The Progression of LNCaP Human Prostate Cancer
Cells to Androgen Independence Involves Decreased
FOXO3a Expression and Reduced p27KIP1
Promoter Transactivation
Rebecca L. Lynch, Bruce W. Konicek, Ann M. McNulty, Kimberly R. Hanna,
Jason E. Lewis, Blake Lee Neubauer, and Jeremy R. Graff
Lilly Research Labs, Cancer Research Division, Eli Lilly and Company, Indianapolis, Indiana
Abstract
The progression of human prostate cancer from the
initial androgen-dependent phase to androgen
independence involves diminished apoptosis and a
release from the cell cycle block triggered by androgen
ablation therapy. FOXO transcription factors play a
central role in promoting expression of proapoptotic and
cell cycle regulatory genes (e.g., FasL and p27 KIP1 ).
Reduced FOXO function might, therefore, play a role in
androgen-independent progression of human prostate
cancer. Herein, we show that FOXO function is
compromised in androgen-independent prostate cancer
cells (LNAI) versus androgen-dependent LNCaP cells.
The FOXO3a protein, the most highly expressed
FOXO family member in prostate cancer cells, is
hyperphosphorylated in LNAI cells. FOXO3a expression
is also markedly reduced in these androgenindependent LNAI cells when compared with parental
LNCaP cells. Together, reduced FOXO3a expression
coupled to FOXO3a hyperphosphorylation would
suppress FOXO transcriptional activity. Accordingly,
activity of the FOXO-responsive p27 KIP1 promoter is
reduced 60% in these LNAI cells when compared with
LNCaP cells. Moreover, mutation of a conserved FOXO
response element suppresses p27 KIP1 promoter activity,
substantiating a regulatory role for this FOXO response
element in p27 KIP1 promoter transactivation. Finally, we
show that the activity of a distinct FOXO-responsive
promoter, the 3X-IRS promoter, is also reduced in LNAI
cells. Collectively, these data show that reduced
FOXO3a expression coupled to increased FOXO3a
phosphorylation coincide with reduced FOXOresponsive promoter activity in androgen-independent
LNAI cells when compared with androgen-dependent
LNCaP cells. To the extent that this model reflects
Received 9/22/04; revised 12/20/04; accepted 1/17/05.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: R.L. Lynch and B.W. Konicek contributed equally to this work.
Requests for reprints: Jeremy R. Graff, Cancer Division, Lilly Research Labs,
Eli Lilly and Company, Lilly Corporate Center, Drop Code 0546, Indianapolis,
IN 46285. Phone: 317-277-0220; Fax: 317-277-3652. E-mail: [email protected]
Copyright D 2005 American Association for Cancer Research.
human disease, these data suggest that FOXO function
may be compromised with androgen-independent
progression of human prostate cancer.
(Mol Cancer Res 2005;3(3):163 – 9)
Introduction
The progression of human prostate cancer from the initial
androgen-dependent phase to androgen independence heralds
the onset of lethality. An estimated 32,000 to 40,000 men
die per year of androgen-independent, metastatic prostate
cancer (1). The emergence of androgen-independent prostate
cancer has been associated with a blunted apoptotic
response and a release from the p27KIP1-mediated cell cycle
arrest that follows androgen withdrawal (2-4), although the
mechanisms controlling these phenotypic alterations are not
yet fully understood.
Recent evidence has implicated protein kinase B/AKT in
prostate cancer progression (1, 5, 6). Expression of the tumor
suppressor PTEN, which normally restrains the phosphoinositide-3 kinase/AKT pathway, is lost in advanced prostate cancer,
correlating with increased Gleason score and pathologic stage of
prostate cancer (7-11). Moreover, AKT activation is dramatically
up-regulated in the highest-grade prostate cancer (Gleason grade
8-10) relative to normal prostate or lower-grade prostate cancer
(12). In the androgen-dependent/sensitive prostate cancer cell
line LNCaP, progression to androgen independence involves
increased AKT activity in concert with diminished p27KIP1
expression (5, 6). In addition, overexpression of a constitutively
active AKT (AKTDD) in LNCaP cells substantially reduced
p27KIP1 protein expression as well. These data implicate
enhanced AKT activity in the control of p27KIP1 expression in
prostate cancer cells.
