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Endocrinology Vol. 145, No. 2 613-619
Copyright © 2004 by The Endocrine Society

Involvement of Cholesterol-Rich Lipid Rafts in Interleukin-6-Induced Neuroendocrine Differentiation of LNCaP Prostate Cancer Cells

Jayoung Kim, Rosalyn M. Adam, Keith R. Solomon and Michael R. Freeman

The Urologic Laboratory (J.K., R.M.A., M.R.F.), Department of Urology, Department of Orthopaedic Surgery (K.R.S.), Children’s Hospital Boston; and the Department of Surgery, Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Michael R. Freeman, Ph.D., John F. Enders Research Laboratories, Room 1161, Children’s Hospital Boston, 300 Longwood Avenue, Boston, Massachusetts 02115. E-mail: michael.freeman{at}tch.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-6 is an inflammatory cytokine that has been linked to aggressive prostate cancer (PCa). Previous studies have demonstrated that IL-6 can enhance the differentiation of PCa cells toward a neuroendocrine (NE) phenotype, a possible indicator of hormone-refractory disease. In this report, we present evidence that the mechanism of IL-6-stimulated NE differentiation employs a detergent-resistant (lipid raft) membrane compartment for signal transduction in LNCaP PCa cells. Signal transducer and activator of transcription (STAT)3, a mediator of IL-6 signaling, was rapidly phosphorylated and translocated to the nucleus in LNCaP cells treated with IL-6. Both processes were inhibited by filipin, a cholesterol-binding compound that disrupts plasma membrane lipid rafts. Isolation of Triton X-100-insoluble raft fractions from LNCaP cells by discontinuous sucrose gradient centrifugation demonstrated that the 80-kDa IL-6 receptor localized almost exclusively to the raft compartment. Although STAT3 was located predominantly in the Triton X-100-soluble subcellular fraction in exponentially growing cells, abundant phosphorylated STAT3 was detected in the raft fraction after stimulation with IL-6. Increases in expression of the NE marker, neuron-specific enolase, and neuron-specific enolase promoter activity after IL-6 treatment were reduced after membrane rafts were disrupted by filipin treatment. LNCaP cells expressed the raft-resident proteins flotillin-2 and Gi{alpha}2, but notably not caveolins, the predominant structural protein present in caveolar membrane rafts in many tissues and tumor cells. These results are the first to define a role for lipid raft membrane microdomains in signal transduction mechanisms capable of promoting the NE phenotype in PCa cells, and they demonstrate that the raft compartment is capable of mediating such signals in the absence of caveolins. Our results also suggest a mechanistic role for membrane cholesterol in cell signaling events relevant to PCa progression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEUROENDOCRINE (NE) PROPERTIES expressed by tumor cells can indicate poor prognosis or the likelihood of the presence of aggressive disease in a variety of solid tumor types, including prostate cancer (PCa) (1, 2). Tumor cells that express NE characteristics can be post-mitotic (3); however, they typically produce cytokines capable of stimulating growth and survival of neighboring adenocarcinoma cells in a paracrine manner (4, 5). In addition, evidence that tumor cells exhibiting NE-like properties can remain capable of proliferation has been obtained in cell culture models (6) and with in vivo model systems (7). These findings indicate that tumor progression coinciding with NE differentiation can result in the emergence of an inherently more aggressive tumor cell population.

