Molecular Endocrinology. First published ahead of print December 18, 2012 as doi:10.1210/me.

2012-1260

ORIGINAL RESEARCH

Androgens Promote Prostate Cancer Cell Growth

through Induction of Autophagy

Yan Shi, Jenny J. Han, Jayantha B. Tennakoon, Fabiola F. Mehta,

Fatima A. Merchant, Alan R. Burns, Matthew K. Howe, Donald P. McDonnell,
and Daniel E. Frigo
Center for Nuclear Receptors and Cell Signaling (Y.S., J.J.H., J.B.T., F.F.M., D.E.F.), Departments of
Biology and Biochemistry (Y.S., J.J.H., J.B.T., F.F.M., D.E.F.) and Engineering Technology (F.A.M.), and
College of Optometry (A.R.B.), University of Houston, Houston, Texas 77204; and Department of
Pharmacology and Cancer Biology (M.K.H., D.P.M.), Duke University Medical Center, Durham, North
Carolina 27710

Androgens regulate both the physiological development of the prostate and the pathology of
prostatic diseases. However, the mechanisms by which androgens exert their regulatory activities
on these processes are poorly understood. In this study, we have determined that androgens
regulate overall cell metabolism and cell growth, in part, by increasing autophagy in prostate
cancer cells. Importantly, inhibition of autophagy using either pharmacological or molecular
inhibitors significantly abrogated androgen-induced prostate cancer cell growth. Mechanistically,
androgen-mediated autophagy appears to promote cell growth by augmenting intracellular lipid
accumulation, an effect previously demonstrated to be necessary for prostate cancer cell growth.
Further, autophagy and subsequent cell growth is potentiated, in part, by androgen-mediated
increases in reactive oxygen species. These findings demonstrate a role for increased fat metab-
olism and autophagy in prostatic neoplasias and highlight the potential of targeting underex-
plored metabolic pathways for the development of novel therapeutics. (Molecular Endocrinology
27: 0000 – 0000, 2013)

oth the basic physiology of the prostate and the de-
B velopment of diseases such as benign prostatic hyper-
plasia and prostate cancer are influenced by androgens
(1). Androgens are steroid hormones that function by
binding to and activating their target protein, the andro-
gen receptor (AR). Ligand-mediated activation of AR
controls the expression of a large number of genes, which
together enable the same AR-ligand complex to exert reg-
ulatory activities over a wide range of cellular processes
(2). Although AR and androgens are involved in the de-
velopment and functioning of the normal prostate and are
causally involved in prostate cancer progression, the
pathways responsible for these activities remain to be
determined.
Macroautophagy, herein referred to as autophagy, is a
cellular process, which occurs in all eukaryotic cells and
has been linked to numerous pathologies, including infec-
ISSN Print 0888-8809 ISSN Online 1944-9917
Printed in U.S.A.
Copyright © 2013 by The Endocrine Society
doi: 10.1210/me.2012-1260 Received August 7, 2012. Accepted November 2, 2012.
tions, neurodegeneration, cancer, and aging (3, 4). Au-
tophagy is a process that involves the de novo formation
of a limiting membrane, called an autophagosome,
around cytosolic components destined for degradation.
Degradation occurs when the autophagosome fuses
with a lysosome to create an autophagolysosome or
autolysosome. Subsequently, the contents of the au-
tolysosome are degraded by numerous acidic lysosomal
hydrolases. In addition to removing damaged organ-
elles and proteins, this process can also facilitate the
cellular remodeling that is necessary for cells to carry
out specialized functions (5, 6).
Abbreviations: ACC, Acetyl-CoA carboxylase; ACL, ATP citrate lyase; AMPK, AMP-acti-
vated protein kinase; AR, androgen receptor; ATG7, autophagy-related protein 7; AV,
autophagic vesicle; CM-H2DCFDA, chloromethyl-2⬘,7⬘-dichlorodihydrofluorescein diace-
tate; CoA, coenzyme A; CS-FBS, charcoal-stripped FBS; DAPI, 4⬘,6-diamidino-2-phenylin-
dole; DHT, dihydrotestosterone; Dox, doxycycline; E-64-d, (2S,3S)-trans-epoxysuccinyl-L-
leucylamido-3-methylbutane ethyl ester; eGFP, enhanced green fluorescent protein;
FACS, fluorescence-activated cell sorting; FAS, fatty acid synthase; FBS, fetal bovine se-
rum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent pro-
tein; LC3BI/II, microtubule-associated light chain 3 ␤ I/II; 3-MA, 3-methyladenine; qPCR,
quantitative RT-PCR; R1881, methyltrienolone; ROS, reactive oxygen species; shRNA,
short hairpin RNA; siRNA, small interfering RNA; TEM, transmission electron microscopy;
TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl; TG, triglyceride.

