithiothreitol; FAS, fatty acid synthase; HMG-CoA, hydroxymethylglutaryl-coenzyme A; HSV, herpes simplex virus; LPDS, lipoprotein-deficient serum; PMSF, phenylmethylsulfonyl fluoride; 18S, 18S rRNA probe; SCAP, SREBP cleavage activating protein; SRE, sterol-regulatory element; SREBP, SRE-binding protein; S1P, site 1 protease; S2P, site 2 protease.

Received for publication November 15, 2000. Accepted for publication June 11, 2001.


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