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Departments of Molecular Pharmacology and Experimental Therapeutics (H.M.), Urology Research (H.M.,, H.H., L.J.S., D.J.T.), Laboratory Medicine and Pathology (D.I.S.), and Biochemistry and Molecular Biology (D.J.T.), Mayo Clinic and Foundation, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: D. J. Tindall, Ph.D., Department of Urology Research, Guggenheim 1701, Mayo Foundation, 200 First Street SW, Rochester, Minnesota 55905. E-mail: tindall.donald{at}mayo.edu
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Abstract |
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Introduction |
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Growth factors and their cognate receptors have long been implicated in PCa progression (7, 8). In fact, androgen removal promotes the increased expression of epidermal growth factor receptor family members and ligands, among others (7), thereby contributing to the formation of the abnormal autocrine loops seen in advanced PCa. In addition, loss of androgenic regulation of important metalloproteases such as neutral endopeptidase and maspin promote multiple growth factor and growth factor receptor signaling, which can contribute to progression (9, 10, 11, 12). Thus, androgen removal may increase growth factor signaling, which could contribute to acquisition of the androgen-independent phenotype.
Additional evidence for the importance of growth factor-dependent support for the progression and survival of PCa cells comes from the findings that the phosphatase PTEN, a major regulatory mechanism of PI3K action, is commonly lost through inactivating mutations or loss of expression in PCa cell lines and tumors (13, 14, 15, 16). PTEN loss in malignant cells facilitates dysregulated PI3K activity (15, 16). In addition, it is conceivable that the increased abnormal growth factor loops seen in PCa can enhance growth and mitogenic signaling through PI3K and other effector pathways even in the presence of wild-type PTEN (7, 8). Thus, a reliance on growth factor support in the absence of androgens may be a favored pathway (17) for PCa progression, which may be mediated at least in part through PI3K signaling. However, there are no studies that show direct evidence for such a hypothesis during the process of progression.
In the presence of androgen ablation, successful progression by PCa requires an ability to overcome two major roadblocks. The most important one is the survival of at least a portion of malignant cells during the acute onset of androgen deprivation. The second is the ability to proliferate in an androgen-independent manner, thereby overcoming the growth arrest instituted by the androgen withdrawal upon androgen-responsive PCa cells (18). Given the association of abnormal growth factor signaling and PCa progression, we investigated the role of the PI3K signaling pathway in the human PCa cell line LNCaP (19) during progression to the androgen-independent state. Here we show that removing androgen support from LNCaP cells triggers a series of events, including cell cycle arrest and increased PI3K-Akt activity, culminating in the eventual acquisition of the androgen-independent phenotype. Under such conditions, despite the presence of growth factor support, inhibition of PI3K signaling during the acute phase of androgen deprivation completely blocked progression and hypersensitized relapsed cells to spontaneous apoptosis. In the absence of androgens, LNCaP cells became hyperdependent on PI3K for growth, survival, and progression to and maintenance of the androgen-independent state.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CSS was prepared as follows. A solution containing 5% (wt/vol) activated charcoal (Sigma, St. Louis, MO) and 0.5% (wt/vol) dextran T70 (Pharmacia Biotech, Piscataway, NJ) was prepared in 1 mM HEPES buffer (pH 7.4). The mixture was stirred gently at 4 C for 3 h and centrifuged at 2500 x g for 10 min. At room temperature, 5 g dextran-treated charcoal were added to 500 ml FBS, mixed gently for 1 h, and centrifuged at 2500 x g for 10 min. The collected supernatant (serum) was subjected to another cycle of dextran-treated charcoal treatment, filtered through 0.2-µm porous membranes (Nalgene, Rochester, NY), aliquoted, and maintained at -20 C until used.
Chemical reagents
Drug stocks (20–50 mM) of R1881 (DuPont Merck Pharmaceutical Corp., Boston, MA), Cx (Zeneca Pharmaceuticals), LY294002
[2-(4-morpholinyl-4H-1-benzopyran-4-one)], PD98059
(2'-amino-3'-methoxyflavone), H89
[N-(2-[(p-bromocinnamyl)amino]ethyl)-5-isoquinoline-sulfonamide]
from Calbiochem (La Jolla, CA), and cycloheximide
(Sigma-Aldrich Corp., St. Louis, MO) were dissolved
in ethanol. Stocks of wortmannin (1 mM)
(Calbiochem) were prepared in dimethylsulfoxide. All drugs
were kept in the dark at -20 C, except for wortmannin, which was kept
at -80 C.
