[Cell Cycle 7:14, 2257-2267; 15 July 2008]; ©2008 Landes Bioscience
Report
Caveolin-1 interacts with a lipid raft-associated population
of fatty acid synthase
Dolores Di Vizio,1,2,* Rosalyn M. Adam,1,2 Jayoung Kim,1,2 Robert Kim,4 Federica Sotgia,5 Terence Williams,5 Francesca
Demichelis,4 Keith R. Solomon,1,6 Massimo Loda,7 Mark A. Rubin,4 Michael P. Lisanti5 and Michael R. Freeman1-3,*
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1The Urological Diseases Research Center and the Departments of 2Surgery, 3Biological Chemistry and Molecular Pharmacology; Children’s Hospital Boston; Harvard Medical
School; Boston, Massachusetts USA; Department of 4Pathology; Brigham and Women’s Hospital; Dana Farber Cancer Institute; Harvard Medical School; Boston, Massachusetts
USA; Kimmel Cancer Center; Departments of 5Cancer Biology, and Biochemistry & Molecular Biology; Thomas Jefferson University; Philadelphia, USA; Program in Molecular
Biology and Genetics; Department of 6Orthopaedic Surgery; Children’s Hospital Boston; Harvard Medical School; Boston, Massachusetts USA; 7Medical Oncology; Dana-Farber
Cancer Institute and Pathology Department; Brigham and Women’s Hospital; Harvard Medical School; Boston, Massachusetts USA
cells, Cav-1C133/143/156S caused a reduction of both Src and Akt
levels, as well as of their active, phosphorylated forms, in comparison with wild type Cav-1. These findings suggest that FASN
and Cav-1 physically and functionally interact in PCa cells. They
also imply that palmitoylation within this complex is involved in
tumor growth and survival.
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Fatty Acid Synthase (FASN), a cytoplasmic biosynthetic
enzyme, is the major source of long-chain fatty acids, particularly palmitate. Caveolin-1 (Cav-1) is a palmitoylated lipid raft
protein that plays a key role in signal transduction and cholesterol
transport. Both proteins have been implicated in prostate cancer
(PCa) progression, and Cav-1 regulates FASN expression in a
mouse model of aggressive PCa. We demonstrate that FASN and
Cav-1 are coordinately upregulated in human prostate tumors
in a hormone-insensitive manner. Levels of FASN and Cav-1
protein expression discriminated between localized and metastatic
cancers, and the two proteins exhibited analogous subcellular
locations in a tumor subset. Endogenous FASN and Cav-1 were
reciprocally co-immunoprecipitated from human and murine
PCa cells, indicating that FASN forms a complex with Cav-1.
FASN, a cytoplasmic enzyme, was induced to associate transiently
with lipid raft membranes following alterations in signal transduction within the Src, Akt and EGFR pathways, suggesting that
co-localization of FASN and Cav-1 is dependent on activation of
upstream signaling mediators. A Cav-1 palmitoylation mutant,
Cav-1C133/143/156S, that prevents phosphorylation by Src, did not
interact with FASN. When overexpressed in Cav-1-negative PCa
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Key words: caveolin-1, fatty acid synthase, lipid raft membranes, prostate cancer
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Abbreviations: Ab, antibody; pAb, polyclonal antibody; mAb, monoclonal antibody; Cav-1, caveolin-1; Cav-3, caveolin-3; Co-IP,
co-immunoprecipitation assay; DAB, 3-3' diaminobenzidine; DAPI, 4',6'-diamidino-2-phenylindole; DMSO, dimethylsulfoxide; DRMs,
detergent-resistant membranes; EGFR, epidermal growth factor receptor; FACS, fluorescence activated cell sorting; FASN, fatty acid
synthase; IF, immunofluorescence; Mets, metastases; OCG, octylglucoside; PCa, prostate cancer; PFA, paraformaldehyde; PI3K, phosphoinositide-3' kinase; SREBP, sterol regulatory binding protein; TMA, tissue microarray
*Correspondence to: Dolores Di Vizio; The Urological Diseases Research Center;
Enders Research Laboratories; Rm 1149; Children’s Hospital Boston; 300
Longwood Ave.; Boston, Massachusetts 02115 USA; Tel.: 617.919.2641;
Fax: 617.730.0238; Email: [email protected]/ Michael R.