The FOXO/Forkhead transcription factors (FOXO1/FKHR,
FOXO3a/FKHRL1, and FOXO4/AFX, hereafter collectively
called FOXO) serve as transcriptional regulators for proapoptotic
genes, such as BIM-1 (13) and FasL (14), and the cell cycle
regulatory genes p27 KIP1 (15-17), cyclin B, polo-like kinase (18),
p130 (19), and cyclin D1 (17). FOXO3a, in particular, has been
implicated in the transcriptional regulation of p27 KIP1 (16, 20, 21).
FOXO activity is negatively regulated by AKT, which
phosphorylates FOXO at multiple sites, forcing FOXO to the
cytoplasm and preventing transcription of FOXO-responsive
genes, including p27 KIP1 (22-26). In addition, AKT-mediated
phosphorylation can promote proteosomal degradation
of FOXO3a (27), thereby suppressing FOXO3a function.
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Disruption of FOXO function may contribute to the suppression
of apoptosis and the release from cell cycle control that
characterize the progression of prostate cancer to androgen
independence.
We have, therefore, examined whether FOXO function may be
altered in the androgen-independent prostate cancer cells (LNAI)
cells when compared with the androgen-dependent/sensitive
LNCaP parental cells. We now show that expression of FOXO3a
is hyperphosphorylated in LNAI cells consistent with the
increased AKT activity of these cells (5). In addition, expression
of FOXO3a is markedly reduced in the androgen-independent
LNAI cells. Accordingly, the activity of the FOXO-responsive
promoters, 3X-IRS and p27 KIP1 , is also reduced in these
androgen-independent LNAI cells when compared with the
androgen-dependent LNCaP cells. Further, disruption of a FOXO
response element in the p27 KIP1 promoter suppresses promoter
activity, supporting the importance of FOXO in the transcriptional
regulation of p27 KIP1 . These data provide evidence that FOXO
function is compromised with androgen-independent progression
of the LNCaP human prostate cancer cell line.
Results and Discussion
FOXO3a Phosphorylation and Expression in LNCaP and
LNAI Cells
The FOXO protein, FOXO3a, was recently shown to be
the most highly expressed FOXO protein in LNCaP cells as
well as other human prostate cancer cell lines. Overexpression
of FOXO3a in these cancer cells robustly induces apoptosis
(28). The activity of FOXO3a and other FOXO transcription
factors is negatively regulated by AKT. Enhanced AKT
activity can, therefore, suppress the expression of FOXO-
responsive genes, such as p27 KIP1 (16, 17, 20). We and others
have established that AKT activity is enhanced in concert with
reduced p27KIP1 expression in androgen-independent derivatives (LNAI cells) of the androgen-dependent/sensitive
prostate cancer cell line LNCaP (5, 6). To evaluate whether
the reduction in p27KIP1 expression in these LNAI cells may
be related to AKT-mediated suppression of FOXO activity, we
examined the phosphorylation and expression of the dominant
FOXO family member, FOXO3a, in both LNCaP and in
LNAI cells.
Consistent with the increased AKT activity in LNAI cells
(5), the phosphorylation of FOXO3a at Thr32 in LNAI cells
was increased f2-fold when compared with that in the
parental LNCaP cells (Fig. 1A). Strikingly, FOXO3a protein
expression was also substantially reduced (f80% when
normalized to h-actin expression; Fig. 1B) in both LNAI
and LNAI-2 cells. Thus, in the androgen-independent LNAI
cells, there is a marked decrease in FOXO3a protein
expression as well as increased phosphorylation. Together,
these changes would indicate that FOXO3a function may be
compromised in the androgen-independent prostate cancer
cells when compared with the androgen-dependent parental
LNCaP cells.