The molecular mechanisms by which PCa cells acquire an NE phenotype are incompletely understood; however, evidence indicates that this process involves the action of one or more of several possible signal transduction pathways. Previous studies have used the human LNCaP cell line to study the manner in which human PCa cells acquire features of NE differentiation (2, 3, 6, 8, 9, 10). LNCaP cells express a marginal NE phenotype under basal conditions, but NE characteristics are substantially enhanced in this cell line by certain culture manipulations, including prolonged androgen depletion (11), pharmacological elevation of intracellular cAMP (12), and exposure of cells to the epidermal growth factor receptor tyrosine kinase (ErbB family) ligand heparin-binding epidermal growth factor-like growth factor (HB-EGF) (6, 13) or to the cytokine IL-6 (3). The majority of published reports have focused on IL-6 as the primary inducer of this phenotype, in part, because elevated circulating IL-6 levels are associated with aggressive PCa and may be an indicator of hormone-refractory disease (14, 15, 16). NE differentiation in LNCaP and other cell lines can be evoked as a result of the activation of multiple, sometimes independent, signal transduction mechanisms. For example, in LNCaP cells, IL-6 triggers phosphorylation and nuclear import of the transcription factor, signal transducer and activator of transcription (STAT)3 and induces cell cycle arrest (10, 17), whereas HB-EGF induces NE differentiation by a STAT3-independent, ERK-MAPK-dependent process that coincides with continued cell cycle transit (6).

STATs are well known as cytosolic proteins that are recruited to the plasma membrane by members of the JAK family of tyrosine kinases (18). After activation, STATs dimerize through SH2-phosphotyrosyl interactions and migrate to the nucleus, where they are incorporated into transcriptional complexes (18, 19, 20). The manner in which STATs are sequestered before activation is still incompletely understood. Recent studies have indicated that a significant fraction of latent and active STATs reside in detergent-resistant membrane domains generally referred to as lipid rafts (21, 22). Lipid rafts are liquid-ordered membrane assemblies that are rich in cholesterol and glycosphingolipids relative to the substantially more abundant liquid-disordered phase of the plasma membrane (23). Lipid rafts sequester signaling proteins of many kinds, including heterotrimeric G protein subunits, receptor tyrosine kinases, and Src-like kinases. Although their biological function is still poorly understood, rafts have been shown to act as membrane platforms for regulating signal transduction in many cell types, including tumor cells (24).

The potential relevance of lipid raft membrane domains to PCa was first suggested by observations that caveolin-1, a raft-resident structural protein thought capable of directly regulating a variety of signal transduction molecules, is a marker of aggressive disease in PCa and other cancers (25, 26, 27, 28, 29). Caveolin-1 is capable of promoting hormone-mediated tumor cell survival and metastatic dissemination in model systems of PCa (26). Furthermore, caveolin-1 may be a mediator of androgenic signaling by a mechanism involving a direct protein-protein interaction with the androgen receptor (30). Lipid rafts also seem to be capable of processing signals relevant to tumor progression even in the absence of caveolin-1. Regulation of the Akt/protein kinase B pathway and cell survival signaling by a cholesterol-dependent mechanism involving lipid rafts was also recently demonstrated in caveolin-negative LNCaP cells (31).

In this study, we present evidence that signal transduction through lipid rafts is involved in IL-6- and STAT3-mediated induction of NE properties in LNCaP cells. These results support a role for lipid raft microdomains as essential mediators of disease progression in PCa. They also suggest that the presence of caveolins in the raft membrane compartment is not necessary for transmission of certain cellular signals involved in the promotion of aggressive disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Human recombinant IL-6 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies against caveolin-1, caveolin-2, pan-caveolin, flotillin-2, and neuron-specific enolase (NSE) were purchased from BD Biosciences (San Diego, CA). Anti-phospho-STAT3 (Tyr705) and anti-STAT3 antibodies were purchased from Cell Signaling Technology (Beverly, MA). Reagents for sucrose gradient ultracentrifugation were the highest possible grade and were obtained from Sigma (St. Louis, MO). Antibodies against IL-6 receptor (IL-6R), and Gi{alpha}2 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against NSE were from Neomarkers (Fremont, CA). The Micro BCA protein assay kit was used for protein measurement (Pierce Chemical Co., Rockford, IL). Polyfect transfection reagents were purchased from Qiagen (Valencia, CA). Infinity cholesterol determination reagents and filipin were obtained from Sigma.