Northern analysis
Primers for RT-PCR of neuron-specific enolase (NSE; GenBank
X51956), prostate-specific antigen (PSA; GenBank M26663), neurotensin
(NT; GenBank U91618), and p27kip1 (GenBank
AY004255) were as follows: NSE: forward, 5'-GTTCTGAACGTCTGGCTAAATAC-3';
reverse, 5'-CATTGAGTTATGGGGAAATGA-3'; PSA: forward,
5'-AGCCACAGCTTCCCACAC-3'; reverse, 5'-CAGTATTCCCCAGGACACAG-3'; and NT:
forward, 5'-CGGACTTGGCTTGTTAGAA-3'; reverse,
5'-TTGTAGAAGAGACAGATAAGTGTGTT-3'. For p27kip1
Northern analysis, an 881-bp insert from the image clone 854668
(GenBank AA630082) in pBlueSK was used after sequence confirmation.
Similarly, the RT-PCR products for NSE (662 bp), PSA (657 bp), and NT
(651 bp) were cloned into the pCR-Blunt vector
(Invitrogen, Carlsbad, CA), and confirmed by sequencing.
Total cellular RNA was isolated using the TRIzol reagent (Life Technologies, Inc., Grand Island, NY), and aliquots (10–15
µg) were electrophoresed on 1.2% denaturing formaldehyde-agarose
gels and transferred onto positively charged nylon membranes. Filters
were hybridized with [32P]deoxy-CTP-labeled
probes (NSE, NT, PSA, p27kip1, and GAPDH) for
Northern analysis.
Western immunoblotting
Protein samples were prepared by lysing cells over ice in
ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40,
0.1% SDS, 0.25% sodium deoxycholate, 150 mM NaCl, 1
mM EGTA, 1 mM PMSF, 1 µg/ml leupeptin, 1
µg/ml aprotinin, 1 µg/ml pepstatin, 1 mM
Na3VO4, and 1
mM NaF). Cell lysates were passed 3-times through a
27-gauge needle and centrifuged at 14,000 x g at 4 C
for 15 min. Protein content was determined using the Bio-Rad Laboratories, Inc. DC-protein assay kit
(Bio-Rad Laboratories, Inc., Hercules, CA). Equal amounts
of protein (15–50 µg) from cell lysates were denatured in sample
buffer, subjected to LDS/NuPAGE on 4–20% gels
(Novex/Invitrogen, San Diego, CA), and
transferred to nitrocellulose membranes. The blots were probed with
specific primary antibodies as recommended by the suppliers.
Appropriate HRP-conjugated secondary antibodies were used (1:5,000) and
visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). Antibodies were purchased from the
following sources: PY20, CDK4, ERK2, and P27Kip1
mouse monoclonal antibodies from Santa Cruz Biotechnology, Inc.; p16INK4a and
p21cip1 mouse monoclonal antibodies from
Calbiochem (La Jolla CA); anti-whole Akt and Phospho-Akt
(Ser473) from New England Biolabs, Inc. (Beverly, MA); anti-androgen receptor (AR) monoclonal
antibody was purchased from PharMingen (San Diego, CA);
and anti-PI3K p85 subunit was from Upstate Biotechnology, Inc. (Lake Placid, NY).
PI3K kinase assay
The PI3K activity assay was performed as described by Endemann
et al. (20) and Huang et al.
(21). Briefly, cells were washed twice with ice-cold PBS
and lysed in RIPA buffer. The lysates were centrifuged, and the protein
content was determined. Five-hundred µg aliquots from each sample
were immunoprecipitated with 40 µl aliquots of pre-conjugated,
monoclonal anti-phosphotyrosine (PY20) agarose beads (Santa Cruz, CA)
by incubating overnight at 4 C in 500 µl of immunoprecipitation
buffer (190 mM NaCl, 50 mM
Tris-HCl pH 7.4, 6 mM EDTA, 2.5% Triton X-100).
All subsequent steps were exactly as described (21). 30
µl aliquots from the kinase reaction assays were separated on thin
layer Silica Gel 60 chromatography plates (EM Separations Technology),
dried at room temperature, and autoradiographed.