Freeman; The Urological Diseases Research Center; Enders Research Laboratories;
Rm 1161; Children’s Hospital Boston; 300 Longwood Ave.; Boston, Massachusetts
02115 USA; Tel.: 617.919.2644; Fax: 617.730.0238; Email: michael.freeman@
childrens.harvard.edu
Submitted: 04/25/08; Revised: 05/12/08; Accepted: 05/12/08
Previously published online as a Cell Cycle E-publication:
http://www.landesbioscience.com/journals/cc/article/6475
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Introduction
Signals entering and leaving a tumor cell must cross a lipid
barrier that is still incompletely understood in terms of structure
and regulatory functions. Abundant evidence has accumulated
that cholesterol- and sphingolipid-enriched membrane patches,
termed lipid rafts, sequester and exclude signaling proteins, harbor
pre-formed signal transduction complexes, and play an important
role in signal transduction.1,2 Caveolae are one form of lipid raft
that is enriched in the membrane protein Cav-1 or its musclespecific paralog, Cav-3.3 The high content of glycosphingolipids
and cholesterol in these membrane microdomains confers a “liquid
ordered” structure that distinguishes them from other regions of the
membrane that are relatively liquid disordered. Most recent theoretical and experimental work suggests that flat (non-caveolar) rafts
are likely to be transient structures that are between 6–20 nm in
diameter, while caveolar rafts, which were first observed by electron
microscopy in the 1950s,4 are much larger, in the range of 80 nm. In
addition to cell signaling, other key cellular processes in which lipid
rafts are thought to play a role include intracellular trafficking, cell
polarization and cell migration.5 Although the non-caveolar form
of lipid raft is less well-defined than are caveolae, the biophysical
properties of the two microdomains are similar if not identical, and
they sequester similar classes of proteins.6 Recent evidence has accumulated suggesting that lipid rafts provide a platform where signals
are processed that are essential to tumor cell growth, resistance to
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Cav-1 and FASN are coordinately expressed in human PCa. In
a published study we showed that Cav-1 ablation interferes with
tumor progression in an animal model of aggressive PCa,30 in which
both Cav-1 and FASN are naturally overexpressed downstream from
oncogenic activation.22,30 More recently we found that genetic ablation of Cav-1 in TRAMP/Cav-1-/- mice coincides with a dramatic
reduction of FASN levels.15 This result, in an in vivo model, suggests
that Cav-1 and FASN may be coordinately regulated at one or more
levels. To determine whether a similar coordinate expression pattern
exists in human tumors, and whether such coordinate expression
might relate to tumor progression, a tissue microarray (TMA) was
evaluated using antibodies for FASN and Cav-1 that have been wellcharacterized for staining on paraffin embedded tissues. The TMA
contains specimens corresponding to benign prostate tissue (n = 18),
localized prostate adenocarcinoma (n = 36) distributed broadly across
the Gleason score categories, hormone responsive metastasis (n = 18),
and hormone refractory metastasis (n = 18). FASN levels, as measured
quantitatively by the Chromavision system, were altered with tumor
progression in a manner that paralleled changes in Cav-1 expression
(Fig. 1A). Levels of both proteins increased from the normal to the
malignant state and with tumor progression, with significant differences between localized and metastatic cancers (p = 0.05 and 0.01 for
FASN and for Cav-1, respectively). Expression of FASN and Cav-1
demonstrated a high degree of coordination within the adenocarcinoma and within the metastatic PCa groups (p < 0.01 and p = 0.03,
respectively) as well as significant correlation with Gleason grade. For
both FASN and Cav-1, there was a striking increase in expression
from Gleason pattern 4 + 3 to Gleason pattern 4 + 4 (p = 0.04 for
FASN and p = 0.01 for Cav-1) (Fig. 1B). No significant difference
in immunostaining pattern was noted between FASN and Cav-1
in hormone responsive versus hormone resistant metastatic tumors
(not shown). This result indicates that overexpression of both FASN
and Cav-1 can occur in the hormone-independent phase of disease
progression. Multivariate regression analysis showed that Cav-1 alone
was a better predictor of PCa progression, compared to FASN and
Cav-1 in combination. Representative examples of immunostaining
in normal tissue, PCa and PCa metastases from the TMA are shown
in Figure 1C. Examination of the histologic sections of the human
tumors showed that FASN, which predominantly localized to the
cytoplasm (Fig. 1D, right), exhibited focal enrichment at or near
the plasma membrane in a subset of tumors (Fig. 1D, left). In those
samples, FASN subcellular localization resembled Cav-1 localization
observed on consecutive sections (Fig. 1E), suggesting that cell trafficking mechanisms in the tumor cells may be permissive for direct
interaction between the two proteins.
FASN and Cav-1 interact in human and mouse PCa cells. To
determine whether direct evidence for such an interaction could be
obtained in vitro, we performed immunofluorescence (IF) imaging
experiments using Cav-1-positive DU145 human PCa cells. Both
Cav-1 (Fig. 2A) and FASN (Fig. 2B and Suppl. Fig. 1, left) were
shown to colocalize with the plasma membrane ganglioside GM1, a
lipid raft marker detected using the cholera toxin B subunit (CTxB).