Cultured cells
The human PCa cell line LNCaP and PC3 were purchased from American Type Culture Collection (Rockville, MD) and cultured in RPMI 1640 or DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc., Grand Island, NY). These supplements were used in all media unless otherwise indicated. Cells were cultured in a humidified atmosphere of 5% CO2 at 37 C.

Sucrose gradient ultracentrifugation
LNCaP cells were lysed in 1% Triton X-100 in 25 mM 2-(N-morpholino)-ethanesulfonic acid, 150 mM NaCl (pH 6.5), 1 mM Na3VO4, and 1 mM phenylmethylsulfonylfluoride (PMSF) followed by mechanical disruption with eight strokes of a Dounce homogenizer. Cell lysates were diluted 1:1 with 60% sucrose (final sucrose concentration of 30%) and layered on a 40% sucrose cushion, followed by successive 2-ml additions of 25%, 20%, 15%, 10%, 5%, 0% (20 mM KCl) sucrose solution. Ultracentrifugation was performed at 100,000 x g for 20 h in a Beckman SW28 rotor (Beckman Coulter, Fullerton, CA). All experimental steps were performed on ice or at 4 C.

Lipid raft isolation by successive detergent extraction
Extraction of Triton-soluble and -insoluble membrane components was performed as described (32). Briefly, LNCaP cell lysates were prepared in buffer A [25 mM 2-(N-morpholino)-ethanesulfonic acid, 150 mM NaCl (pH 6.5)], and an equal volume of the same buffer with 2% Triton X-100, 2 mM Na3VO4, and 2 mM PMSF was added. After 30 min of incubation, lysates were centrifuged, and supernatants (containing the Triton-soluble fraction) were removed. Insoluble pellets were resuspended with buffer B [1% Triton X-100, 10 mM Tris-Cl (pH 7.6), 500 mM NaCl, 2 mM Na3VO4, 60 mM ß-octylglucoside, and 1 mM PMSF] for 30 min on ice. Triton-insoluble and octylglucoside (OCG)-soluble supernatants were collected after 20 min of centrifugation at 15,000 x g.

Cholesterol measurements
Cholesterol determinations were performed on 300-µl fractions harvested from sucrose gradients described above or in total cell membranes prepared by resuspending cells in a hypotonic buffer [50 mM HEPES, pH 7.4; 10 mM NaCl; 5 mM MgCl2; 0.1 mM EDTA; and protease inhibitors] followed by mechanical disruption (12 strokes with a Dounce homogenizer) and centrifugation (9,000 x g for 10 min). Lipids were solubilized in chloroform, extracted two times through H2O, dried, and subjected to cholesterol determination using the Infinity cholesterol determination assay kit (Sigma).

Lipid raft disassembly
Lipid raft disruption was accomplished by treating cells with filipin, a cholesterol-binding polyene macrolide that has been shown to disassemble lipid rafts by dispersing cholesterol in the membrane, thereby interfering with transmission of raft-dependent signals (24, 33, 34). Cells were treated with varying concentrations of filipin (see figures) at 37 C in serum-free culture medium before assay.

Western blot analysis
Proteins isolated from cell fractions as described above were subjected to SDS-PAGE and electroblotted onto nitrocellulose. Blots were stained with Ponceau S to verify uniform transfer and equal protein loading (where appropriate) and subjected to immunoblotting with various antibodies as previously described (6).

Indirect immunofluorescence cell staining
Low-density LNCaP cells were serum starved for 12 h, followed by challenge with 100 ng/ml IL-6 for 15 min. In some cases, cells were pretreated with 2 µg/ml filipin before IL-6 treatment. Cells were fixed with ice-cold methanol and incubated with anti-phospho-STAT3 antibody in 2% BSA solution, followed by secondary antibody conjugated to fluorescein isothiocyanate. After mounting and incubation with 4'-containing mounting agent, cells were analyzed by fluorescence microscopy.