Cell transfection
Wild-type PTEN cDNA (a kind gift from Dr. C. David James, Mayo
Clinic and Foundation) was subcloned into the pcDNA3.1(+) vector
(Invitrogen, Carlsbad, CA). The PTEN open reading frame
was confirmed by sequencing of both strands. Transient transfections of
LNCaP cells and LNCaP-Rf cells were performed by electroporation.
Briefly, total plasmid DNA was kept constant (20 µg) for all
transfections by the addition of empty vector (pcDNA-3.1(+) or pCMV6)
to pcDNA-PTEN or pCMV6-CA-Akt. The DNA was ethanol- precipitated and
resuspended in 50 µl of serum-free media. Cells (1 x
107 per transfection) in 350 µl of
serum-containing media were mixed with the plasmid DNA to a total of
400 µl. The DNA-cell mixture was transferred to a 4-mm cuvette (BTX
Inc., San Diego, CA) and electroporated with a single 10-ms, 300-V
pulse using a BTX T820 square wave electroporator (BTX Inc., San Diego,
CA). Transfection efficiency was assessed by enhanced green fluorescent
protein expression (CLONTECH Laboratories, Inc. Palo Alto,
CA) cotransfected with each sample.
Cell cycle analysis
Control and treated cells were collected by trypsinization,
fixed in freshly prepared ice cold 70% ethanol for 30 min, and placed
at -20 C overnight. After washing with 1x PBS, the cells (1 x
106) were stained with 10 µg/ml propidium
iodide and 50 µg/ml RNase A, and incubated for 30 min before flow
cytometry analysis using a FACSCalibur flow cytometer (Becton Dickinson and Co., San Jose, CA). Data were analyzed using
ModFit V.1.2 software.
Morphometric cell death assay
Adherent and floating cells were collected and fixed with a
solution of 1.5% formaldehyde, 40% methanol, and 10% acetic acid.
Bis-benzimide (Sigma-Aldrich Corp.) was added to a final
concentration of 1 µg/ml, and the cells were incubated for 10 min at
room temperature. Ten- microliter aliquots were placed on slides
and viewed under UV and phase contrast illumination (Carl Zeiss Axiophot). Cells scored as "dead" exhibited signs of
hyperfluorescent chromatin (condensation) and/or nuclear fragmentation
(apoptotic), or hypofluorescence with cell lysis and/or karyolysis
(necrotic). Cells in three to four field quadrants representative of
the sample were identified as normal, apoptotic, or necrotic and
quantified to obtain the percent of dead vs. normal cells in
each sample in three independent experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
This model, like those of others (22, 23, 24), mimics many of
the features of PCa progression, and gives rise to androgen-independent
cell sublines.
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) and
neuroendocrine morphological changes that included soma compaction and
the growth of two or more long slender processes connecting with
neighboring cells (Fig. 1A
). The same was seen in cells treated with
WS+Cx (Fig. 1A). In contrast, cells receiving CSS+R1881 showed none of
these changes and behaved like those receiving WS alone (Fig. 1A).
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, B and C). In
addition, androgen removal resulted in the induction of the neuronal
marker, NSE mRNA (10), which correlated with the
morphological changes seen in response to androgen removal (Fig. 1, B
and C). Moreover, the induction of NT mRNA was observed with both Cx
and CSS treatment (Fig. 1, B–D). The latter was in agreement with
previous reports of NT protein induction under similar conditions
(26). Also, just as the cell cycle arrest and
neuroendocrine changes could be blocked by adding R1881, the changes in
PSA, NSE, and NT mRNA levels could also be blocked by the addition of
R1881 to the medium (Fig. 1D). Therefore, in this model the growth,
morphological, and molecular changes triggered by the removal or
antagonism of androgens were due specifically to the ablation of
androgen action.
The dramatic cell cycle arrest and neuroendocrine changes are
appreciable within 72 h of androgen deprivation. We call cells at
this stage LNCaP-NEL, for LNCaP with
neuroendocrine-like features. However,
after prolonged culture in the absence of androgens (>10 wk), cells
exhibited intermediate neuroendocrine changes and reestablished normal
PSA and AR expression (data not shown). In addition, the cell fraction
traversing the cell cycle increased (i.e. S-phase 
6–12%
vs. 