CTxB binds GM1 with a high level of specificity.31 We further
analyzed FASN and Cav-1 subcellular localization in DU145 by dual
IF imaging experiments. Our results indicate that the two proteins
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apoptotic stimuli, and other aggressive characteristics of cancer
cells.1,7,8
Cav-1 is a 22 kDa protein, harboring an unusual transmembrane
domain with a hairpin turn, that deforms membranes into the
characteristic invaginated and vesicular shapes assumed by caveolae.9 Cav-1 also acts as a scaffold to organize multiple molecular
complexes that regulate a variety of cellular events.10,11 Genetic
evidence from the study of cav-1 (-/-) null mice, along with human
breast cancer mutations, indicate that Cav-1 plays a role as a negative
modulator of cell transformation and mammary tumorigenesis.12,13
In contrast, Cav-1 promotes tumor progression in PCa and is capable
of mediating signals through the PI3K/Akt pathway by sustaining
Akt activation.14
We have recently demonstrated that Cav-1 expression is required
for upregulation of fatty acid synthase (FASN) in tumor cells and in
adipose tissue in the TRAMP autochthonous PCa model, providing
the first molecular genetic evidence that Cav-1 functions upstream of
FASN.15 FASN is a large (~265 kDa) homodimeric enzyme16 that is
responsible for de novo fatty acid synthesis in mammals.17 Normal cells
express low endogenous levels of FASN because they obtain significant
amounts of fatty acids from the diet. In contrast, FASN expression
and activity in cancer cells can be extremely high because of increased
requirements for long chain fatty acids. Many cancer types characteristically exhibit increased levels of FASN. Recent evidence points to
a link between FASN overexpression and dysregulation of membrane
composition. Once synthesized, saturated fatty acids are incorporated
into membrane lipids and used as substrates for the post-translational
modification of proteins via thioester linkages.18 Importantly, FASN
inhibitors induce apoptosis of cancer cells19,20 and decrease the size
of PCa xenografts and of TRAMP prostate tumors that overexpress
the enzyme.21,22 An FDA approved drug, Orlistat, is able to induce
apoptosis in FASN-overexpressing prostate tumors by inhibiting the
thioesterase domain of FASN, the enzymatic pocket that liberates
newly synthesized palmitate, and exogenous introduction of palmitate reverts this effect.23 FASN may be a metabolic oncogene and a
variety of oncogenic changes (amplification of H-ras, erbB-2, EGFR,
etc.,)24 result in FASN-catalyzed lipogenesis.25,26 While a consensus
has been reached on a pro-tumorigenic role for both FASN and
Cav-1 in PCa, the potential functional relationship between these two
proteins is obscure. Importantly, the presence in FASN of a caveolinbinding motif (1506YRDGAWGAF1514) allows for the possibility that
the two proteins might interact directly.27 This motif is located within
a region of low complexity, between the methyltransferase (a.a.12431342) and alcohol dehydrogenase (a.a.1667-1816) domains in FASN,
suggesting its availability for interaction with caveolin. Notably,
mutation of the caveolin-binding motif in the EphB1 receptor and
the angiotensin II receptor, AT1, ablated plasma membrane targeting
of both proteins,28,29 consistent with a role for this motif in localizing
proteins to membrane microdomains.
In this study we demonstrate that FASN and Cav-1 are coordinately upregulated in human PCa and that the two proteins
physically and functionally interact in PCa cells. We also provide
evidence that a subpopulation of FASN associates with lipid raft
membranes in PCa cells following a variety of pharmacological and
genetic manipulation of upstream signals. Our results also suggest
that FASN/Cav-1 interaction is dependent on palmitoylation of
Cav-1 and results in the modulation of signal transduction.
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Caveolin-1 and fatty acid synthase in prostate cancer
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Figure 1. Coordinate expression of Cav-1 and FASN in human prostate tissue. (A) Relative levels of Cav-1 and FASN as assessed by ChromaVision analysis
of TMA containing benign, adenocarcinoma, and metastatic PCa. The red box highlights PCa and MET groups. Error bars denote 95% confidence interval.
(B) Correlation of FASN (left) and Cav-1 (right) levels with Gleason grade. (C) Selected images of tissue microarray cores showing immunohistochemical
staining for Cav-1 and FASN in PCa progression. (D) 63X magnification of membrane (left) and cytoplasm (right) FASN immunostaining in prostate tumors.
(E) 40X magnification of Cav-1 and FASN immunostaining, showing the similar subcellular expression patterns exhibited by the two proteins (insets, 63X).
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colocalize predominantly at the plasma membrane, although colocalization occurs at internal membranes as well (Fig. 2C and Suppl.
Fig. 1, right). These data are consistent with the apparent co-localization observed in the human tumors. To determine whether FASN
and Cav-1 interact, immunoprecipitation (IP) experiments were
performed in DU145 (human) and TRAMP C1 (mouse) cells. IP of
endogenous Cav-1 resulted in co-precipitation of endogenous FASN.
Co-IP of Cav-1 and FASN was also seen in the reciprocal experiment
(Fig. 2D).
A population of FASN transiently associates with lipid rafts
and interacts with Cav-1. High levels of FASN and Cav-1 have
been linked to the activation of the PI3K/Akt pathway in the PTEN
null LNCaP PCa cell line,25,32 and positive feedback regulation
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between Akt activation and FASN expression has been reported in
PTEN-negative ovarian cancer cell lines.33 In experiments directed
toward manipulating this pathway we used a series of agents that
alter signal transduction and assessed FASN location with respect
to lipid raft membranes. During this analysis we discovered that
LY294002, a selective PI3K inhibitor, induced a transient association of FASN with lipid raft-enriched membrane preparations. This
effect was observed using two independent biochemical methods
of lipid raft isolation (Fig. 3A, raft fractions & C, compare relative levels of FASN in the sucrose gradients +/- LY294002), as well
as fluorescence imaging (Suppl. Fig. 2). Efficacy of the inhibitor
was monitored by its ability to block phosphorylation of Akt on
Ser473, while levels of total Akt were not changed (Fig. 3B). At
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Figure 2. Cav-1 and FASN co-localization. (A) Lipid rafts in DU145 cells were stained with 0.5 μg/ml FITC-CTxB for 10 min before staining with anti-Cav-1
pAb (1:50) or (B) anti-FASN pAb (1:100) and Cy3-conjugated secondary antibody (1:250). Nuclei were counterstained with DAPI before imaging. Original
magnification 63X. (C) Immunofluorescence cell staining with anti-Cav-1 pAb (1:50) and anti-FASN mAb (1:200) followed by anti-rabbit FITC conjugated
(1:100) and anti-mouse Cy3-conjugated (1:250) secondary antibodies. Nuclei were counterstained with DAPI before imaging. Original magnification 63X.