Construction of NSE-luciferase promoter reporter plasmid and measurement of promoter activity
A fragment of approximately 1.3-kb encoding the NSE promoter was amplified from genomic DNA (Promega Corporation, Inc., Madison, WI) using the Advantage-GC Genomic PCR reagent (BD Biosciences Clontech, Palo Alto, CA) and the primers: 5'-GCGGCTAGCTGTATGCAGCTGGACCTAGGAGAGAAGCAG-3' and 5'-GCGAGATCTCGGTGGTAGTGGCGGTGGCGGTGGCGGTGG-3'. The primers incorporated restriction sites for NheI and BglII, respectively (underlined). The PCR product was digested with NheI and BglII and subcloned into the reporter vector, pGL3-Basic (Promega Corporation Inc.) to generate pNSE-Luc. The integrity of the construct was confirmed by DNA sequencing. NSE promoter activity was determined after transient transfection with the NSE promoter-luciferase reporter plasmid, using the Polyfect reagent. Experiments using filipin and IL-6 were performed 24 h after transfection. At the end of treatment, medium was removed, and cells were lysed with 100 µl reporter lysis buffer. Lysates were prepared by freeze-thaw (three times), and insoluble material was pelleted with high-speed centrifugation. Luciferase activity was normalized to ß-Gal activity.

Statistical analysis
Experimental data were compared using Student’s two-tailed t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To determine the role of lipid raft microdomains in IL-6 signaling independently of the contribution of caveolin proteins, we used LNCaP cells that do not express detectable levels of caveolins. The results shown in Fig. 1Go demonstrate that LNCaP cells contain a verifiable lipid raft membrane fraction that does not contain caveolins, consistent with our previous report (31). In the experiment shown, this detergent-resistant membrane fraction contained the raft protein markers, flotillin-2 and Gi{alpha}2, but did not contain detectable caveolin-1 and -2, as demonstrated by immunoblotting with antibodies to caveolin-1 and -2 or with a pan-caveolin antibody (Fig. 1Go). In contrast to LNCaP cells, the more aggressive PC-3 PCa cell line expressed caveolin-1 and -2, which partitioned, as expected, into the raft fraction obtained from these cells. The caveolin-negative LNCaP cells used for the experiments shown in Fig. 1Go were used throughout these studies.



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  		FIG. 1. LNCaP PCa cells are caveolin-negative. LNCaP and PC3 cells in serum-containing medium were harvested, and cell lysates were separated into Triton-soluble (TS) and Triton-insoluble/OCG-soluble fractions. Localization of flotillin-2, Gi{alpha}2, and caveolin isoforms was determined by Western blot. The OCG fraction is the detergent-resistant membrane (lipid raft) fraction.

 
Lipid raft disruption inhibits IL-6-mediated STAT3 phosphorylation and nuclear translocation
IL-6 stimulates NE differentiation in PCa cell lines through a STAT3-dependent pathway (10). To study the possible functional contribution of plasma membrane rafts to expression of the NE phenotype, we disrupted the rafts (see Materials and Methods) before treatment of LNCaP cells with IL-6. Raft disruption with the cholesterol-binding compound, filipin, blocked STAT3 phosphorylation induced by 100 ng/ml IL-6 (Fig. 2AGo), indicating that STAT3 activation is a raft-dependent event. Immunofluorescence cell staining with an anti-phospho-STAT3 antibody demonstrated an increase in STAT3 phosphorylation and migration of phosphorylated STAT3 to the nucleus after IL-6 treatment (Fig. 2BGo, middle). These events were inhibited when the rafts were disrupted (Fig. 2BGo right).



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  		FIG. 2. Disruption of plasma membrane rafts inhibits IL-6-induced STAT3 phosphorylation and nuclear translocation. Quiescent LNCaP cells were preincubated with filipin at the indicated concentrations for 1 h, followed by treatment with IL-6 for 15 min. A, Phosphorylation of STAT3 determined by Western blot. B, Phosphorylated STAT3 observed by immunofluorescence staining using anti-p-STAT3 antibody. Nuclei were counterstained with 4',65-diamidino-2-phenylindole.