3% in the LNCaP-NEL stage) in an androgen-independent
manner (data not shown). We call cells at this stage LNCaP-Rf for
hormone refractory LNCaP. Because the system is
amenable to manipulation and exhibits many of the features seen in PCa
progression (19), it allows for studies aimed at defining
the temporal role of the PI3K signaling pathway in the
androgen-independent growth and survival of PCa cells during
progression.
PI3K and Akt activity are increased during and after acute and
chronic androgen ablation
To determine the role of PI3K during progression to the
androgen-independent state, we first asked what effects, if any, acute
or chronic androgen deprivation had on the PI3K activity of LNCaP or
LNCaP-Rf cells, respectively. As hormone-depleted medium (CSS) can
mimic the antiandrogenic effects of Cx, we used CSS-medium to deprive
LNCaP cells of androgenic support for the remaining studies. We
assessed levels of PI3K activity both directly and indirectly. For
direct measurements, we assayed PI3K activity in antiphosphotyrosine
(PY20) immunoprecipitates from cells under various treatment
conditions. As shown in Fig. 2, A and B,
comparison of PI3K activity in LNCaP cells in the presence of WS or
CSS+R1881 (control) vs. the absence of androgens (CSS)
consistently showed a 50% increase within the first 96 h of acute
androgen ablation. This activity was highest in LNCaP-Rf cells
(
4.3-fold compared with parental; Fig. 2, A and B). Importantly, the
increased PI3K activity during the acute or chronic culture conditions
could be inhibited by addition of R1881. Furthermore, this activity was
sensitive to the PI3K inhibitor LY294002 (Fig. 2, A and B).
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, C and D,
removal of androgens resulted in increased levels of active
phosphorylated Akt in both LNCaP and LNCaP-Rf cells. Furthermore, the
increase in active Akt under acute androgen deprivation was time
dependent and consistent with the 50% increase in PI3K activity seen
at 4 d of androgen deprivation (Fig. 2, E and F). No changes were
seen in Akt protein levels during the acute phase period examined.
Taken together, these findings show that acute androgen deprivation of
LNCaP cells triggered an increase in PI3K and Akt activity. The
heightened PI3K-Akt activation was maintained throughout the
progression and during the refractory, androgen-independent state of
LNCaP-Rf cells.
PI3K is required for growth and survival of LNCaP cells during
acute androgen ablation
Next, we asked whether this increased PI3K-Akt activity is
functionally important for the survival of LNCaP cells undergoing acute
androgen ablation. To address this question, we subjected LNCaP cells
to the androgen ablation protocol and treated them with the PI3K
inhibitor LY294002. We assessed cell growth, survival, and the
stereotypical progression of LNCaP cells to the NEL stage. Under acute
androgen deprivation, PI3K inhibition triggered apoptotic cell death,
as determined by morphological changes in chromatin condensation and
nuclear fragmentation (Fig. 3A, inset). As shown in Fig. 3A, LY294002 treatment resulted in
a dose-dependent increase in spontaneous apoptosis within 48 h in
either the presence or absence of androgens. However, LY294002 had a
more potent killing effect on cells subjected to the androgen ablation
protocol. These effects were also time dependent. As shown in Fig. 3B, most of the cells subjected to the CSS protocol and treated with
LY294002 (20 µM or more) succumbed by 96 h
(98% cell death). Cells cultured in CSS+R1881 plus LY294002 had 20%
or less cell death (Fig. 3B). Therefore, increased PI3K-Akt activation
during the acute phase of androgen ablation is functionally significant
for the survival of LNCaP cells. Under these conditions, inhibition of
PI3K hypersensitizes LNCaP cells to apoptosis and blocks their
transition beyond the NEL stage. This effectively blocked the emergence
of androgen-independent cells in these cultures (Fig. 3B).
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, unlike the effects of PI3K inhibition, pharmacological
inhibition of MAPK or PKA with PD98059 and H89, respectively, had
minimal effects on the survival of LNCaP cells under acute androgen
deprivation. Cell cultures receiving CSS+R1881 (controls) were also
minimally affected by MAPK or PKA inhibition. Therefore, these findings
suggest that the survival of LNCaP cells upon acute androgen
deprivation is predominantly due to the PI3K signaling pathway and not
to the MEK-ERK or PKA pathways.