Mol Endocrinol, February 2013, 27(2):0000 – 0000 mend.endojournals.org 1

Copyright (C) 2012 by The Endocrine Society

2 Shi et al. Autophagy Increases Prostate Cell Growth
While autophagy is commonly thought of as a cata-
bolic process that is activated either during times of star-
vation or upon elevation of misfolded proteins, it is clear
that autophagy is also required for both normal and,
more recently, tumorigenic processes that require ana-
bolic activities (5–9). Initially, autophagy was thought to
function solely as a tumor suppressor largely because of
data indicating that the genetic deletion of Beclin1, a com-
ponent of the autophagic machinery, increases the inci-
dence of lung cancers, hepatocellular carcinomas, and
lymphomas (10, 11). Further, the commonly mutated tu-
mor suppressors p53 and PTEN have both been demon-
strated to activate autophagy (12, 13). Conversely, it has
recently been demonstrated using both pharmacological
and molecular inhibition that autophagy is required for
the growth and viability of multiple types of cancers, in-
cluding breast, pancreatic, colon, ovarian, cervical, brain,
lung, skin, and lymphomas (14 –27). For example, chlo-
roquine, an inhibitor of autophagic flux, decreased tumor
formation in both xenograft and transgenic mouse mod-
els of pancreatic cancer (26). Additionally, genetic dele-
tion of either atg5 or atg7, core components of the au-
tophagic machinery, suppressed the growth of Ras-driven
tumors (17). Thus, depending on the circumstances, au-
tophagy may function either as a tumor suppressor or a
tumor promoter. As a result, our understanding of
whether to target autophagy for therapeutic purposes,
and if so how, is limited.
It is currently unclear how exactly autophagy could
promote cancer. The prevailing thought is that autophagy
induces a dormant state similar to that of a stem cell (7, 8,
28). This state enables a level of independence that makes
the cell resistant to outside insults, such as hypoxia or
even chemotherapeutic drugs. Alternatively, autophagy
may take on a more active role in cell growth. Since its
initial discovery in the 1960s, the major focus of studies
on autophagy has been on its role in protein and organelle
turnover. However, several groups have recently demon-
strated a role for autophagy in differentiation and corre-
sponding anabolic processes, such as skeletal muscle
growth and lipogenesis (29 –35). Interestingly, altered
lipid metabolism has been demonstrated by multiple
groups to play an important role in prostate cancer (36 –
40). Fatty acid synthase (FAS), a rate-limiting enzyme in
de novo lipogenesis, is frequently overexpressed in pros-
tate cancers (41– 44). Correspondingly, pharmacological
or molecular inhibition of either FAS or other lipogenic
enzymes like acetyl-coenzyme A (CoA) carboxylase
(ACC) and ATP citrate lyase (ACL) suppresses both in
vitro and in vivo tumor growth (45, 46). Although andro-
gens promote prostate cancer cell growth in part by in-
creasing the expression of several of these lipogenic en-
Mol Endocrinol, February 2013, 27(2):0000 – 0000
zymes (36, 47, 48), it is not known whether androgens
may promote the formation of these lipid reservoirs by
additional mechanisms that may therefore also be critical
for AR-mediated tumorigenesis. The goal of this study
was to determine whether autophagy has a role in AR-
mediated prostate cancer cell growth and, if so, mecha-
nistically how this occurs. By identifying novel processes
that modulate prostate cancer cell growth, we hoped to
discover new targets that could be exploited with future
therapeutics.
Materials and Methods
Reagents
Fetal bovine serum (FBS), L-glutamine, RPMI 1640 medium,
DMEM, and chloromethyl-2⬘,7⬘-dichlorodihydrofluorescein di-
acetate (CM-H2DCFDA) were obtained from Life Technologies
(Carlsbad, CA). Charcoal-stripped FBS (CS-FBS) was purchased
from HyClone (Logan, UT). Polyclonal antibodies recognizing p27
Kip1, p15 INK4B, long-chain acyl-CoA synthetase, ACL, lipin1,
microtubule-associated light chain 3 ␤ I/II (LC3BI/II), FAS, and
monoclonal antibodies recognizing cyclin D1, cyclin D3, p21
Waf1/Cip1, CDK4, p18 INK4C, acetyl-CoA synthetase, and ACC
were from Cell Signaling (Danvers, MA). Antiglyceraldehyde-3-
phosphate dehydrogenase (GAPDH) antibody, N-acetyl cysteine,
4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL),
doxycycline (Dox), chloroquine, 3-methyladenine (3-MA), bafilo-
mycin A1, pepstatin A, and (2S,3S)-trans-epoxysuccinyl-L-leucyl-
amido-3-methylbutane ethyl ester (E-64-d) were from Sigma (St.
Louis, MO). Dihydrotestosterone (DHT) and methyltrienolone
(R1881) were purchased from Steraloids (Newport, RI) and
PerkinElmer (Waltham, MA), respectively.
Cell culture and plasmid transfection
Androgen-sensitive LNCaP, VCaP, CWR22, and 22Rv1 hu-
man prostate cancer cell lines were obtained from American
Type Culture Collection (Manassas, VA) and maintained as
recommended. Androgen-sensitive LAPC4 cells were a gift from
Charles L. Sawyers (Memorial Sloan-Kettering Cancer Center,
New York, NY) and maintained as previously described (49).
All experiments were carried out with cells of passage less than
25. All cells were authenticated by morphological inspection
and mycoplasma testing. Their response to androgens was au-
thenticated using growth and reporter gene assays. Unless oth-
erwise noted, for all experiments, cells were steroid starved for
72 h in steroid-reduced medium, that is, phenol red-free medium
containing 8% CS-FBS (15% CS-FBS for LAPC4 cells). For
transfections, cells were transfected with the mCherry-green flu-
orescent protein (GFP)-LC3 construct (50) using Lipofectamine
2000 (Life Technologies) according to the manufacturer’s
instructions.
Cell proliferation assay
Cell proliferation assays were carried out as previously de-
scribed (49) by measuring the cellular DNA content using a
FluoReporter Blue fluorometric double-stranded DNA Quanti-
Mol Endocrinol, February 2013, 27(2):0000 – 0000
tation kit (Life Technologies) following the manufacturer’s
protocol.
Cell viability assay
Cell viability assays were carried out as previously described
(51) using a CellTiter-Blue Cell Viability Assay (Promega, Mad-
ison, WI) following the manufacturer’s protocol.
Small interfering RNA (siRNA) transfection of
human prostate cells
Stealth siRNA (Life Technologies) transfections were carried
out as previously described (52). The sequences of all siRNAs
used in this study are listed in Supplemental Table 1, published
on The Endocrine Society’s Journals Online web site at
http://mend.endojournals.org.
Western blot analysis
Western blottings were conducted as previously described
(53). Densitometry was performed in Figure 2 using ImageJ
software (National Institutes of Health, Bethesda, MD). Here,
LC3BII levels (functional readout of levels of autophagy) were
normalized to GAPDH (loading control) and not LC3BI, be-
cause the low immunoreactivity of LC3BI can lead to erroneous
results. Hence, it is highly recommended that LC3BII levels be
normalized only to loading control to compare samples (54, 55).
RNA isolation, cDNA preparation, and quantitative
RT-PCR (qPCR)
RNA isolation, cDNA preparation, and qPCR were carried
out as previously described using 36B4 as a control (49). The
sequences of the primers were as follows: autophagy-related
protein 7 (ATG7) forward, 5⬘-GATCCGGGATTTCTT-
TCACG-3⬘ and reverse, 5⬘-CAGCAATGTAAGACCAGT-
CAAGT-3⬘; and FAS forward, 5⬘-CGCTCTGGTTCATCT-
GCTCTG-3⬘ and reverse, 5⬘-TCATCAAAGGTGCTCTCG-
TCTG-3⬘. The sequences of all other qPCR primers used in this
study have been previously described (49).
Transmission electron microscopy (TEM) and
immunogold TEM
For basic TEM, cells were plated in six-well plates at 500,000
cells/well and treated as described above. Cells were then
washed twice in serum-free media and fixed for 3–5 min at room
temperature with 4% glutaraldehyde in 0.1 M cacodylate buffer
(pH 7.0). Cells were gently scraped and centrifuged at 5000 ⫻ g
for 3 min in a swinging bucket rotor followed by additional
fixing for 1 h. Pellets were then embedded and sectioned by the
Duke University Department of Pathology’s Electron Micros-
copy Core. Grids were viewed using a Philips/FEI CM 12 Trans-
mission EM with AMT 2k x 2k digital camera.
For immunogold TEM, cells were plated in 24-well plates at
100,000 cells/well and treated as described above. Preembed-
ding immunogold labeling was performed on samples fixed in
4% paraformaldehyde/0.1% glutaraldehyde after free alde-
hydes were quenched in 0.1 M sodium borohydride in PBS for 15
min, blocked with 3% BSA, incubated with LC3 antibody (1:
100) overnight at 4 C, washed extensively, and incubated with
the gold-conjugated secondary antibody (1:100) overnight at 4
C. After further extensive washing, samples were fixed a second
mend.endojournals.org 3
time for 10 min in 2% gluteraldehyde, washed, and stained with
1% uranyl acetate for 15 min. Samples were then embedded and
sectioned by the MD Anderson Cancer Center High Resolution
Electron Microscopy Facility (Houston, TX). All grids were
viewed on a JEOL 100CX II TEM at 80 kV.
Creation of stable cell lines using retrovirus
LNCaP and VCaP enhanced GFP (eGFP)-LC3B stable cells
were created using a previously described GFP-LC3 retroviral
vector (56) and previously described approach (53).
Fluorescence microscopy
Cells were seeded on coverslips coated with poly-L-lysine,
cultured and treated with vehicle (ethanol), DHT (10 nM), or
R1881 (10 nM) for 72 h. To visualize punctae in mCherry-GFP-
LC3 or eGFP-LC3-expressing cells, cultures were fixed in 4%
paraformaldehyde, washed with PBS, and then stained with 50
nM LysoTracker or 200 nM 4⬘,6-diamidino-2-phenylindole
(DAPI) as a counter stain and mounted in ProLong antifade
(Life Technologies). Lipid droplets were stained using HCS Lip-
idTOX 1:1000 dilution from stock (Life Technologies). Of cau-
tionary note, BODIPY 493/503 stain was initially used to detect
intracellular lipids but yielded false positive staining (data not
shown). This commonly used fluorescent marker of neutral lip-
ids is highly susceptible to bleed through into the other fluores-
cent channels, unlike the LipidTOX stain that has a narrow
emission spectrum. Therefore, BODIPY 493/503 should be used
with caution when performing costains (especially in the green
and red spectra). Stable cells were visualized using an Olympus
FV-1000 confocal microscope (Olympus, New York, NY) un-
der ⫻63 objective or a DeltaVision core deconvolution micro-
scope (Olympus IX-71) with a ⫻60 oil immersion objective lens
and analyzed using ImageJ software (National Institutes of
Health). The Pearson correlation coefficient for lysosome and
autophagosome colocalization was determined using the Soft-
Worx software (Applied Precision, Issaquah, WA).
For rapid characterization of the tet-inducible ATG7 short
hairpin RNA (shRNA) stable cell line or reactive oxygen species
(ROS) imaging described below (ROS measurement), fluores-
cence microscopy was performed on plated and treated cells
using a Zeiss Axiovert 40 CFL fluorescence microscope with a
⫻10 objective (Zeiss, Oberkochen, Germany).
Lipogenesis assay
For lipogenesis assays, cells were plated in CS-FBS-contain-
ing media at 20,000 cells/well in poly-L-lysine-coated clear bot-
tom/black walled 96-well plates. Cells were then either trans-
fected with or without indicated siRNAs as described above and
then treated for 3 d with vehicle, R1881, or chloroquine. Intra-
cellular triglyceride (TG) levels were then determined using an
AdipoRed Nile Red-based fluorometric assay according to the
manufacturer’s instructions (Lonza, Allendale, NJ). The relative
fluorescence intensity was corrected for the background and
normalized to cell number using duplicate plates and the fluo-
rescent DNA-binding dye assay described above. For stable LN-
CaP cells with inducible ATG7-targeting shRNA, cells were
plated (10,000 cells/well) and then treated ⫾800 ng/ml Dox and
⫾R1881 in 96-well plates containing CS-FBS. Neutral lipid con-
tent was then quantitated as described above.
4 Shi et al. Autophagy Increases Prostate Cell Growth
Creation of inducible stable cell lines using
lentivirus
Commercially available shRNAs (sequences are listed in Sup-
plemental Table 1) in the pGIPZ backbone were obtained from
Thermo Scientific (Lafayette, CO). An shRNA that we deter-
mined in preliminary experiments (data not shown) to give the
best knockdown of ATG7 was used to produce lentivirus. For
inducible RNA interference experiments, shRNAs were sub-
cloned into the pINDUCER11 Dox-inducible lentiviral expres-
sion system by XhoI and MluI double digestion and ligation
(57). Lentiviruses were generated by cotransfecting the appro-
priate shRNA constructs along with the packaging plasmids
pMDL, pRSV-Rev, and pCMV-VSV-G into 293T cells. Viral
supernatants were harvested 48 h after transfection and com-
bined with 8 ␮g/ml polybrene to increase infection efficiency.
Virus was then used to infect target cells, and 96 h later, target
cells were GFP sorted using flow cytometry. Induction of
shRNA expression was done using 800 ng/ml Dox.
ROS measurement
Intracellular ROS levels were determined using CM-
H2DCFDA and subsequent fluorescence-activated cell sorting
(FACS) analysis (58) or fluorescence microscopy described
above. Briefly, for FACS analysis, treated cells were rinsed in
PBS buffer and exposed to 5 ␮M CM-H2DCFDA in the dark for
30 min at 37 C. Subsequently, the cells were trypsinized and
resuspended in PBS buffer. Intracellular fluorescence was then
quantified using a BD Aria II flow cytometer (Becton Dickinson,
Franklin Lakes, NJ).
To visualize intracellular ROS levels, cells were plated and
treated as described above for immunofluorescence microscopy.
Cells were then incubated with 2.5 ␮M CM2-H2DCFDA for 30
min at 37 C and imaged according to previously described meth-
ods (59, 60).
Statistical analysis
Two-sample comparisons were performed using Student’s t
tests. Multiple comparisons were performed using a one-way
ANOVA and post hoc Dunnett’s test with GraphPad Prism,
Version 4 (GraphPad Software, Inc., San Diego, CA). Unless
otherwise noted, statistically significant changes were deter-
mined at the P ⬍ 0.05 level.
Results
Functional autophagy is required for maximal
androgen-mediated prostate cancer cell growth
There has been a recent shift in our understanding of
the role of autophagy in cancer that suggests that au-
tophagy plays a major role in the progression of many
types of cancers (14 –27). Given the importance of andro-
gens and AR signaling in both the development of the
prostate and the progression of prostate cancer (61), we
sought to determine whether autophagy was important
for androgen-mediated cell growth in cellular models of
androgen-sensitive prostate cancer. Using LNCaP or
Mol Endocrinol, February 2013, 27(2):0000 – 0000
VCaP human prostate cancer cells, which express endog-
enous AR, we determined the effects of three different
inhibitors of autophagy (3-MA blocks autophagosome
formation; chloroquine and bafilomycin A1 block au-
tophagic flux) on androgen-mediated cell growth using
inhibitor concentrations that are both recommended and
widely used in the field of autophagy (14 –19, 26, 34, 56,
62– 66). In both cell models, all three inhibitors sup-
pressed androgen-mediated cell growth in a dose-depen-
dent manner (Fig. 1A). Although there were significant
effects of the higher concentrations of chloroquine and
bafilomycin A1 on basal cell growth, these compounds
had a more dramatic effect on androgen-mediated
growth, in most instances suppressing growth to vehicle-
alone-treated levels. Additional assays using these phar-
macological inhibitors suggest that some of the effects on
cell growth, particularly at the higher concentrations, are
due to changes in cell viability (Supplemental Fig. 1A).
Like most pharmacological modulators, potential off-
target effects of the autophagy inhibitors are a concern.
Therefore, to complement our pharmacological studies,
we also blocked autophagy using molecular techniques.
To determine the effects of molecular inhibition on an-
drogen-mediated prostate cell growth, we used siRNAs
targeting three separate regions of ATG7, a core compo-
nent of the autophagic machinery that is required for LC3
lipidation, a hallmark of functional autophagy (67). Dur-
ing autophagy, the cytoplasmically diffuse form of LC3B,
LC3BI, is conjugated to a phospholipid to create a mod-
ified form, LC3BII, which localizes to autophagosomes
and autolysosomes. LC3BII, despite having a phospho-
lipid conjugate, migrates faster on an SDS-PAGE gel and
hence can be distinguished from LC3BI using Western
blot analysis. Depletion of ATG7 levels (Supplemental
Fig. 1B) not only blocked the conversion of LC3BI to
LC3BII (Fig. 1B, right) but also inhibited androgen-me-
diated cell growth approximately 50% (Fig. 1B, left). Im-
portantly, molecular inhibition of autophagy had mini-
mal effect on cell viability (Supplemental Fig. 1C).
Collectively, these findings indicate that autophagy is
needed for maximal prostate cell growth and, more spe-
cifically, for androgen-mediated prostate cancer cell
growth.
Androgens increase autophagy
Because both molecular and pharmacological inhibi-
tors suppressed androgen-mediated prostate cancer cell
growth, we wanted to know whether androgens could
induce autophagy. To assess cellular autophagy, three
classical approaches are primarily used: 1) TEM (still of-
ten considered the gold standard), 2) LC3B immunofluo-
rescence microscopy to assess LC3B relocalization, and 3)
Mol Endocrinol, February 2013, 27(2):0000 – 0000 mend.endojournals.org 5