 
IL-6R is associated with lipid rafts in replicating LNCaP cells
To investigate the localization of STAT3 and the 80-kDa ligand-binding IL-6R in LNCaP cells under exponentially growing conditions, we evaluated the buoyant density of these proteins in discontinuous sucrose gradients. Cells growing at subconfluent density in serum (in the absence of IL-6) were solubilized with Triton X-100-containing lysis buffer, homogenized, and resuspended in the same volume of 60% buffered sucrose solution. Samples were loaded onto step gradients containing 0–40% sucrose. After ultracentrifugation (20 h), fractions were removed sequentially from the top of the gradient and examined by immunoblot and cholesterol measurement. Light buoyant density fractions that contained the majority of the raft-associated proteins, flotillin-2 and Gi{alpha}2, were identified and were verified to contain high levels of cholesterol (Fig. 3Go), indicating that these fractions correspond to lipid raft membranes. In the experiment shown in Fig. 3Go, fraction 11 contained essentially all of the raft protein Gi{alpha}2, the majority of flotillin-2, and the highest levels of cholesterol. This fraction also contained essentially all of the detectable IL-6R, indicating that the IL-6R localizes exclusively to the lipid raft membrane compartment in LNCaP cells under exponential growing conditions. STAT3 was also detected in fraction 11, although the majority of STAT3 was present in the heavy fractions.



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  		FIG. 3. Lipid rafts in LNCaP cells contain IL-6R and STAT3. Exponentially growing LNCaP cells (in the absence of IL-6) were lysed in Triton-containing buffer and prepared for sucrose gradient ultracentrifugation as described in Materials and Methods. Expression of STAT3, IL-6R, flotillin-2, and Gi{alpha}2 were analyzed by Western blot. Relative cholesterol levels in each fraction are shown below the lower panel. A.U., Arbitrary units.

 
Phosphorylated STAT3 accumulates in the lipid raft fraction after IL-6 stimulation
To investigate whether IL-6 employs lipid raft microdomains to transmit a cellular signal, the subcellular location of phosphorylated STAT3 was evaluated after IL-6 treatment. The majority of phosphorylated STAT3 was detected in the Gi{alpha}2-enriched fraction, 15 min after stimulation with IL-6, as evaluated by sucrose density centrifugation and immunoblot (Fig. 4AGo). A similar result was observed using a separate method that employs differences in detergent solubility to isolate raft membranes (32) (Fig. 4BGo). These results indicate that IL-6 signaling to STAT3 occurs through a lipid raft membrane domain. Interestingly, the data shown in Fig. 4BGo also indicate that phosphorylated STAT3 molecules (i.e. actively signaling molecules) are relatively enriched in the raft fraction, in comparison with the nonraft subcellular compartments, where the majority of STAT3 resides (consistent with the data shown in Fig. 3Go).



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  		FIG. 4. Lipid rafts in LNCaP cells are enriched in phosphorylated STAT3 after treatment with IL-6. Quiescent cells were stimulated with IL-6 in serum-free medium. A, After 15 min, LNCaP cell lysates from vehicle (-IL-6) and IL-6-treated (+IL-6) conditions were prepared for sucrose gradient ultracentrifugation. p-STAT3 and Gi{alpha}2 were detected by Western blot. B, At the indicated times, IL-6-treated LNCaP cells were harvested, and lysates were separated into TS and Triton-insoluble/OCG-soluble (OCG) fractions. Phosphorylated and total STAT3 were detected by Western blot.