Transactivation of the p27 KIP1 Promoter Is Reduced in
LNAI Cells
Because LNAI cells showed increased phosphorylation and
decreased expression of FOXO3a, we next sought to determine
whether the transactivation of FOXO-responsive promoters,
such as the p27 KIP1 promoter, might be suppressed in LNAI
cells. We, therefore, cloned a fragment of the human p27 KIP1
FIGURE 1. FOXO3a phosphorylation and expression. A.
Western blot analysis for phosphorylation of FOXO3a at Thr32
(anti-phospho-Thr32, UBI 1:250)
in LNCaP and LNAI cells. Blots
were reprobed for h-actin to
control for loading and transfer
variations. Signal intensities
were normalized to that of hactin and then divided by the
FOXO3a total protein/h-actin ratio from a parallel blot to yield the
normalized FOXO3aphosphoT32
values depicted graphically and
numerically (inset ). Columns,
mean of LNCaP and LNAI
lysates shown in the inset Western blots; bars, SE. B. Western
blot analysis of FOXO3a protein
expression in androgen-dependent LNCaP and the androgenindependent derivatives, LNAI
and LNAI-2, using antiFOXO3a/FKHRL1 antibody
(UBI 1:250). The blot was
reprobed for h-actin (1:10,000,
Sigma, St. Louis, MO) to control
for loading and transfer. Blots
(A and B ) are representative
of more than five independent
Western blot experiments.
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FOXO Function Is Compromised with Androgen Independence
promoter region (bases 2,873-3,552, Genbank accession
no. AB003688; ref. 29) into pGL3 to assess p27KIP1 promoter
transactivation using the firefly luciferase reporter gene (p27KIP1
promoter). The murine p27KIP1 promoter is specifically
activated by inhibition of the phosphoinositide-3 kinase/AKT
pathway (15, 16). Likewise, the activity of this human p27KIP1
promoter fragment is also specifically activated when the
phosphoinositide-3 kinase/AKT pathway is blocked by pharmacologic inhibition of phosphoinositide-3 kinase or by
cotransfection with wild-type PTEN (but not by cotransfection
with the c124s and g129e nonfunctional PTEN mutants; data not
shown). The activity of this promoter was reduced by f60% in
the androgen-independent LNAI cell lines (LNAI and LNAI-2)
when compared with that in the parental, androgen-dependent
LNCaP cells (Fig. 2A), suggesting that the basal activity of the
p27KIP1 promoter is suppressed in LNAI cells. Cotransfection
with wild-type PTEN, which encodes the lipid phosphatase that
suppresses phosphoinositide-3 kinase/AKT signaling (7), induced p27KIP1 promoter activity to a similar extent in both
LNCaP and LNAI, indicating that forced expression of PTEN
can restrain the phosphoinositide-3 kinase/AKT signaling
pathway in both LNCaP and LNAI cells (Fig. 2B). Consistent
with the marked reduction of FOXO3a expression in the LNAI
cells, these data indicate that the basal activity of the FOXOresponsive p27KIP1 promoter is reduced, paralleling the reduced
expression of p27KIP1 protein in these LNAI cells (see Fig. 7 in
ref. 5).
A FOXO Response Element Regulates Transactivation of
the p27 KIP1 Promoter
We next examined whether a putative FOXO response
element (24), located at 57 relative to translation start,
might regulate the transcriptional activity of this human
p27KIP1 promoter construct. This putative FOXO response
element is absolutely conserved in both the mouse and
human p27KIP1 sequences, supporting a potential regulatory
role for this sequence (30). We created two distinct constructs
(p27KIP1 mut1 and p27KIP1 mut2) incorporating point
mutations designed to disrupt this FOXO response element
in the p27KIP1 promoter (31). Both constructs were transfected into LNCaP cells and transcriptional activity was
compared with that of the wild-type p27KIP1 promoter. The
activity of both mutant promoter constructs was reduced by
40% to 55% when compared with the activity of the wildtype p27KIP1 promoter (Fig. 3A). The activity of both mutant
promoters was induced by PTEN cotransfection, although to
a slightly lesser extent than that induced by PTEN
cotransfection with the wild-type p27KIP1 promoter (Fig.