(D) Cav-1 and FASN were immunoprecipitated (IP) from whole cell lysates using anti-Cav-1 pAb and anti-FASN pAb. IP eluates were blotted using the indicated Abs. An irrelevant Ab was used as an IP control.
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these concentrations of LY294002, no overt signs of toxicity or
apoptosis were observed (Fig. 3D), and levels of other proteins
such as β-actin, β-tubulin and Cav-1 were unaffected (Fig. 3A).
Consequently, FASN localization to lipid rafts is not related to
potential membrane changes associated with apoptosis. Consistent
with this interpretation, the same phenomenon was not elicited by
cisplatin, an inducer of apoptosis in DU145 cells34 (Fig. 4A). After
48 h treatment at concentrations of LY294002 that fully blocked
activation of Akt (30 μM), FASN expression was completely abolished (Fig. 4B). This result is consistent with the reported induction
of FASN levels by activation of the PI3K/Akt pathway,32 and with
reported positive regulation of FASN at the transcriptional level
through the activation of SREBP.26
In an attempt to determine whether Cav-1 could be implicated
in FASN redistribution to the raft membranes, we analyzed FASN
subcellular localization after treatment of Cav-1 negative LNCaP
cells with LY294002. Interestingly, the PI3K inhibitor, used at the
time and dose that elicited the strongest effect in DU145 cells, did
not induce FASN localization to the raft membranes in absence of
Cav-1 (Fig. 4C). Because Cav-1 is principally a lipid raft-resident
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protein, we next investigated whether FASN/Cav-1 interaction
occurs at a specific subcellular site. Co-IP experiments were therefore
performed in cytosolic (C), membrane (M) and Triton-insoluble,
lipid raft-enriched fractions isolated from DU145 cells. IP of endogenous FASN failed in co-precipitation of endogenous Cav-1 in C
and M fractions (data not shown), while the reciprocal Co-IP of
Cav-1 and FASN in the raft fractions revealed complex formation
(Fig. 4D). These experiments strongly suggest that Cav-1/FASN
complexes exist in lipid rafts.
Src mediates FASN association to lipid rafts. In the experiments
described above, we also observed that treatment of DU145 cells
with LY294002 resulted in a transient enrichment of activated Src
(P-Tyr416) in lipid raft-enriched fractions, paralleling the enrichment of FASN seen under the same conditions (Fig. 5A, part i).
These data suggest that inhibition of the PI3K/Akt pathway induces
activation of Src. A similar result was obtained using Wortmannin, a
structurally unrelated inhibitor of PI3K (Fig. 5A, part ii). Consistent
with these results, we also co-immunoprecipitated FASN and Src
from DU145 cell lysates (Fig. 5A, part iii). Imaging data showed
that Src focally co-localized with the lipid raft probe CTxB (Fig. 5B),
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fractions (Fig. 5C, part ii and D). These findings
indicate that alteration of signal transduction
mechanisms can induce a dynamic localization of FASN to cell membranes. Importantly,
activation of the EGFR and of Src, which lies
downstream from EGFR, induces this change in
FASN subcellular localization.
A Cav-1 palmitoylation mutant fails to
interact with FASN. Recent studies suggest
that post-translational modification of certain
proteins with lipid moieties, and palmitoylation
in particular, not only results in the targeting
of proteins to rafts/caveolae but may also
function to modulate protein-protein interactions occurring within caveolar membranes.36
FASN is the predominant intracellular source
of palmitate, and Cav-1 palmitoylation at a
single site (Cys-156) is required for Cav-1
phosphorylation by Src.37 The biological function of Cav-1 palmitoylation is not known. To
test the possibility that palmitoylation of Cav-1
might be required for FASN/Cav-1 interaction, Cav-1-WT or the triple palmitoylation
mutant Cav-1C133/143/156S,37 were expressed
in Cav-1-negative LNCaP cells and interaction
with FASN was assessed. We confirmed the
lack of Cav-1 in this background to rule out
the possibility that endogenous Cav-1 could
interfere with the binding to the antibody
used for IP (not shown). FASN co-immunoprecipitated with Cav-1-WT but did not
immunoprecipitate with the Cav-1 palmitoylation mutant, suggesting that FASN/Cav-1
interaction requires the integrity of one or
more of the cysteine residues at the C-terminus
of Cav-1 (Fig. 6A). This result suggests that
binding of Cav-1 to FASN may be implicated
in Cav-1 palmitoylation, which has been shown
to be a relevant determinant of Src-dependent
Figure 3. Inhibition of PI3K causes redistribution of FASN within PCa cells. (A) DU145 cells
oncogenic signaling.37
treated with LY294002 (10 μM) or vehicle (DMSO) for the indicated times were fractionated into
Palmitoylated Cav-1 seems to play a role
cytosolic (C), non-raft membrane (M) and raft (R) components, and fractions were blotted with the
indicated Abs. Arrow highlights transient association of FASN with lipid raft membranes. (B) Nonin signal transduction. In an attempt to invesraft membrane fractions, treated as in (A), were blotted with the indicated Abs. p-Akt recognizes tigate the significance of Cav-1 palmitoylation
phospho-Ser473. (C) Lysates of DU145 cells treated with LY294002 (10 μM) or vehicle (DMSO)
in signal transduction in PCa cells, we assessed
for 6 h were subjected to sucrose density gradient centrifugation. Fractions were blotted with the
the effect of expression of Cav-1C133/143/156S
indicated Abs. Fractions 3–5 contain the highest enrichment for lipid rafts, based on Giα2 level. (D)
with regard to the activation of Src and PI3K/
Flow cytometric analysis of DU145 cells treated with LY294002 (10 μM) for the indicated times.