FIG. 1. Autophagy is required for androgen-mediated prostate cell growth. A, LNCaP or VCaP cells were treated for 7 d with vehicle or the
synthetic androgen R1881 in combination with vehicle or increasing concentrations of various inhibitors of autophagy (chloroquine: 1, 10, and 40
␮M; 3-MA: 1 and 10 mM; bafilomycin A1: 1, 10, and 100 nM). Cells were then lysed, and the relative number of cells was quantified using a
fluorescent DNA-binding dye. Each sample was performed in triplicate, and results from a representative experiment are shown. Results are
expressed as mean relative cell number ⫹ SE (n ⫽ 3). *, Significant changes from vehicle (no R1881)-treated cells; #, significant changes from
vehicle (no autophagy inhibitor)-treated cells. B, LNCaP cells were transfected with mock, a control siRNA or siRNAs targeting three separate
regions of the core autophagy molecule ATG7 and then treated for 7 d ⫾ R1881. Relative cell numbers were then quantitated as in A. Each
sample was performed in triplicate, and results from a representative experiment are shown. Results are expressed as mean relative cell number ⫹
SE (n ⫽ 3). *, Significant changes from vehicle (no R1881)-treated cells; #, significant changes from mock-transfected cells. D, Western blot analysis
of transfected LNCaP cell lysates to demonstrate ATG7 knockdown blocks autophagy as assessed by the decrease in the levels of LC3BII.