3B). These data indicate that the FOXO response element
located at 57 relative to translation start is involved in the
regulation of p27KIP1 promoter activity, particularly the basal
activity of the p27KIP1 promoter.
The Activity of FOXO-Responsive Promoters Is Specifically Reduced in LNAI Cells
We next sought to determine whether the activity of other
FOXO-responsive promoters might be reduced in LNAI cells.
We evaluated transactivation of an artificial promoter construct
containing three insulin response sequences/FOXO response
p27 KIP1 promoter activity is reduced in LNAI cells
compared with LNCaP cells. A. The p27 KIP1 promoter was transfected
into LNCaP, LNAI, and LNAI-2 cells along with the pRLSV40 (Renilla
luciferase). To control for variations in transfection efficiency, the p27 KIP1
firefly luciferase activity was normalized to the Renilla luciferase activity
from each experimental well (presented in relative luciferase units). The
percent reduction relative to the activity in LNCaP is noted. B. LNCaP and
LNAI cells were transfected with the p27 KIP1 promoter reporter and either
the empty pcDNA vector control or a plasmid vector encoding the wild-type
PTEN tumor suppressor, which restrains AKT pathway activity and
activates FOXO-mediated transcription (15). Data are presented as the
fold induction relative to that from transfection with the pcDNA empty
vector control. All transfections were run in triplicate. Columns, mean;
bars, SE. Data presented are representative of more than six independent
experiments.
FIGURE 2.
elements (CAAAA[C/T]AA, the 3X-IRS promoter; refs.
24, 31). Like the p27KIP1 promoter, the activity of the 3XIRS promoter was decreased by f50% in LNAI cells when
compared with that in LNCaP cells. The activity of this 3XIRS promoter was stimulated in both LNAI and LNCaP cells
by cotransfection with wild-type PTEN, although to a lesser
extent in LNAI cells (Fig. 4B). These data indicate that, like
the FOXO-responsive p27KIP1 promoter, the activity of the
FOXO-responsive 3X-IRS promoter was reduced in the LNAI
cells.
To ensure that diminished activity of the FOXO-responsive
p27KIP1 and 3X-IRS promoters in LNAI cells does not reflect a
generalized reduction in promoter activity in LNAI cells, we
transfected LNAI and LNCaP cells with a promoter reporter
that is not FOXO responsive, the sterol response element
binding protein (SREBP) promoter. Unlike the FOXO-responsive p27KIP1 and 3X-IRS promoters, SREBP promoter activity
was not reduced in LNAI cells when compared with LNCaP.
In fact, there was an increase in SREBP promoter activity
in LNAI cells when compared with LNCaP. Further, in both cell
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putative FOXO response element in this human p27KIP1
promoter reduced the activity of this promoter, indicating that
this FOXO response element plays a role in the regulation of
the p27KIP1 promoter.
We also show that the activity of a distinct FOXOresponsive promoter, the 3X-IRS promoter, is similarly
suppressed in the LNAI cells when compared with parental
LNCaP cells. In contrast, the activity of the SREBP promoter,
which is not FOXO responsive, is increased in LNAI cells,
indicating that the decrement in FOXO promoter activity does
not reflect a generalized decrease in transcriptional activity in
these LNAI cells. Taken together, these data show that FOXO
transcriptional activity is specifically suppressed in androgenindependent LNAI cells in concert with reduced FOXO3a
expression and increased FOXO3a phosphorylation.