The apoptotic fraction (% of cells in subG1) is indicated.
Akt pathways. After transfection of Cav-1-WT
and Cav-1C133/143/156S into LNCaP cells, we
indicating that Src is present in raft membranes. Collectively, these examined the levels of total Akt, activated Akt (P-Ser473), Src and
data suggest that Src may be a mediator of the effect of LY294002 on active Src (P-Tyr416). Interestingly, we observed an increase in levels
FASN redistribution. To test this hypothesis, we activated Src in two of activated Src and Akt in LNCaP overexpressing Cav-1-WT, in
ways: (1) with EGF, a known upstream regulator of Src and (2) by comparison to parental cells. Moreover, a significant reduction of
overexpression of a constitutively activated Src construct (Y529F).35 total and activated Src and Akt was detected in cell overexpressing
The responsiveness of EGFR to its ligand, and to ZD1839 (Iressa), Cav-1133/143/156S in comparison to Cav-1-WT (Fig. 6B). This result
a potent inhibitor of EGFR activation, was demonstrated by immu- supports the published findings that Cav-1 plays an oncogenic role
noblot analysis using a specific P-Tyr1068 antibody (Fig. 5C, part i). in PCa,30,38-40 and also suggests that palmitoylation of Cav-1 may
Both these manipulations resulted in enrichment of FASN in the raft mediate signal transduction in PCa cells.
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Figure 4. FASN and Cav-1 complex formation in raft membranes. (A) DU145 cells treated with cisplatin (10 μM) for the indicated times, fractionated as in
Figure 3A, and fractions blotted with the indicated Abs. (B) Whole cell lysates of DU145 treated with different doses of LY294002 or DMSO were blotted
with the indicated Abs. (C) LNCaP cells treated with LY294002 (10 μM) or vehicle (DMSO) for the indicated times were fractionated into cytosolic + non-raft
membrane (C + M) and raft (Raft) components, and fractions were blotted with the indicated Abs. (D) Cav-1 and FASN were immunoprecipitated from raft
fractions using anti- Cav-1 pAb and anti-FASN pAb. IP eluates were blotted using the indicated Abs. An irrelevant Ab was used as an IP control.
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Discussion
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In this study we provide the first evidence that FASN and Cav-1
are coordinately overexpressed in human PCa tissues and that the
two proteins interact. Quantitative evaluation of FASN and Cav1 protein levels in human tumors showed that overexpression of
both FASN and Cav-1 can be correlated with tumor grade and is
predictive of metastatic potential. In the tumor series we examined,
FASN and Cav-1 overexpression was independent of the hormone
responsive state. Although previous studies have examined either
Cav-1 or FASN expression in human PCa tissues and in an animal
model,14,22,30,41,42 ours is the first to address the question of the
potential for coordinate expression of FASN and Cav-1 in human
cancers.
In a subset of the tumors, we saw a similar pattern of subcellular co-localization of the two proteins, suggesting that they can
co-localize in vivo, and allowing for the possibility that they interact
in human prostate cancer. Interaction between Cav-1 and FASN
was shown directly using cultured human and mouse PCa cells.
Significantly, we found that the Cav-1/FASN complex is resistant
to the detergent octylglucoside (Fig. 4D), which completely solubilizes rafts,43 providing strong biochemical evidence that the two
endogenous proteins physically interact (i.e., this result is unlikely
to arise from independent and unrelated association of Cav-1 and
FASN with raft membranes). Interestingly, we discovered that a
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s­ ubpopulation of FASN is redistributed within the subcellular
compartment and is enriched at the raft membranes in response to
activation of the EGFR via Src, as well as by other means of pharmacological and genetic manipulations of oncogenic cell signaling.
This result indicates that FASN and Cav-1 may operate coordinately
to regulate cell signaling. Our findings further suggest that Cav-1
palmitoylation is required for FASN/Cav-1 interaction, and that
palmitoylation, which is indispensable for phosphorylation of Cav-1
on Tyr14 by Src,37 plays a role in signaling events downstream from
Cav-1. Collectively, our data suggest that FASN and Cav-1 may
collaborate to regulate signal transduction pathways that mediate
tumor growth and survival in PCa. They also imply that FASN and
Cav-1 could be potential mediators of compositional alterations of
raft membranes seen in tumor cells.2
Cav-1 is a mediator of vesicular transport, cholesterol homeostasis
and signal transduction, and has been implicated in cancer, although
oncogenic alterations in Cav-1 expression exert organ-specific effects
that are still poorly understood. Cav-1 is consistently downregulated
in some tumors, such as mammary, ovarian, lung carcinomas and
sarcomas, whereas it is upregulated in bladder, esophagus, papillary
carcinoma of the thyroid and PCa.3,9,38,44 Genetic evidence from
studies on cav-1 (-/-) null mice and human breast cancer mutations indicate that ablation or haploinsufficiency of Cav-1 promotes
mammary cell transformation and tumorigenesis.12,13,44 In contrast,
Cav-1 is a marker of aggressive disease in human PCa and genetic
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Figure 5. Src and FASN physically interact, and activation of Src coincides with FASN relocalization to lipid rafts. (A) Raft fractions from DU145 treated
with LY294002 (10 μM) (i) or Wortmannin (ii) for the indicated times were blotted with the indicated Abs. (iii) Src & FASN were immunoprecipitated from
DU145 whole cell lysates using anti-Src mAb and anti-FASN rAb. IP eluates were blotted using the indicated Abs. An irrelevant antibody was used as an
IP control. (B) IF using FITC-CTxB, and Src antibody (Cy3) in DU145. (C) (i) Equal amounts of whole lysates of DU145 cells treated with EGF (50 ng/ml,
30 min) or EGF and Iressa (ZD1839) (10 μM), were blotted with the indicated antibodies. (ii) Equal amounts of whole lysates or cytosolic + non-raft membrane (C + M) and raft fractions of DU145 cells treated with EGF (50 ng/ml, 30 min) were blotted with Abs to FASN and β-actin. (D) Cytosolic + non-raft
membrane (C + M) and raft fractions of DU145 transfected with no plasmid, p-USE empty vector, p-USE-Src-WT and p-USE-Src (Y529F) were blotted using
the indicated antibodies.