LC3B Western blot analysis to assess LC3BII levels.
Therefore, we treated LNCaP cells for 3 d with vehicle or
R1881 and assessed the formation of autophagic vesicles
(AVs) (autophagosomes ⫹ autolysosomes) using TEM
followed by blinded scoring (Fig. 2A). Using this rela-
tively low-throughput approach, we determined there to
be a roughly 4-fold increase in AV levels after androgen
treatment. To further corroborate and expand our find-
ings, we created, using a retroviral approach, both LN-
CaP and VCaP prostate cancer cells that stably expressed
an eGFP-LC3B construct. We then treated these cells for
3 d with vehicle, R1881, or the physiological androgen
DHT and quantified GFP-LC3 punctae formation using
immunofluorescence microscopy (Fig. 2B). In either LN-
CaP- or VCaP-GFP-LC3 cells, both the synthetic andro-
gen R1881 or the physiological androgen DHT signifi-
cantly increased GFP punctae formation, indicating
LC3B relocalization and hence autophagosome forma-
tion. Interestingly, in our hands, it took at least 2 d to
detect androgen-mediated autophagy, indicating an indi-
rect induction of autophagy by androgens (data not
shown). Next, we wanted to confirm that androgens ex-
erted a similar effect on autophagy in other androgen-
sensitive prostate cancer cell models. Using Western blot
analysis to assess the formation of the lipid-modified
LC3BII form, it was determined that androgens increased
autophagy in LNCaP, VCaP, LAPC4, CWR22, and
22Rv1 cells after R1881 treatment (Fig. 2C). To demon-
strate the role of AR, LNCaP cells were treated with in-
creasing concentrations of R1881 or DHT, and it was
observed that either androgen increased LC3BII levels in
a dose-dependent manner (Fig. 2D). This androgen-me-
6 Shi et al. Autophagy Increases Prostate Cell Growth Mol Endocrinol, February 2013, 27(2):0000 – 0000