Requirement of PI3K for growth and survival during the chronic,
androgen-deprived, refractory phase
We next examined whether the heightened PI3K-Akt activity in the
androgen-independent LNCaP-Rf cells was likewise required for their
growth and survival in the chronic state of androgen deprivation. We
further considered whether inhibition of PI3K by transfection of
wild-type PTEN in the LNCaP-Rf cells had the same effects as
pharmacological inhibition with LY94002. As shown in Fig. 4A, transfection of PTEN into LNCaP and
LNCaP-Rf cells cultured in CSS-medium resulted in a dose-dependent
increase in apoptosis. However, LNCaP-Rf cells were more sensitive to
the killing effects of PI3K inhibition by PTEN. Similar results were
observed with the pharmacological inhibition of PI3K with LY294002
(Fig. 4B). The slightly lower cell death in the PTEN-transfected cells
(Fig. 4A) compared with LY-treated cells (Fig. 4B) may be due to
transfection efficiency (70%, as determined by cotransfected
enhanced green fluorescent protein expression). Taken together, these
findings demonstrate that intact PI3K signaling is functionally
important for the growth and survival of LNCaP-Rf cells during the
chronic stages of androgen deprivation as it is during the acute phase
(LNCaP-NEL).
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, inhibition of PI3K is
growth inhibitory to LNCaP cells regardless of whether androgens are
present in the medium. The absence of androgens hypersensitizes cells
to spontaneous apoptosis induced by LY294002 treatment. These
characteristics are not limited to in vitro-derived LNCaP
sublines. As shown in Fig. 4, D and E, the in vivo-derived
LNCaP-C4-2 subline (24), when cultured under conditions of
androgen ablation, exhibits similar dependence on PI3K signaling and
susceptibility to apoptosis in the absence of androgens.
Modulation of p27kip1 levels by PI3K during
androgen-independent proliferation of LNCaP-Rf cells
A remarkable change in the LNCaP-Rf compared with LNCaP-NEL cells
is the ability to proliferate in an androgen-independent manner.
Because androgen-independent proliferation would be expected to be a
rate-limiting step in PCa progression, we next examined the role of
PI3K in the androgen-independent proliferation of LNCaP-Rf cells.
Studies have shown that androgen ablation has dramatic growth
inhibitory effects in PCa cells and have implicated the
cyclin-dependent kinase inhibitor p27kip1 as a
major mediator of this growth inhibition (18, 22, 23).
During acute androgen deprivation, cell cycle arrest in our model
correlated with increased p27kip1 protein levels
and PI3K activation in LNCaP-NEL cells (Figs. 2C and 5A). However, during progression to
androgen-independent proliferation (LNCaP-Rf), PI3K activation remained
highly elevated, yet p27kip1 protein levels were
markedly diminished (Figs. 2C and 5A). This occurred despite the fact
that LNCaP-Rf cells have a relatively slower proliferation rate than
LNCaP cells (data not shown). Therefore, we sought to determine the
relationship of p27kip1 protein levels and PI3K
action in the refractory state. First, we examined the effects of
inhibiting PI3K on the cell cycle in LNCaP-Rf cells. As shown in Fig. 5B, 20 µM LY294002 induced cell cycle arrest in LNCaP-Rf
cells within 24 h of treatment. The cell cycle arrest due to
LY294002 treatment is distinctly different from that induced by
androgen deprivation (compare Table 1 and Fig. 5B). The
LY294002-induced arrest is rapid and characterized by
G1- and G2/M-enriched
fractions. Although a similar effect is seen in parental LNCaP cells in
the presence of androgens, the kinetics are slower (40 h), and they
do not exhibit the hypersensitivity to apoptosis seen in LNCaP-Rf cells
(
Figs. 3–5 and data not shown). Thus, we next examined the effects of
PI3K inhibition on the levels of cyclin-dependent kinase inhibitors in
LNCaP-Rf cells. As shown in Fig. 5, C and D, PI3K inhibition with
LY94002 triggered an increase in p27kip1 levels
in a dose- and time-dependent manner in exponentially growing
LNCaP-Rf cells. In contrast to changes in
p27kip1 levels, p21cip1 and
p16INK4a levels decreased with PI3K inhibition.
As expected, the blocked phosphoactivation of Akt after LY treatment
confirmed the inhibition of PI3K. Taken together, these findings show
that changes in the levels of p27kip1 are finely
regulated by PI3K activity in LNCaP-Rf cells.