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ablation of Cav-1 inhibits progression of the extremely
aggressive TRAMP autochthonous prostate carcinoma
in the mouse.30
FASN overexpression in tumor cells can lead to
increased fatty acid turnover, oxidation and clearance,
and may represent an essential element for tumor
cell survival.45 FASN inhibitors specifically induce
apoptosis in cancer as opposed to normal cells.23
This observation suggests that FASN is a promising
therapeutic target. Recent lines of evidence point to a
link between FASN overexpression and dysregulation
of membrane composition. It has been proposed that
activation of FASN reflects an epigenetic dysregulation
of the lipogenic pathway in response to poor oxygenation and possible reduction in dietary fatty acids. In Figure 6. Mutation of Cav-1 palmitoylation sites ablates Cav-1/FASN interaction.
(A) Immunoblotting of myc immunoprecipitates from LNCaP expressing myc-tagged Cav-1this abnormal metabolic situation, FASN upregulation WT, or myc-tagged Cav-1C133/143/156S using anti-myc mAb & anti-FASN pAb. (B) Whole
could represent a strategy to maintain high proliferation cell lysates of parental LNCaP, and LNCaP expressing Cav-1-WT and Cav-1C133/143/156S
rates of surviving cells.46
were blotted with the indicated Abs. Samples were blotted with myc mAb to confirm expresThe discovery that FASN and Cav-1 interact is sion of the transfected Cav-1 construct.
consistent with the finding that both proteins are
overexpressed in PCa22,30,32,42 and with our recent demonstra- rafts have recently been implicated in cell signaling events relevant
tion that Cav-1 is upstream of the control of FASN expression.15 to PCa progression that are independent of Cav-1,7,52 indicating a
FASN is a cytoplasmic protein that produces long chain fatty acids dysregulation of signal transduction under conditions where cholesin the form of palmitate, myristate, oleate and stearate. FASN has terol content of cell membranes is altered in tumors. Consequently,
not been shown previously to associate with cell membranes or it is possible that recruitment of FASN to raft membranes may be of
membrane components. Long-chain saturated fatty acids are used in wider significance and not limited to situations where Cav-1 plays a
the synthesis of sphingolipids, which predominantly partition into functional role.
In summary, our findings provide the first evidence for functional
raft-like membranes,18 and are used as substrates for lipid modification of proteins, including Cav-1, which is palmitoylated on three cooperation between the membrane protein, Cav-1 and FASN, an
different residues (Cys-133,143,156) in a hydrophobic domain near abundant metabolic enzyme responsible for the production of longthe C-terminus. Notably, Cav-1 is phosphorylated by Src,37 and the chain fatty acids. Our analysis of human prostate tumors provides
subsequent product, Cav-1(Y14-P), localizes within caveolae near a physiologic context where collaboration between Cav-1 and
focal adhesions and, through an interaction with Grb7, augments FASN might occur, based on the observation that the two proteins
anchorage-independent growth and EGF-stimulated cell migra- co-localize in a subset of tumors. Further studies on the stability
tion.47 Palmitoylation of Cav-1 at a single site (Cys-156) is required and/or transient nature of the FASN/Cav-1 complex we describe
for coupling Cav-1 to Src.37 In addition, palmitoylation of Cav-1 may provide insight into the downstream effects of alterations in
on residues 143 and 156, but not residue 133, is required to restore membrane composition seen in tumor cells, including changes in
cell surface expression of GPI-linked proteins, that is lost in Cav-1 cholesterol and lipid content as well as tumor-specific lipid modificanull cells.48 Interestingly, palmitoylation of Cav-1, irrespective of the tion of membrane proteins.
specific site, is necessary for binding of cholesterol, formation of a
Materials and Methods
caveolin-chaperone transport complex, and rapid, direct transport
49
of cholesterol to caveolae. Whether Cav-1 palmitoylation plays a
Antibodies and reagents. The antibodies used in this study
role in cancer is not known; however beside its function as a signal include anti-FASN pAb (from Assay Designs), anti-Cav-1 (N-20)
transduction molecule, Cav-1 might play a role in PCa as a choles- pAb, anti-Giα2 subunit pAb, anti-β-tubulin mAb (clone D10) (all
terol transporter.50 Our data suggest that Src is an intermediate in from Santa Cruz Biotechnology, Santa Cruz, CA); anti β-actin mAb
Cav-1-dependent signaling and that membrane FASN is mechanisti- AC-15 (Sigma), anti-Akt1 mAb 2H10 (#2967), anti-phospho-Akt
cally coordinated with Src activation. Further studies are in progress (Ser473) pAb (#9271), anti-Src mAb and Tyr416 p-Src pAb (all from
to reveal the mechanistic basis of this process.