 
Lipid raft disruption antagonizes IL-6-mediated stimulation of NE properties
IL-6 treatment stimulates an enhancement of NE properties in LNCaP cells (3). To determine whether this effect is mediated by lipid rafts, cells in which the rafts were disrupted were compared with raft-competent control cells, with respect to the expression of NE properties after IL-6 treatment under the conditions used for the experiment shown in Fig. 2Go. Expression of the NE marker NSE increased about 6-fold with IL-6 treatment at 5 d, an effect significantly inhibited when rafts were disrupted with filipin (Fig. 5AGo). The inhibitory effect of filipin on IL-6 expression was dose dependent. A similar effect was demonstrated at the transcriptional level when luciferase activity produced from a transiently transfected NSE promoter-reporter construct was analyzed (Fig. 5BGo). These findings indicate that IL-6 stimulation of NE properties is raft-dependent. Consistent with this conclusion, addition of cholesterol-cyclodextrin complexes to the cell cultures, a procedure that increases levels of membrane cholesterol and the size of the raft compartment, stimulated NSE promoter activity about 20% in the absence of IL-6 (data not shown).



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  		FIG. 5. NSE expression and NSE promoter activity are inhibited by disruption of plasma membrane rafts. Quiescent cells were preincubated with filipin for 1 h, then medium was changed to serum-free RPMI 1640 containing IL-6. A, To analyze the effect of filipin on NSE protein expression, total cell lysates were prepared 5d after IL-6 addition with or without the indicated quantity of filipin as shown, and Western blot was performed with anti-NSE and {alpha}-actin antibodies. Results shown in the graph are the means ± SD of NSE/actin ratios from three independent experiments (*, P < 0.05; **, P < 0.005). C (control), Serum-containing conditions (no IL-6). B, To determine the effect of filipin on NSE-promoter activity, LNCaP cells were transiently transfected with an NSE promoter-luciferase construct. Luciferase activity was measured under indicated conditions (-IL-6, +IL-6, and +IL-6+filipin) and at different times (1 and 2 d after IL-6 stimulation). Results are the means ± SD (n = 6; *, P < 0.005; **, P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have identified a novel role for detergent-resistant plasma membrane rafts in IL-6-mediated promotion of NE differentiation in LNCaP PCa cells. IL-6, a cytokine that has been linked to human PCa progression (15), has been identified previously as being capable of enhancing the NE characteristics of LNCaP and other PCa cell lines (3, 10). The evidence we present in support of a role for lipid rafts in NE differentiation in LNCaP cells is the following: 1) raft disruption by dispersion of cholesterol in the plasma membrane inhibited STAT3 phosphorylation and translocation of STAT3 to the nucleus in LNCaP cells treated with IL-6; 2) the 80-kDa IL-6R localized essentially entirely (and STAT3 localized partially) to a lipid raft compartment in LNCaP cells under exponential growth conditions; 3) the majority of activated STAT3 localized to the raft compartment in response to treatment of cells with IL-6; and 4) raft disruption inhibited NSE protein accumulation, and induction of NSE promoter activity, in response to IL-6. Our findings provide the first evidence that the IL-6 signaling mechanism employs a cholesterol-rich membrane raft compartment for transmission of the cytokine-mediated signal in PCa cells. The LNCaP cell line is used widely for in vitro and in vivo studies of PCa because it expresses a range of properties characteristic of differentiated human prostate epithelial cells (e.g. prostate-specific antigen). LNCaP cells respond to androgen by modulating growth and altering gene expression. Consequently, these studies may be relevant to mechanisms of NE differentiation in vivo, where NE properties in prostate tumors have been associated with aggressive or hormone-refractory disease (1). Although the role of plasma membrane events in the activation of STATs is still poorly understood, our results are consistent with recent reports that STATs employ raft-dependent signaling mechanisms in other cell types (22, 35). An important distinction, however, is that these previous studies employed cells that express the raft-resident protein, caveolin-1. In contrast, our experiments have employed caveolin-negative cells, thereby demonstrating that caveolins are not an obligatory component of the STAT3-mediated pathway described here.