FIG. 2. Androgens increase autophagy in an AR-dependent manner in prostate cancer cells. A, LNCaP cells were treated for 3 d with vehicle
(ethanol) or 10 nM R1881. Cells were then fixed and examined by TEM. Left, AVs, autophagosomes and autolysosomes. Right, Quantification of
the number of AVs in vehicle- and R1881-treated cells. Data are presented as the mean number of AVs/cell (n ⫽ 19 cells each). *, Significant
changes from vehicle-treated cells. B, LNCaP or VCaP cells stably expressing an eGFP-LC3B fusion were treated for 3 d with vehicle, R1881 (10 nM),
or the endogenous androgen DHT (10 nM). Redistribution of eGFP-LC3B (green) was covisualized with a DAPI nuclear stain (blue) using
immunofluorescence confocal microscopy. Left, Representative pictures are shown. Scale bar, 20 ␮m. Right, Average number of punctae/cell (n ⫽
30 cells) was analyzed and plotted. *, Significant changes from vehicle-treated cells. C, Indicated prostate cancer cells were treated for 3 d with
vehicle or 10 nM R1881 and then subjected to Western blot analysis to determine LC3BI/II and GAPDH (loading control) levels. Below the images
are the average LC3BII densitometry values normalized to GAPDH ⫾ SE (n ⱖ 3). D, LNCaP cells were treated for 3 d with vehicle (⫺), 40 ␮M
chloroquine (CQ) (blocks autophagic flux leading to build up of LC3BII), or increasing doses (0.1, 1, and 10 nM) of R1881 or DHT. Cells were then
subjected to Western blot and densitometry analysis as in C. E, LNCaP cells were treated for 3 d ⫾ 10 ␮M Casodex and ⫾10 nM R1881. Cells were
then subjected to Western blot and densitometry analysis as in C.
Mol Endocrinol, February 2013, 27(2):0000 – 0000
diated effect could be blocked by cotreatment with the
antiandrogen Casodex, indicating that LC3B conversion
is AR dependent (Fig. 2E).
Androgens increase autophagic flux in prostate
cancer cells
Although LC3 lipidation and relocalization is a marker
for autophagosome formation, it does not report on the
rate of autophagic flux. This is because LC3BII is de-
graded over time by lysosomal enzymes and hence the
accumulation of LC3BII can result from either increased
autophagic entry or decreased autophagic flux (i.e. failure
of autophagosomes to fuse with lysosomes) (68). There-
fore, we took three different approaches to assess the
impact of androgens on autophagic flux in prostate can-
cer cells. First, if functional autophagy is occurring, au-
tophagosomes need to fuse with existing lysosomes to
degrade their contents. To test this, we used our LNCaP
and VCaP stably expressing eGFP-LC3B cell lines to dem-
onstrate that androgens increased LC3B and lysosome
colocalization (Fig. 3A and Supplemental Fig. 2A). A sec-
ond, newer assay uses a tandem mCherry-GFP fusion pro-
tein linked to LC3 (69). This GFP signal is sensitive to the
acidic and/or proteolytic environment within the lyso-
some, unlike mCherry, which is more stable. Thus, punc-
tate colocalization (yellow) of both GFP and mCherry
indicates the formation of an early autophagosome that
has not yet fused with a lysosome, whereas mCherry (red)
punctate staining alone suggests LC3 has been delivered
to the lysosomes, marking late autophagy. After transient
transfection of the mCherry-GFP-LC3 construct and an-
drogen treatment in either LNCaP or VCaP cells, we im-
aged cells and quantitated the different LC3 punctate
populations (Fig. 3B and Supplemental Fig. 2B). In both
cell lines, R1881 or DHT treatment lead to a significant
increase in GFP⫺mCherry⫹ (red) punctate formation, in-
dicating that androgens promote autophagic flux. Fi-
nally, to complement our immunofluorescence studies,
we assessed autophagic flux using a third approach that is
based on Western blot analysis of LC3BII levels before
and after lysosomal inhibition (Fig. 3C and Supplemental
Fig. 2C). Here, cells were pretreated with a cocktail of
E-64-d and pepstatin A (Fig. 3C), specific inhibitors of
lysosomal proteases, or chloroquine (Supplemental Fig.
2C) before androgen treatment. After inhibitor treat-
ment, LC3BII levels accumulated due to lysosomal turn-
over being blocked. Importantly, androgen treatment fur-
ther increased LC3BII levels despite impaired lysosomal
function, implying that AR-signaling promotes entry into
autophagy rather than blocking the autophagic flux.
mend.endojournals.org 7
Autophagy enhances androgen-mediated
intracellular lipid accumulation
Although our data here, as well as studies performed
by other groups, indicate that autophagy can contribute
to cancer cell growth, it is still not fully understood mech-
anistically how autophagy could augment prostate cancer
cell growth. We initially tested whether blocking au-
tophagy could influence the levels of various cell cycle
regulatory proteins (Supplemental Fig. 3). In our hands,
suppression of autophagy did not consistently alter the
levels of cyclin D1, cyclin D3, p21 Waf1/Cip1, p27 Kip1,
CDK4, p15 INK4B, or p18 INK4C. Although this is cer-
tainly not a comprehensive list of all potential growth
regulatory proteins, it suggested that perhaps autophagy
was not directly influencing the cell cycle machinery.
Recently, several groups have demonstrated that pros-
tate cancers have elevated levels of intracellular lipids and
that blocking the accumulation of these lipids inhibits
tumorigenesis (36 – 40). Interestingly, although au-
tophagy is classically thought of as a catabolic process, it
has also become apparent that autophagy plays a role in
certain anabolic processes, such as lipogenesis (31–35).
Therefore, we tested the hypothesis that androgen-medi-
ated autophagy contributes to prostate cancer cell growth
through altering lipid metabolism. Androgen treatment
of either LNCaP or VCaP cells leads to a significant AR-
dependent increase in intracellular levels of lipids as de-
termined using either TEM or a Nile Red-based neutral
lipid assay (Fig. 4A and Supplemental Fig. 4). Further,
using immunofluorescence confocal microscopy with a
HCS LipidTOX stain highly specific for neutral lipids, we
determined that, although there was not a high level of
lipid and LC3 punctae colocalization, the androgen-me-
diated rise in intracellular lipid levels correlated with the
appearance of autophagy (Fig. 4B and Supplemental Fig.
5). This is consistent with what has been observed by
other laboratories studying the link between autophagy
and fat metabolism (35, 65, 70, 71). To get a better idea
of the subcellular targets of autophagy, we performed
LC3 immunogold TEM with androgen-treated cells (Fig.
4C). After androgen treatment, LC3 immunogold stain-
ing could be detected in discrete regions adjacent to lipid
droplets (left two pictures), consistent with findings from
other laboratories (65), or localized to AVs (far right pic-
tures). In vehicle-treated cells, LC3 staining was difficult
to find and did not localize to lipid droplets since vehicle-
treated prostate cancer cells do not accumulate lipid drop-
lets (data not shown). Unexpectedly, we did not observe a
robust localization of LC3 density in the proximity of fat
droplets, a consequence we believe of the ultrathin sec-
tions required for TEM or the fixing procedure. There-
fore, to complement these qualitative studies, we used the
8 Shi et al. Autophagy Increases Prostate Cell Growth Mol Endocrinol, February 2013, 27(2):0000 – 0000

FIG. 3. Androgens increase autophagic flux in prostate cancer cells. A, LNCaP (representative pictures shown on left) or VCaP (representative
pictures shown in Supplemental Fig. 2A) cells stably expressing an eGFP-LC3B fusion were treated for 3 d with vehicle or 10 nM R1881. Cells were
then fixed and stained with LysoTracker to identify acidic organelles. eGFP-LC3B and LysoTracker colocalization was determined using
immunofluorescence confocal microscopy. Scale bars, 10 ␮m. Quantification of colocalization (right) was done using ImageJ software. B, LNCaPs
(representative pictures shown in Supplemental Fig. 2B) or VCaPs (representative pictures shown on left) were transfected with an mCherry-GFP-
LC3B construct and treated for 3 d with vehicle, DHT (10 nM), or R1881 (10 nM) to assess autophagic flux. Cells were then fixed and visualized
using a confocal immunofluorescence microscope. Scale bars, 10 ␮m. Right, Quantification of average total, GFP⫹mCherry⫹ (yellow, early
autophagy/autophagosome) and GFP⫺mCherry⫹ (red, late autophagy/autolysosomes) punctae/cell ⫾ SE for both LNCaP and VCaP cells (n ⬎ 10
cells each condition). *, Significant changes (P ⬍ 0.05) from vehicle-treated cells. **, Significant changes (P ⬍ 0.01) from vehicle-treated cells. C,
LNCaP cells were treated for 3 d ⫾ 10 ␮g/ml each of E-64-d and pepstatin A (specific lysosomal protease inhibitors) and ⫾ 10 nM R1881. Cells
were then subjected to Western blot analysis as in Fig. 2.