The mechanism underlying reduced FOXO3a expression in
these LNAI cells is currently unclear. Recent reports have
shown that FOXO gene expression can be influenced by
hormones and nutritional status (32, 33). Certainly, the
FIGURE 3. Disruption of the FOXO response element in the p27KIP1
promoter reduces promoter activity. A. LNCaP cells were transfected with
pRLSV40 vector, the pcDNA empty vector control, and either the p27KIP1
wild-type plasmid, the mutant p27 KIP1 promoter with two point
mutations (p27 KIP1 mut1), or the mutant p27 KIP1 harboring three point
mutations (p27 KIP1 mut2) designed to disrupt the FOXO response
element (31) located at 57 relative to the translation start site (Genbank
accession no. AB003688). Data are normalized to Renilla luciferase
activity to control for transfection efficiency and are presented in relative
luciferase units. The experiment depicted is representative of six
independent experiments. Columns, mean; bars, SE. B. LNCaP cells
were transfected with a plasmid vector encoding the wild-type PTEN tumor
suppressor, which restrains AKT pathway activity and activates FOXOmediated transcription (15). Data are presented as the fold induction
relative to the values obtained by transfection with the pcDNA empty
vector control (these data are from the same experiment depicted in A). All
transfections were run in triplicate. Columns, mean; bars, SE.
lines, the activity of the SREBP promoter construct was
essentially unaffected by cotransfection with wild-type PTEN
(Fig. 5). These data, therefore, indicate that transactivation of
FOXO response elements is selectively affected in the
androgen-independent LNAI cells when compared with
LNCaP cells.
The data presented in this study implicate compromised
FOXO function in the progression of LNCaP human prostate
cancer cells to androgen independence. Expression of the
dominant FOXO family member in human prostate cancer
cells, FOXO3a (28), is markedly reduced. In addition,
FOXO3a is also hyperphosphorylated. Together, reduced
expression and hyperphosphorylation of FOXO3a would
conspire to suppress FOXO activity. Indeed, transactivation
of the p27KIP1 promoter, which has been repeatedly linked to
FOXO3a function (16, 19, 20), is suppressed in these
androgen-independent LNAI cells when compared with the
androgen-dependent LNCaP cells. Moreover, disruption of a
FIGURE 4. FOXO-responsive 3X-IRS promoter activity is reduced in
LNAI cells versus LNCaP cells. A. The 3X-IRS promoter, which harbors
three insulin response sequences/FOXO response elements (24), was
transfected into LNCaP and LNAI cells along with the pRLSV40 (Renilla
luciferase). To control for variations in transfection efficiency, the 3X-IRS
firefly luciferase activity was normalized to the Renilla luciferase activity
from each experimental well (presented in relative luciferase units). The
percent reduction in 3X-IRS promoter activity in LNAI cells versus LNCaP
cells is noted. B. LNCaP and LNAI cells were transfected with the empty
pcDNA control vector or a plasmid vector encoding the wild-type PTEN
tumor suppressor, which restrains AKT pathway activity and activates
FOXO-mediated transcription (15). Data are presented as the fold
induction relative to the values obtained by transfection with the pcDNA
empty vector control for each cell line. All transfections were run in
triplicate. Columns, mean; bars, SE. These data are representative of
more than three independent experiments.
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FOXO Function Is Compromised with Androgen Independence
FIGURE 5.
SREBP promoter
activity is similar in LNCaP and
LNAI cells. The SREBP promoter,
which is not FOXO responsive, was
transfected into LNCaP and LNAI
cells along with the pRLSV40 and
either the pcDNA empty vector
control or a plasmid vector encoding the wild-type PTEN tumor suppressor. To control for variations in
transfection efficiency, the
SREBP1 promoter firefly luciferase
activity was normalized to Renilla
luciferase activity (shown in relative
luciferase units). All transfections
were run in triplicate. Columns,
mean; bars, SE. These data are
representative of two independent
experiments.
progression of human prostate cancer cells to androgen
independence involves altered hormonal responsiveness (1).