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, A and B), whereas
parallel samples used for immunoblot analysis consistently showed
increased p27kip1 protein levels with PI3K
inhibition (Fig. 6, A and B). Likewise, increasing active Akt had no
effect on p27kip1 message levels.
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, C and D, PI3K inhibition
increased the half-life of p27kip1 protein from
4 h to greater than 8 h, whereas the half-life of
p21cip1 was not affected by the treatment. Taken
together, these findings suggest that p27kip1
protein levels are regulated posttranscriptionally by PI3K at a step
involving its rate of degradation. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our in vitro androgen ablation modeling triggers changes that are specifically due to the ablation of androgen action. Two critical components of this study system are the androgen and serum deprivation for 48 h, followed by the addition of hormone-depleted medium (or WS and AR antagonism) that provides growth signals. However, because PCa cells require androgens to effectively overcome the G1 cell cycle restriction (18), cells eventually accumulate at the G1 phase of the cell cycle. This androgen deprivation induced cell cycle arrest differs from that induced in these cells by PI3K inhibition. The latter is characterized by enriched G1 and G2/M fractions from which cells will undergo apoptosis if the PI3K inhibition is not relieved.
In our model, as in those used by others (18, 22, 23), the
CDI p27kip1 seems to be the major mediator of the
G1 arrest, because its changes among the
fluctuating cyclin-dependent kinase inhibitors correlated with the
arrest or resumption of cell cycling throughout the progression
process. However, it is possible that the arrest due to acute androgen
ablation is different from that due to PI3K inhibition. The acute
androgen ablation is mainly a
G0/G1 arrest, whereas the
PI3K inhibition-mediated arrest is a mix of late
G1 and G2/M arrest.
Consistent with such a possibility is the finding that
p16INK4a, which acts in early
G1 (38), does not decrease during
the acute androgen ablation G1 arrest, but is
decreased beyond its already low levels by the PI3K inhibition-mediated
arrest in the chronic and refractory state. In contrast, the cell cycle
arrest due to PI3K inhibition appears to be a late
G1 event, because p27kip1,
which exhibits broader inhibitory roles (39), accumulates,
whereas p16INK4a and
p21cip1 levels are further diminished. In this
context, the decrease in p16INK4a (and possibly
p21cip1) allow cells beyond the early
G1 phase at the time of PI3K inhibition
initiation, to continue cell cycle progression. However, even those
cells escaping p16INK4a restrains are affected by
the increasing p27kip1 levels and are eventually
arrested (i.e. G2/M). For example, the
G2/M and S-phase fractions due to acute androgen
ablation are equally diminished (<4%). In contrast, the PI3K
inhibition-mediated growth arrest in LNCaP-Rf cells exhibited a large
G2/M fraction (>16%) and negligible S-phase
(1%). In either scenario, the resulting cell cycle arrest is
distinctively different and consistent with the overall fluctuation in
cyclin-dependent kinase inhibitors.
The morphological and molecular neuroendocrine changes observed under our experimental manipulations are not solely dependent on intact PI3K signaling (this report and our unpublished data). However, neuroendocrine changes in LNCaP cells can be triggered by a myriad of stimuli (30, 31, 40) and may be a generalized stress response by these cells. These changes are not limited to in vitro-derived, androgen-independent LNCaP sublines because the C4- 2 subline (24) can also be induced to an NEL stage by the in vitro androgen ablation protocol (our unpublished data). Nevertheless, the contribution of neuroendocrine changes to progression remain to be clarified, as PCa tumors exhibit similar changes when androgen deprived (40).
Our findings show that PI3K is required for the growth of LNCaP cells regardless of whether androgens are present. However, the absence of androgens hypersensitizes cells to PI3K inhibition-dependent apoptosis. This latter effect was dependent upon the length of androgen deprivation before PI3K inhibition, as noted by others (41). These effects may be due to the long-acting effects of androgens. The finding that pharmacological or molecular inhibition of PI3K results in apoptotic hypersensitivity under androgen-deprived conditions strongly suggests that increased PI3K activation is functionally significant for the ability of these PCa cells to survive and thus progress to the androgen-independent state.