Cell Signaling Technology, Beverly, MA); protein G Sepharose was
We believe it is significant that lipid raft membranes were found obtained from Amersham Biosciences (Piscataway, NJ). Where indito be sites of interaction between Cav-1 and FASN. Raft membranes cated, the PI3K inhibitor LY294002, purchased from Calbiochem,
are heavily enriched in Cav-1 in Cav-1-expressing cells, and are the was added from a 1000-fold concentrated stock in DMSO. Control
presumptive sites of signaling events in which Cav-1 plays a major cultures received similar amounts of DMSO only. Final DMSO
role.44 Lipid rafts serve as membrane platforms for signal transduc- concentration did not exceed 0.1%. OptiMEM reduced serum
tion mechanisms that mediate cell growth, survival, and a variety of medium was from Invitrogen Corporation (Carlsbad, CA). All other
other processes relevant to cancer.1,2,51 Cholesterol accumulates in chemicals were obtained from Sigma Chemical Co., (St. Louis, MO).
solid tumors and cholesterol homeostasis breaks down in the prostate Cav-1 (WT and Cav-1C133/143/156S) and Src constructs (c-Src WT
with aging and with the transition to the malignant state.2 Lipid and c-Src Y529F) were previously described.35,37 FuGENE6 reagent
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Immunofluorescence microscopy. For imaging experiments,
DU145 cells were seeded in chamber slides and immunostained
using the following primary antibodies: anti-FASN pAb (from Assay
Designs), anti-FASN mAb (from Transduction Laboratories), antiCav-1 (N-20) pAb, (Santa Cruz Biotechnology, Santa Cruz, CA);
anti-Cav-1 mAb (Transduction Laboratories), anti-Src mAb (Cell
Signaling Technology, Beverly, MA), at dilutions of 1:100, 1:50,
1:100 and 1:100, respectively. Live cells were washed once with
ice-cold PBS and incubated on ice with FITC-conjugated cholera
toxin B subunit (CTxB) (Sigma) diluted in medium. Cells were then
fixed in 4% paraformaldehyde (PFA) and non-specific binding sites
blocked in PBS/0.1% BSA for 1 h at room temperature (RT), prior
to incubation with primary antibodies. The immune reaction for
each primary antibody was detected by Cy3-conjugated secondary
antibodies (1:250) for 30 min at RT. For dual immunostaining of
FASN and Cav-1, we used the above mentioned anti-FASN mAb
and the anti-Cav-1 (N-20) pAb. FASN was identified using an
anti-mouse cy3 conjugated secondary antibody, and Cav-1 by an
anti-rabbit FITC conjugated secondary antibody. For dual immunostaining of FASN and Cav-1 after FITC-CTxB, FASN was detected
with a anti-mouse Cy5-conjugated Ab, Cav-1 with an anti-rabbit
Cy3 conjugated Ab. Slides were mounted in Vectashield medium
containing DAPI (Vector Laboratories, Inc., Burlingame, CA) and
analyzed using an Axioplan 2 microscope (Carl Zeiss MicroImaging,
Inc., Thornwood, NY). The above instrument is composed of
an ApoTome system, which allows generation of optical sections
through fluorescence samples on the basis of fringe projection. In the
resulting processed image, out-of-focus elements of the images are no
longer visible, and the sharpness, contrast and resolution in the axial
direction have all been increased.
Immunohistochemistry. The human prostate tissue microarray
(TMA) consists of human normal and tumor prostate tissues from
Brigham and Women’s Hospital, Boston, MA and is composed of
90 samples in quadruplicate. The cohort contains cases of normal
prostate tissue (n = 10), benign prostate hyperplasia (n = 8), localized PCa cases distributed broadly across Gleason score categories
(n = 36), and metastases (n = 36). The latter group comprises
18 hormone responsive metastases, and 18 hormone refractory
metastases. Sections from the paraffin embedded TMA block were
mounted on charged glass slides and baked at 60°C for 1 h, de-waxed
and re-hydrated. For antigen retrieval, the sections were heated in
citrate buffer (pH 6.0) in a microwave for 15 min. Slides were incubated for 12 h at 4°C with anti-FASN rabbit pAb (1:500 dilution)
or with anti-Cav-1 rabbit pAb (1:400 dilution). The sections were
then incubated with HRP-conjugated anti-rabbit IgG for 30 min at
RT, and 3-3' diaminobenzidine (DAB) substrate chromogen solution
(Envision Plus kit, Dako Corp.,) was applied for 5 s. The reaction
was monitored at the microscope. Nuclei were counterstained with
Meyer’s Hematoxylin.
Evaluation and statistical analysis of human immunohistochemistry data. The Automated Imaging System (ACIS; ChromaVision
Medical System, Inc., San Juan Capistrano, CA) was used for
semi-quantitative analysis of FASN and Cav-1 expression in the
progression array.56 The image analysis system provides intensity
expression values (range from 0 to 255) for each TMA core. For each
protein, intensity values were transformed, mean centered and standard deviation set to 1, to standardize the variables to the same scale.