A recent report has now indicated that expression of the fulllength FOXO1 protein can be regulated by androgens in
prostate cancer cells. Androgens, which protect prostate cells
from apoptosis, can trigger the proteolytic processing of the
full-length FOXO1 protein into a smaller protein product,
which can then act as a dominant-negative inhibitor of FOXO
transcriptional activity (34). Additionally, there is also evidence
that AKT-mediated phosphorylation can trigger the proteosomal degradation of the FOXO3a protein (27). Decreased
expression of the FOXO3a protein in the androgen-independent
LNAI cells may reflect altered androgen responsiveness and,
therefore, androgen-regulated processing, although anti-androgens
and protease inhibitors (i.e., MG132) do not restore expression
of full-length FOXO3a in these LNAI cells (data not shown).
Alternatively, the enhanced AKT-mediated FOXO3a phosphorylation in these cells may trigger proteosomal degradation of
full-length FOXO3a. Unraveling the complex mechanistic
nature underlying the reduced expression of FOXO3a in these
LNAI cells will necessitate further exploration.
The progression of human prostate cancer from the initial
androgen-dependent phase to androgen independence has been
associated with both a diminished apoptotic response and a
release from the p27KIP1-mediated cell cycle block initially
triggered by androgen withdrawal (2-4). The FOXO/Forkhead
transcription factors control the transcription of both proapoptotic genes (e.g., FasL, Bim-1) and cell cycle regulatory genes
(e.g., p27 KIP1 ; ref. 26). Indeed, reduced p27KIP1 expression has
been repeatedly linked to prostate cancer progression in human
prostate cancer samples (35, 36). It is interesting to speculate that,
as prostate cancer cells emerge from androgen ablation, there
would be a significant selective advantage in undermining the
function of FOXO and FOXO3a in particular. Reduced FOXO
function would compromise the normal apoptotic response and
diminish the expression of cell cycle regulatory genes, such as
p27 KIP1 , thereby enabling the outgrowth of these cells in the
absence of androgens. To the extent that these LNAI cells reflect
androgen-independent human prostate cancer, the data in this
report support this hypothesis. Diminished FOXO function may,
therefore, be a powerful means by which the prostate cancer cells
override the normal apoptotic signals and the cell cycle block
elicited by androgen withdrawal. With data from other recent
reports (17, 28, 37), the data in this report underscore the
importance of FOXO activity in prostate cancer cell biology
and suggest that enhancing FOXO function may be a
compelling strategy to combat prostate cancer progression.
Materials and Methods
Plasmids
Sequences incorporating key promoter elements within the 5V
regulatory region of the p27 KIP1 gene (29) corresponding to
nucleotides 2,873 to 3,552 (Genbank accession number
AB003688) were PCR amplified and cloned into pGL3
(Promega, Madison, WI) to generate the p27KIP1 promoter,
firefly luciferase plasmid construct (p27KIP1 promoter), using the
following primers: (upstream) 5V-TCTATAGCTAGCAGAGCAGGTTTGTTGGCAGC-3V; (downstream) 5V-CATATCAAGCTTCACGACCGCCTCTCTCGC-3V (Invitrogen Life
Technologies, Carlsbad, CA). This promoter contains an IRS
sequence (TTGTTTTG), the binding site for FOXO transcription
factors (24, 25), at position 57 relative to the translation start
site of p27KIP1 . Two mutant constructs were generated to disrupt
this site using the Quickchange XL site-directed mutagenesis kit
(Stratagene, La Jolla, CA). Mutants were generated to disrupt
FOXO responsiveness based on previously published work (31).