Addition of androgens within a discrete window of time affords cells
some protection from PI3K inhibition-dependent apoptosis. This could
also be appreciated in LNCaP-Rf cells despite their growth inhibitory
sensitivity to 1 nM R1881, as has been shown in similar
LNCaP sublines (22, 23). In this regard, the LNCaP-Rf
cells, like similarly derived in vitro cell sublines
(22, 23), differ from in vivo-derived LNCaP
sublines such as C4-2 (24) in that they are growth
inhibited by nanomolar concentrations of androgens. The in
vivo-derived, androgen-independent PCa cell sublines and
xenografts are apparently not subsequently growth inhibited by
androgens (42). However, when C4-2 cells are subjected to
androgen ablation conditions as in our studies, they do exhibit some
growth inhibition after the addition of androgen, as well as a
dependence and sensitivity to PI3K signaling inhibition. Furthermore,
prolonged culturing of LNCaP-Rf cells in CSS-medium eventually (1
yr) gives rise to androgen-independent cells that are not growth
inhibited by the addition of 1 nM R1881 (our
unpublished data), in agreement with findings in similarly evolved
sublines (22). This paradoxical result may in part be
related to changes in hormone sensitivity and the bimodal effect on
cell proliferation in response to androgens as well as AR expression
and stability changes (42).
The PI3K-Akt signaling axis has been described recently as a dominant growth factor survival pathway in PCa cells, which at best can only be partially compensated by a MAPK-sensitive step (43). A similar possibility was suggested by others in LNCaP cells (41). Our studies show that PI3K signaling plays multiple roles throughout the progression and maintenance of the androgen-independent state. Both acute and chronic androgen deprivation triggered an increase in PI3K and Akt activation, which was sustained throughout the progression process. Although it is unclear how the increased PI3K activation ensues in our model, removal of androgen support from PCa cells has been shown to promote the abnormal establishment of autocrine growth factor loops and enhance the engagement of already present growth factor signaling pathways (7, 10). Consistent with this possibility, we saw the induction of neurotensin after androgen ablation (10, 26). It is conceivable that in PCa cells under such conditions, neurotensin could synergize with other growth factors, such as epidermal growth factor and IL-6 (44), thereby heightening PI3K activation. Alternatively, the loss of PTEN, which often occurs in PCa, may also potentiate PI3K activation (16).
Recent studies have shown antagonism between PTEN and the AR’s transcriptional activity as well as AR-dependent growth and apoptosis in PCa cells (45). Our findings are in agreement with such a possibility. Together, these studies suggest that PI3K activity contributes to the AR transcriptional activity and androgen-dependent growth, possibly by increasing overall gene transcription and expression (46). Inhibition of PI3K activity in PTEN-null PCa cells can antagonize androgen action (45). Indeed, an apparently antagonistic relationship between PTEN and androgen action is consistent with our findings of increased PI3K activity upon androgen ablation. Likewise, reintroduction of androgens in the context of androgen ablation decreases PI3K activity, yet does not cause cell death as does direct PI3K inhibition by PTEN or LY294002. The latter is in agreement with the idea that PI3K is required for LNCaP cell growth regardless of androgen status. Although no effects of androgen addition on phospho-Ser473-Akt levels were found by Li et al., (45), this apparent difference from our findings may be due to the differences in experimental conditions used, which include cell culture and the time frames examined. For example, under our experimental conditions, it is the adaptive response of the cells to the androgen ablation stress that increases PI3K-Akt activity above basal levels. It is this relative increase in PI3K-Akt that is decreased or blocked from taking place by the reintroduction of androgen for 96 h. In addition, our findings suggest that in the context of androgen ablation stress, the relationship between the PI3K pathway and androgens is likely to be indirect, through enhanced or antagonized autocrine growth factor loops (7).
Whereas PI3K action is necessary for PCa cell progression as seen in this study model, it remains unclear which PI3K effector(s), in the context of a PCa cell, mediates the critical actions needed for progression. Major individual effectors, such as Akt alone, may not suffice. For example, during the acute ablation of androgen action in this model, PI3K and Akt activities are increased. However, during this very critical period, cell cycle progression is arrested rather than driven forward. This cell cycle quiescence is maintained for months despite the heightened PI3K and Akt activity levels. In addition, the expression of a constitutively active Akt protein does not promote cell cycle progression in the absence of androgens (our unpublished data). In agreement with such findings, Graff et al. (47) reported that LNCaP cells stably expressing a constitutively active form of Akt cannot form tumors in castrated mice. These findings suggest that other major PI3K effectors, the combination of Akt and other PI3K-dependent actions, or a complex interaction of PI3K action and that of other, as yet unknown, signaling mechanisms cooperate in providing the necessary growth and survival support for PCa cells to proliferate in an androgen-independent manner.