Nile Red-based assay to quantitate total neutral lipid levels
after androgen treatment with or without molecular (ATG7
siRNA) or pharmacological (chloroquine) inhibition of au-
tophagy. Both siRNAs targeting ATG7 (Fig. 4D and Sup-
plemental Fig. 6A) or increasing concentrations of chloro-
quine (Fig. 4E and Supplemental Fig. 6B) significantly
decreased neutral lipid levels after androgen treatment.
To avoid the potential side effects that can occur with
the use of chemical siRNAs, we created an LNCaP stable
cell line using the pINDUCER system (57). This platform
Mol Endocrinol, February 2013, 27(2):0000 – 0000 mend.endojournals.org 9

Dox, promotes the pol II-driven ex-

pression of an shRNA transcript (in
our case ATG7-targeting) expressed as
a microRNA mimic (Fig. 5A). These
microRNA mimics have the advantage
of minimizing off-target RNA interfer-
ence effects compared with their classi-
cal shRNA predecessors (72). After the
creation and validation of this cell
model (Fig. 5, B and C), we confirmed
that the inducible knockdown of
ATG7 decreased both androgen-medi-
ated lipogenesis (Fig. 5D) and cell
growth (Fig. 5E). Collectively, these
data indicate that autophagy facili-
tated the formation of intracellular fat
depots. Importantly, this was not the
result of shutting down basic cellular
processes like transcription, because
knockdown of ATG7 had no effect on
the expression of classic AR-regulated
genes (Supplemental Fig. 7). Further, in-
hibition of autophagy using either induc-
ible ATG7 shRNA or E-64-d and pepsta-
tin A cotreatment did not prevent the
expression of FAS, a bona fide AR-target
and the rate-limiting step of de novo li-
pogenesis (Supplemental Fig. 8, A and B)
(42, 43, 48, 73, 74). Additionally,
knockdown of ATG7 did not affect the
levels of a sampling of other proteins
classically involved in lipid metabolism,
including ACC, ACL, acetyl-CoA syn-
FIG. 4. Autophagy promotes androgen-mediated lipid accumulation. A, LNCaP cells were thetase, acyl-CoA synthetase, and lipin1
treated for 3 d with vehicle (ethanol) or 10 nM R1881. Cells were then fixed and examined by (Supplemental Fig. 8C), suggesting that
TEM as in Fig. 2. B, LNCaP cells stably expressing an eGFP-LC3B fusion were treated for 3 d
with vehicle, DHT (10 nM), or R1881 (10 nM). Cells were then fixed and costained with autophagy may not regulate the levels of
LipidTOX (neutral lipid depots) and DAPI (nucleus). eGFP-LC3B, LipidHTX, and DAPI lipogenic enzymes.
colocalization was visualized using immunofluorescence confocal microscopy. Scale bars, 20
␮m; 5 ␮m for higher resolution images on far right. C, LNCaP or VCaP cells were treated as in
A and subjected to immunogold TEM to determine LC3B subcellular localization (yellow
Autophagy promotes androgen-
arrows). D, LNCaP cells were transfected with siRNAs as in Fig. 1B and treated for 3 d with
vehicle or 10 nM R1881. Intracellular TG levels were then determined using a fluorescent Nile mediated lipid accumulation
Red-based stain (AdipoRed) and normalized to cell numbers that were determined using independent of FAS expression
duplicate plates that were instead subjected to the fluorescent DNA-binding dye described in Because inhibition of autophagy did
Fig. 1. Each sample was performed in triplicate, and results from a representative experiment
are shown. Results are expressed as TG levels normalized to cell numbers ⫹ SE (n ⫽ 4). *, not alter the levels of FAS or other li-
Significant changes from mock-transfected cells. E, LNCaP cells were treated with vehicle or pogenic enzymes (Supplemental Fig.
increasing concentrations of chloroquine (CQ) ⫾ 10 nM R1881 for 3 d. Intracellular TG levels 8), we asked the reciprocal question of
were then determined as described in D. Each sample was performed in triplicate and results
from a representative experiment are shown. Results are expressed as TG levels normalized to whether de novo lipogenesis could reg-
cell numbers ⫹ SE (n ⫽ 4). *, Significant changes from vehicle (no CQ)-treated cells. ulate autophagy. In other words, we
wanted to know whether the accumu-
is advantageous, because it uses a single vector to consti- lation of intracellular fat was the cue that stimulated au-
tutively express a third-generation reverse Tet transacti- tophagy. To test this, we knocked down FAS levels using
vator that, upon addition of tetracycline or its derivative three separate siRNAs and then treated cells with or with-
10 Shi et al. Autophagy Increases Prostate Cell Growth Mol Endocrinol, February 2013, 27(2):0000 – 0000