In p27KIP1 mut1, two bases within the FOXO response element
were mutated using the following primers: 5V-CAAAACCGGAACACCACAAAGCGCCCCTACGC-3V and (reverse); 5VGCGTAGGGGCGCTTTGTGGTGTTCGGTTTTG-3V (forward). For p27KIP1 mut2, three point mutations within the
FOXO response element were generated using the following
primers: 5V-CAAAACCGAAGACCACAAAGCGCCCCTACGC-3V (reverse) and 5V-GCGTAGGGGCGCTTTGTGGTCTTCGGTTTTG-3V (forward). The SREBP1 promoter construct was generated as described (38) by inserting the sterol
response element/Sp1 sequence from the low-density lipoprotein
receptor in triplicate into pGL3. The 3X-IRS promoter reporter
was generated essentially as described (24). pRLSV40, which
expresses Renilla luciferase from the SV40 promoter, was
purchased from Promega and used to normalize transient
transfections for transfection efficiency.
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Transient Transfections
Cells were plated in six-well tissue culture plates (Falcon,
Becton Dickinson Labware, Franklin Lakes, NJ) and were
transfected 24 hours later with 0.5 Ag of each plasmid using 6 AL
Fugene as in the manufacturer’s protocol (Roche Biochemicals,
Indianapolis, IN). Cells were lysed in 1 passive lysis buffer
(Promega) 72 hours after transfection. To control for transfection
efficiency, the various test promoters linked to firefly luciferase
were always cotransfected with the pRLSV40 plasmid, which
yields expression of Renilla luciferase. Promoter activities were
assayed with the dual luciferase assay kit (Promega) using the
Wallac Victor (Wallac, Turku, Finland). Transient transfections
were always run in triplicate.
Cell Culture and Western Blotting
Androgen-independent derivatives of the androgen-sensitive/dependent human prostate cancer cell line LNCaP were
established through successive rounds of in vivo selection (5).
Briefly, LNCaP tumors were established in male nude mice
supplemented with testosterone. These tumor-bearing mice
were then castrated and the testosterone pellets were removed.
Most of the LNCaP tumors regressed or stopped growing. A
minority continued to grow even in the absence of testosterone.
First-generation cell lines were established from these tumors.
To evaluate the potential for these cell lines to grow in the
absence of androgen, these cells were reinjected into castrated
nude mice. Again, cell lines were established from tumors that
developed in these castrated nude mice. These secondgeneration cell lines formed rapidly growing tumors in castrated
nude mice with nearly 100% frequency (5). This androgenindependent phenotype is stable. Moreover, these androgenindependent sublines (LNAI cells) were independently derived
from different xenograft tumors (LNAI was previously called
LNAI T1.16; ref. 5) and share many of the molecular changes
defined for advanced human prostate cancers, including AKT
activation and reduced p27KIP1 expression (5). These LNAI
cells also show similar phenotypic characteristics to other
androgen-independent sublines derived by depriving LNCaP
cells of androgens in culture (6).
Protein lysates were harvested from cells at f70%
confluence (5) using radioimmunoprecipitation assay buffer,
1 Cell Lysis Buffer (Cell Signaling, Beverly, MA) or cell
extraction buffer (Biosource International, Camarillo, CA)
supplemented with protease inhibitor cocktail (P8340), and
phosphatase inhibitor cocktails I and II (P2850, 5726; Sigma).
Chemiluminescent signal was detected with the Lumi-imager
digital image capture system (Roche).
Acknowledgments
We thank Juan Wang, Teresa Burke, Zhao Hai Lu, Dr. Marcio Chedid, Dr. Richard
Beckmann, Dr. Mei Lai, and Dr. James Thomas (Lilly Research Laboratories) for
providing the plasmids used in this study, and Drs. Richard Gaynor, Homer Pearce,
Jake Starling, John McDonald, and Kerry Blanchard for supporting this work.
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169
The Progression of LNCaP Human Prostate Cancer Cells to
Androgen Independence Involves Decreased FOXO3a
Expression and Reduced p27KIP1 Promoter Transactivation
Rebecca L. Lynch, Bruce W. Konicek, Ann M. McNulty, et al.
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