Although the IL-6/STAT3/NE differentiation mechanism we focus on in the present study is independent of contributions from caveolins, our findings are consistent with previous reports implicating caveolin-1 in PCa progression (26, 27, 28). This is because caveolins localize essentially exclusively to lipid raft membranes, an observation that points to the membrane raft compartment as an important element of the mechanism by which PCa cells survive in the host and progress to a metastatic state. The present study and earlier studies, reporting increases in caveolin-1 expression in aggressive PCa, point to a role for the plasma membrane raft compartment as a potential locus of cell signaling events relevant to PCa progression. Caveolin-1 has been reported to modulate the activity of a number of signaling molecules capable of transiting caveolar lipid rafts, in some cases by direct protein-protein interaction with caveolin proteins (30, 36, 37, 38). Thus, the cholesterol- and sphingolipid-rich raft fraction may serve as an important node for signal transduction events regulating PCa cell growth and survival. Consistent with this idea, our laboratory has recently demonstrated a facilitative role for lipid rafts in Akt pathway signaling and survival in LNCaP cells (31).

A correlation between PCa aggressiveness and levels of STAT3 activation has been reported by several groups (39, 40). STAT up-regulation has also been shown to enhance the growth of LNCaP xenografts in an androgen-independent manner (41), consistent with a role for this signaling protein in hormone-refractory disease. Conversely, enforced down-regulation of STAT3 was shown to trigger apoptosis in PCa cell lines (40). We were able to significantly attenuate IL-6-mediated phosphorylation and nuclear translocation of STAT3, as well as increases in NSE promoter activity and protein levels, by disrupting the membrane raft compartment with filipin, a highly specific cholesterol-binding drug (24, 33, 34). These findings suggest an important role for membrane cholesterol as a mediator of STAT3-derived signal transduction events. The link between tumor cell survival and tumor aggressiveness, STAT3, and cholesterol suggests the possibility that membrane cholesterol may be an important component of the PCa cell’s repertoire of defenses against apoptotic stimuli.

The hypothesis that cholesterol is a mediator of tumor cell survival is consistent with observations made decades ago, that benign and malignant prostate tissues accumulate cholesterol and other fatty deposits (42). Cholesterol synthesis inhibitors have also been demonstrated to induce apoptosis in prostate, mammary, neuroblastoma, and other tumor cell lines, suggesting a role for cholesterol or other downstream products of the mevalonate (cholesterol synthesis) pathway in resistance to apoptotic triggers (43, 44, 45). Furthermore, although the mechanism is still presently unknown, PCa incidence and progression have been linked to high fat diets and/or the consumption of animal products (46, 47). It is interesting that this epidemiological relationship to dietary fat and animal products is a feature of only some malignancies, with PCa and breast cancer being among the most notable (46). Similarly, apoptotic sensitivity to cholesterol synthesis inhibition has been demonstrated to be dependent on cell type or tissue origin (43).

The membrane cholesterol content of somatic cells in primates is partly dependent on low-density lipoprotein levels in the circulation. Long-term cholesterol-lowering drug therapy has been associated with a significant reduction in the incidence of a number of solid tumors, including PCa (48). One potential explanation for this finding is that cell survival signaling mechanisms involving cholesterol-rich membrane domains are affected by pharmacologic intervention to lower circulating cholesterol levels. Although more study is needed to rigorously test this hypothesis, an important corollary is that pharmacologic interventions directed at altering tumor cell cholesterol levels may be a rational means of therapy or chemoprevention for prostate and other solid tumors in which relationships to either cholesterol-rich diets or elevated cholesterol have been established.


   Footnotes
 
This work was supported by grants from the NIH (R37 DK47556, RO1 CA77386, and RO1 DK57691 to M.R.F., and R01 CA101046 to K.R.S.). J.K. and R.M.A. are American Foundation for Urologic Disease Research Scholars.

Abbreviations: HB-EGF, Heparin-binding epidermal growth factor-like growth factor; IL-6R, IL-6 receptor; NE, neuroendocrine; NSE, neuron-specific enolase; OCG, octylglucoside; PCa, prostate cancer (prostate adenocarcinoma); PMSF, phenylmethylsulfonylfluoride; STAT, signal transducer and activator of transcription.

Received June 20, 2003.

Accepted for publication October 10, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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