shown, CM2-H2DCF-DA and subse-

quent FACS analysis demonstrated
that treatment of either VCaP or LN-
CaP cells with androgens increased in-
tracellular ROS levels (Supplemental
Fig. 9). By adapting the CM2-
H2DCF-DA assay, we used basic fluo-
rescence microscopy to more rapidly
and easily assess relative ROS levels.
Using this approach, we demonstrated
that VCaP cells treated for 3 d with
R1881 have elevated ROS levels, an ef-
fect that could be blocked by the anti-
oxidant TEMPOL (Fig. 7A). Further-
more, treatment of prostate cancer
cells with either of the antioxidants
TEMPOL (Fig. 7B) or N-acetyl cys-
teine (Supplemental Fig. 10) was suffi-
cient to block androgen-mediated au-
tophagy. This corresponded with the
FIG. 5. Inducible expression of ATG7-targeting shRNA inhibits androgen-mediated lipid
accumulation and cell growth. A, Diagram of the pINDUCER11 construct used in this study. B
ability of antioxidants to block andro-
and C, To characterize the LNCaP ATG7 shRNA stable cells, cells were treated for 2 d ⫾ 800 gen-mediated lipid accumulation (Fig.
ng/ml Dox and subjected to fluorescence microscopy (B) or Western blot analysis (C). D, TG 7C). Importantly, blocking ROS pro-
levels were quantitated as described in Fig. 4 but using LNCaP ATG7 shRNA-inducible stable
duction, although having minimal ef-
cells cotreated for 3 d ⫾ Dox and ⫾ R1881. Results are expressed as TG levels normalized to
cell numbers ⫹ SE (n ⫽ 2). *, Significant changes from double vehicle-treated cells. E, Stable fect on basal cell growth, inhibited an-
cells were again cotreated but for 7 d followed by quantification of cell numbers using the drogen-mediated cell growth (Fig. 7D),
assay described in Fig. 1. Results are expressed as mean relative cell number ⫹ SE (n ⫽ 2). **, consistent with what has been demon-
Significant change (P ⬍ 0.01) from R1881 alone-treated cells.
strated before in LNCaP cells (48, 58,
76, 77). Taken together, these data
out R1881. Consistent with what others have shown (41– suggest that androgens increase cell growth, in part, by
43, 47, 74), knockdown of FAS severely impaired AR’s activating a complex metabolic shift that is signaled by
ability to increase intracellular fat levels (Fig. 6A) and cell the elevation of intracellular ROS levels.
growth (Fig. 6B). Despite the similarities to the knock-
down of ATG7, knockdown of FAS did not impact au-
tophagy as assessed by the Western blot analysis of Discussion
LC3BII levels (Fig. 6C). These findings suggest that au-
tophagy can promote intracellular lipid accumulation by The work presented here indicates that androgens, via
a mechanism that is at least partially independent of FAS AR, utilize autophagy to promote prostate cancer cell
activity. growth. Initially, these data were counterintuitive to us
because autophagy is usually considered to be a catabolic
Androgens promote autophagy, in part, through process that is activated during times of stress (78). This
the production of ROS contrasts with the established roles of androgens as ana-
Although we established the importance of autophagy bolic steroids. Indeed, previous studies have reported that
for androgen-mediated prostate cancer cell growth, we androgens can block autophagy in either LNCaP cells or
still did not understand how androgens increased au- PC-3 cells transiently transfected with AR (62, 79, 80).
tophagy. Previous work by Scherz-Shouval et al. (75) However, these studies looked at more rapid androgenic
demonstrated that elevated ROS levels led to increased effects under different conditions, during hypoxia or
autophagy. Given that androgens can increase ROS in complete serum starvation, conditions where androgens
prostate cancer cells to a level that potentiates cell growth do not optimally promote cell growth. Conversely, our
(48, 58, 76, 77), we decided to explore whether andro- data clearly indicate in multiple cellular models that when
gens could stimulate autophagy by increasing the intra- androgens promote cell growth, autophagy is activated.
cellular levels of ROS. Consistent with what others have Further, autophagy has been recently shown by multiple
Mol Endocrinol, February 2013, 27(2):0000 – 0000
FIG. 6. Autophagy-mediated lipid accumulation is downstream of
FAS. LNCaP cells were transfected with mock, a negative scramble
control (siControl) or three siRNAs targeting separate regions of FAS
mRNA. Cells were then treated for 3 d with vehicle or 10 nM R1881. A,
Cells were assayed for intracellular TG levels as described in Fig. 4D.
Each sample was performed in triplicate, and results from a
representative experiment are shown. Results are expressed as TG
levels normalized to cell numbers ⫹ SE (n ⫽ 2). *, Significant changes
from mock-transfected cells. B, Cells were transfected with mock, a
control siRNA, or siRNAs targeting three separate regions of FAS and
then treated for 7 d ⫾ R1881. Relative cell numbers were then
quantitated as described in Fig. 1. Each sample was performed in
triplicate, and results from a representative experiment are shown.
Results are expressed as mean relative cell number ⫹ SE (n ⫽ 3). *,
Significant changes from mock-transfected cells. C, Lysates from
LNCaP cells transfected and treated the same as in A were subjected to
Western blot analysis. A representative blot is shown (n ⫽ 4).
groups to be required for the progression of various types
of cancers (14 –27), supporting a progrowth role. It
should be also noted that although Bennett et al. (79)
mend.endojournals.org 11
reported that androgens could block serum starvation-
induced autophagy at up to 24 h, longer treatments (i.e.
72 h) that increased cell survival also increased au-
tophagy. These findings indicate that when the extracel-
lular environment can provide a necessary level of sup-
port, androgen-treated cancer cells can utilize autophagy
for their benefit. Ultimately, we think that cancer cells,
not surprisingly, have the ability to adapt to their envi-
ronment. In the context of prostatic diseases, androgens
activate signaling pathways that facilitate the cell’s poten-
tial to use diverse nutrients, building blocks, and energy
sources for growth and survival. Androgen-mediated au-
tophagy may be one way in which a prostate cell can
redirect its efforts away from nonessential processes to-
ward processes it deems more necessary like growth
and/or survival.
The delayed induction demonstrated here suggests an
indirect mechanism by which AR signaling enhances au-
tophagy. Recent work by Scherz-Shouval et al. (75) show-
ing that elevated ROS levels activate autophagy, com-
bined with the previous work of several groups
demonstrating that androgen-mediated ROS generation
promotes prostate cancer cell growth (48, 58, 76, 77),
provided the rationale for us to test whether androgenic
regulation of autophagy and subsequent cell growth re-
quired ROS signaling (Fig. 7). The regulation of ROS
levels in cells is a double-edged sword. Although too
much ROS can trigger apoptosis, moderate levels pro-
mote cell signaling activities that are needed for both pro-
liferation and survival (81). In our experiments, elevated
ROS levels contributed to the androgen-induced au-
tophagy, intracellular lipid accumulation, and cell growth
(Fig. 7), indicating a role for ROS as a signaling molecule.
While ROS were required for the induction of au-
tophagy, we cannot exclude the possibility that other ini-
tiating mechanisms exist. There are many modulators of
autophagy, some of which are regulated by androgens.
For example, we have previously shown that androgens
increase AMP-activated protein kinase (AMPK) signaling
through the expression of calcium/calmodulin-dependent
kinase kinase ␤ (53). Because AMPK is a known positive
regulator of autophagy (82), our initial hypothesis was
that androgens increased autophagy through the up-reg-
ulation of the calcium/calmodulin-dependent kinase ki-
nase ␤-AMPK cascade. However, preliminary studies us-
ing siRNAs targeting AMPK have yielded inconclusive
results (data not shown). Veeramani et al. (77) have re-
ported that androgens can increase ROS levels and sub-
sequent prostate cancer cell proliferation through an AR
nongenomic mechanism. While androgens may function
through membrane signaling, given the delayed onset of
autophagy after treatment and the stereotypical AR phar-
12 Shi et al. Autophagy Increases Prostate Cell Growth Mol Endocrinol, February 2013, 27(2):0000 – 0000