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was from Roche Applied Science (Indianapolis, IN). Nucleofector
and the Nucleofector kit were from Amaxa Biosystems.
Cell culture and transfections. TRAMP cell line C1, DU145 and
LNCaP cell lines were obtained from the American Type Culture
Collection. TRAMP C1 expresses androgen receptor, E-cadherin,
and is PTEN- and p53-null. TRAMP C1 was cultured in Dulbecco’s
modified Eagle’s medium (DMEM) (high glucose) without sodium
pyruvate, with L-Glutamine, 5% fetal bovine serum (FBS), 5 μg/
ml bovine insulin (Sigma), and 10 nM dehydroisoandrosterone
(Sigma). DU145 was cultured in Dulbecco’s modified Eagle’s
medium (high glucose) with sodium pyruvate, with L-glutamine,
10% FBS. LNCaP cells were cultured in RPMI 1640/10% FBS. All
media were supplemented with 100 μg streptomycin, and 100 units/
ml penicillin (Invitrogen, Carlsbad, CA). Cells were maintained
in a humidified atmosphere of 5% CO2 at 37°C. LNCaP cells in
150 mm dishes at ~80% confluence were transfected with plasmid
vectors using FuGENE6 or by nucleofection.
Preparation of membrane fractions. Lipid raft membrane fractions were isolated using two methods. In the first method, lipid
rafts were isolated from DU145 cells using sucrose gradient ultracentrifugation, as described.53 In the second method, a procedure
involving successive detergent extraction of cell membranes was used
essentially as described.7,8,43,54 In some experiments, the cytosolic
fraction was isolated prior to membrane fractionation. Briefly, cell
pellets were resuspended in 50 mM HEPES, pH 7.4, 10 mM NaCl,
1 mM MgCl2, 1 mM EDTA, 1 mM PMSF and 1 mM Na3VO4
and subjected to mechanical disruption with 12 strokes of a Dounce
homogenizer (1800 rpm). Homogenized samples were centrifuged
at 14000xg for 20 min at 4°C and the supernatant removed as the
cytosolic fraction. Membrane pellets were washed with buffer A, and
lysed as described above to extract Triton-soluble and raft membrane
fractions. The protein content of fractions was determined using the
MicroBCA assay (Pierce Chemical Co., Rockford, IL).
Immunoprecipitations. Equal amounts of protein from whole
cell lysates, or cytosol and non-raft membrane (C + M fraction)
or lipid raft fractions were incubated with appropriate dilutions of
different antibodies, for 2 h at 4°C, and protein A Sepharose resin
beads for 30 min at 4°C.55 Immunoprecipitates were washed four
times with lysis buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
0.5% NP-40, 2.5 mM NaPPi, 1 mM β-glycerophosphate, 1 mM
Na3VO4, 1 μg/ml leupeptin, 1 mM PMSF] and resuspended in 2X
SDS-loading buffer.
Preparation of whole cell lysates and immunoblot analysis. Cells
were washed twice in ice-cold PBS and lysed in a minimum volume
of 1X cell lysis buffer (Cell Signaling Technology) supplemented with
60 mM OCG and 1 mM PMSF. Protein content was determined
using the Micro BCA protein assay reagent as described above.
Cell extracts (10 μg/lane) and immunoprecipitates were resolved
by 4–12% gradient SDS-polyacrylamide gel electrophoresis and
electrotransferred to nitrocellulose membranes. Following transfer,
membranes were stained with Ponceau S to confirm equal protein
loading. Membranes were blocked with PBS/0.1% Tween-20/5%
BSA and incubated with antibodies overnight at 4°C. Following
incubation with species-specific horseradish peroxidase-conjugated
secondary antibodies, signals were detected using SuperSignal chemiluminescent reagent (Pierce Chemical Co., Rockford, IL) followed
by exposure of blots to X-ray film.
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Caveolin-1 and fatty acid synthase in prostate cancer
Quadruplicate data points of each case were averaged, after assessment
of intra-case expression homogeneity with respect to intra-diagnostic
group variability (data not shown).57 Expression level differentiations
among diagnostic groups and Gleason Scores were assessed by t-test
for unpaired data. To assess protein expression correlation, intensity
values were dichotomized with respect to the mean intensity and
Fisher exact test was applied on contingency tables. All p-values were
considered 2-tails and 0.05 was used as upper threshold for statistical
significance. Commercially available software SPSS 15.0 (SPSS Inc.,
Chicago, IL) was used for statistical analysis.
Cell cycle analysis by FACS. Cells were serum starved overnight
and stimulated with 10 μM LY294002 for the indicated times. After
harvesting, at the indicated time points, cells were fixed, stained with
propidium iodide, and visualized by flow cytometry.58
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This study is supported by the American Italian Cancer Foundation
(D.D.V. & M.L.), NIDDK R3747556, P50 DK65298, NCI R01
CA112303 and DAMD17-03-2-0033 (to M.R.F), and NCI R01
CA101046 (to K.R.S.). This project is also funded, in part, under a
grant with the Pennsylvania Department of Health, to M.P.L. The
Department specifically disclaims responsibility for any analyses,
interpretations or conclusions. The authors wish to thank Drs. P. de
Candia and J. Suh for helpful suggestions on co-IP experiments, and
Mr. Paul Guthrie and Mr. Raj Jhaveri for technical assistance.
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