The androgen-independent proliferation in LNCaP-Rf cells was accompanied by a profound decrease in p27kip1 protein levels. Surprisingly, although PI3K-Akt inhibition would be expected to influence p27kip1 mRNA levels, possibly via forkhead transcription factors (35, 36), no induction of mRNA levels were observed. Also, increasing active Akt by exogenous expression of constitutively active Akt (37) had no effect on p27kip1 mRNA levels. However, it has recently been shown that endogenous forkhead transcription factors in PTEN-null cells may be deregulated in their localization, which may hinder their transcription capacity (48). In our studies the critical regulation of diminished p27kip1 protein levels in LNCaP-Rf cells, which presumably allows androgen-independent G1-S transition, seems to be dependent primarily on the heightened PI3K activity. This appears to be at the level of protein turnover, because the p27kip1 protein half-life rapidly increased upon PI3K inhibition. Furthermore, the effects of PI3K inhibition were specific and selective for p27kip1, as shown by the accumulation of p27kip1, but not p21cip1. Consistent with these findings, two recent reports have shown that p27kip1 levels can be regulated by PTEN through the ubiquitin E3 ligase SCFSKP2 and its lipid phosphatase, respectively (49, 50).
Our findings suggest one mechanistic explanation for the changes in p27kip1 protein levels seen in PCa tumors. The initial cell cycle inhibitory effects of acute androgen ablation correlate with increased p27kip1 levels, much as is seen in vivo (51, 52, 53, 54). After progression to the androgen-independent state, the heightened PI3K activity in LNCaP-Rf cells appears to be responsible for the steady state profound decrease in p27kip1 protein. In vivo, PCa tumors also exhibit a decrease in p27kip1 with progression (51). It is tempting to speculate that this process would be enhanced during the progressing of PCa to a hormone refractory state due to increased growth factor action, possibly via cyclin E-CDK2 activation and changes in cyclin-p27kip1 complexes (55, 56). Naturally, loss of PTEN would be expected to contribute to this effect, as has been seen in PTEN-null glioblastoma cells (57). The androgen deprivation model used in our studies exhibits striking similarities to PCa in vivo, where p27kip1 is generally decreased and is emerging as a promising prognostic marker (51, 58, 59, 60).
In conclusion, our findings are consistent with a major steady shift in growth support from androgens to growth factors during progression of LNCaP cells to the androgen-independent state, much as is seen during PCa progression (7, 8, 10). The PI3K signaling axis appears to be a central mediator of this growth factor action, especially under the stress of androgen ablation. The fact that LNCaP cells are PTEN-null may have allowed this event to be more easily revealed in our studies. These findings suggest that prostate cancer cell growth is controlled by a balanced interrelationship of multiple signaling pathways. Interference of androgen-regulated growth and survival pathways by androgen ablation in LNCaP cells is mainly compensated by PI3K signaling, allowing cells to survive androgen deprivation and the eventual emergence of androgen-independent growth. As PI3K signaling alone does not suffice to sustain androgen-independent proliferation, it remains to be determined what additional changes in PCa cells occur during the transitory phase following acute androgen ablation that eventually make androgen-independent proliferation possible. If indeed the PI3K pathway is a favored pathway for PCa progression (17), targeting the PI3K signaling axis alone or in combination with androgen ablation may prove therapeutically useful.
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Acknowledgments |
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Footnotes |
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1 H.M. and H.H. contributed equally. 
Abbreviations: A-I, Androgen-independent; CDI, cyclin-dependent kinase inhibitor; CSS-medium, charcoal-stripped serum medium; Cx, Casodex; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GF, growth factor; LN-NEL, LNCaP with neuroendocrine-like features; LN-Rf, androgen refractory LNCaP; NSE; neuron-specific enolase; NT, neurotensin; PCa, prostate cancer; PSA, prostate-specific antigen; WS-medium, whole FBS-containing medium.
Received April 9, 2001.
Accepted for publication July 12, 2001.
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