the fact that LC3B is typically found in

discrete locations on the lipid droplet
membranes (Fig. 4C) (35, 65). How-
ever, until more dynamic labeling stud-
ies can be performed, this process re-
mains an enigma.
One of the other major questions in
the field is how does AR-mediated fat
accumulation promote cell growth. To
date, several explanations have been
proposed. However, a dearth of exper-
imental evidence has kept this issue
largely unsolved. One theory is that the
elevated levels of fat can help control
cellular redox levels (42). Others have
postulated that the fat droplets may
serve as reservoirs that can be tapped
into whenever the cell needs lipid
building blocks to make new mem-
FIG. 7. Androgens promote autophagy-mediated cell growth, in part, through elevating branes during cell division. Although
intracellular ROS levels. A, VCaP cells were cotreated for 3 d ⫾ 1 mM TEMPOL and ⫾ 10 nM this is an attractive hypothesis, in pros-
R1881. Cells were then assayed for ROS production using CM-H2DCF-DA-based fluorescence
microscopy analysis. B, VCaP cells were treated as in A and subjected to Western blot analysis tate cancer, typically only a fraction of
for LC3B and GAPDH (loading control). C, VCaP cells were treated as in A and B. Cells were the cells are actively proliferating at
then assayed for intracellular TG levels as described in Fig. 4D. Each sample was performed in any one time, indicating that this might
triplicate, and results from a representative experiment are shown. Results are expressed as
TG levels normalized to cell numbers ⫹ SE (n ⫽ 2). *, Significant changes from double vehicle-
be only a part of the answer (41). An
treated cells. D, VCaP cells were again cotreated but for 7 d and then assayed for relative cell alternative hypothesis is that the intra-
numbers as described in Fig. 1. Each sample was performed in triplicate, and results from a cellular lipid accumulation is needed
representative experiment are shown. Results are expressed as mean relative cell number ⫹ SE
for various aspects of signal transduc-
(n ⫽ 3). *, Significant changes from double vehicle-treated cells.
tion. Many secondary messengers,
such as prostaglandins and phosphati-
macology we see (Fig. 2, D and E), it may be premature to dylinositols, are derived from neutral lipid scaffolds. Fur-
say genomic AR signaling does not play any role in the ther, many of the posttranslational modifications (e.g.
regulation of autophagy. Certainly, as our understanding palmitoylation and myristoylation) that are required for
of the regulation of autophagy advances, it may be worth- signal transduction cascades are lipid based. For example,
while to reexamine earlier microarray signatures to deter- the phosphoinositol 3-kinase-Akt signaling pathway,
mine any additional points of AR regulation. which plays a major role in prostate cancers, requires
We demonstrate here that androgen-mediated au- these lipid modifications to properly organize itself at the
tophagy promotes the accumulation of intracellular fats. plasma membrane and thus ensure efficient signaling
Although androgens are known to accumulate intracellu- (83). Additionally, androgens have been demonstrated to
lar fats through the expression of several enzymes in- potentiate prostate cancer cell proliferation by increasing
volved in de novo lipogenesis (36, 45– 48), it appears that the expression of squalene synthase, a lipogenic enzyme
autophagy may function independently of these tran- that contributes to membrane lipid raft formation (84).
scriptional events. Recent work in the field has demon- Another intriguing option is that the fat depots provide
strated a bona fide role for autophagy in fat depot forma- precursors for the production of androgens. Several stud-
tion. Adipose-specific atg5 or atg7 knockout mice are ies have indicated that one of the causes of castrate-resis-
lean and resistant to type 2 diabetes (31–34). In these tant prostate cancer is the up-regulation of enzymes in-
mice, it appears there is a severe defect in normal fat cell volved in local steroid hormone production (85– 87).
differentiation. Further, the MAP1-LC3 conjugation sys- Thus, even though circulating levels of androgens are neg-
tem is required for lipid droplet formation in both hepa- ligible following hormone therapy, the intratumoral con-
tocytes and cardiomyocytes (35). It is possible that the centration of androgens remains high. While the andro-
membrane-forming capabilities of autophagy are used by gen-mediated lipid droplets are thought to be mainly TGs
the cell to help form the lipid depots. This is supported by due to the known AR-regulated lipogenic genes, the neu-
Mol Endocrinol, February 2013, 27(2):0000 – 0000 mend.endojournals.org 13

which ones are benign bystanders. Our

understanding of these signaling path-
ways will ultimately yield new thera-
peutic targets. Thus, as our work indi-
cates, in addition to exploring the use
of antioxidants and inhibitors of fatty
acid synthesis, autophagy may offer a
new therapeutic target for AR-driven
prostatic diseases.

Acknowledgments
We thank members of the Frigo Laboratory
for technical advice, helpful suggestions,
and critical comments on this study and
manuscript. We especially thank Dr. Ana
Maria Cuervo (Albert Einstein College of
Medicine, Bronx, New York, NY) for ad-
vice on various autophagy approaches. We
FIG. 8. Proposed model of how androgen-mediated autophagy promotes prostate cell
growth. Androgens, via AR, promote intracellular lipid accumulation through 1) increasing the also thank Dr. Thomas Westbrook (Baylor
expression of enzymes involved in de novo lipogenesis (e.g. FAS, ACC, and ACL) and 2) College of Medicine, Houston, TX) for
elevating intracellular ROS levels. The increased ROS levels stimulate lipid accumulation providing the pINDUCER system plasmids
through an autophagy-mediated mechanism that is not fully understood but is downstream/ and advice; Dr. Herman Dierick (Baylor
independent of FAS. The intracellular fat depots can then ultimately promote prostate cell
College of Medicine) for help with the
growth and/or survival through potentially altering multiple aspects of cell biology and
metabolism. pGIPZ constructs; Dr. Charles Sawyers
(Memorial Sloan-Kettering Cancer Center,
New York, NY) for providing the LAPC4
tral lipid stains used here could also be detecting increases cells; Dr. Brian Altman (University of Pennsylvania, Philadel-
in cholesterol levels. Finally, one emerging possibility to phia, PA) and Dr. Tso-Pang Yao (Duke University) for provid-
explain the role of fat droplets in cell growth is the au- ing the GFP-LC3 retroviral construct (Altman) and helpful dis-
tophagy-mediated breakdown of fat droplets. This pro- cussions regarding autophagy; Dr. Fei Su (University of
cess, termed lipophagy, can yield free fatty acids that enter Houston) and the Duke Comprehensive Cancer Center Flow
Cytometry Shared Resource for help with cell sorting; both the
␤-oxidation (65, 66, 70), which is an important energy-
Duke University Department of Pathology’s Electron Micros-
producing process in the prostate (88 –92). This suggests copy Core and MD Anderson Cancer Center High Resolution
that lipophagy may provide an alternative fuel source for Electron Microscopy Facility (Houston, TX) for help with TEM
cells. Although inhibition of autophagy led to an overall and immunogold TEM, respectively; and Dr. Sean Hartig (Bay-
decrease in lipid levels, the opposite of what one would lor College of Medicine) for imaging advice and, along with Dr.
expect if high rates of lipophagy were occurring, it is Chin-Yo Lin (University of Houston), critical reading of this
plausible that some of the newly mobilized fats were re- manuscript.
directed and used for fatty acid oxidation, an avenue of
Address all correspondence and requests for reprints to:
research we are currently exploring. Daniel E. Frigo, Center for Nuclear Receptors and Cell Signal-
The data presented here suggest that AR signaling, via ing, Department of Biology and Biochemistry, University of
a ROS signal, promotes the autophagy-mediated mobili- Houston, 3605 Cullen Blvd, Houston, Texas 77204. E-mail:
zation of intracellular lipid depots that are then utilized by frigo@uh.edu.
This work was supported by National Institutes of Health
the cell through various ways to increase cell growth (Fig. Grants K01DK084205 and R03DK092434 (to D.E.F.),
8). This is important because these findings begin to link P30EY007551 (to A.R.B.), and R01CA139818 (to D.P.M.),
together multiple aspects of AR-mediated prostate cell and by a grant from the Texas Emerging Technology Fund.
biology. Despite the known importance of AR signaling Disclosure Summary: The authors have nothing to disclose.
in the prostate, the mechanisms by which AR manifests its
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