Hindawi Publishing Corporation
Obstetrics and Gynecology International
Volume 2013, Article ID 537479, 8 pages
Protein Kinase C훼 Modulates Estrogen-Receptor-Dependent
Alicia M. Thorne,1Twila A. Jackson,1Van C. Willis,2and Andrew P. Bradford1
Transcription and Proliferation in Endometrial Cancer Cells
1Department of Obstetrics and Gynecology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora,
CO 80045, USA
2Division of Rheumatology, Department of Medicine, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora,
CO 80045, USA
Correspondence should be addressed to Andrew P. Bradford; email@example.com
Received 10 May 2013; Accepted 31 May 2013
Academic Editor: Donghai Dai
Copyright © 2013 Alicia M. Thorne et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Endometrial cancer is the most common invasive gynecologic malignancy in developed countries. The most prevalent endo-
metrioid tumors are linked to excessive estrogen exposure and hyperplasia. However, molecular mechanisms and signaling path-
invasion. In this study, we demonstrate that expression of active, myristoylated PKC훼 conferred ligand-independent activation of
potentiate estrogen activation of ER-dependent transcription was attenuated by inhibitors of phosphoinositide 3-kinase (PI3K)
growth and transcription. Thus, PKC훼 signaling, via PI3K/Akt, may be a critical element of the hyperestrogenic environment and
ER positive tumors.
ways underlying their etiology and pathophysiology remain poorly understood. We have shown that protein kinase C훼 (PKC훼) is
aberrantly expressed in endometrioid tumors and is an important mediator of endometrial cancer cell survival, proliferation, and
dependent gene expression and inhibited estrogen-induced proliferation of endometrial cancer cells. The ability of PKC훼 to
PKC훼-dependent pathways may provide much needed prognostic markers of aggressive disease and novel therapeutic targets in
activation of ER that is thought to underlie the development of estrogen-dependent endometrial hyperplasia and malignancy.
ical malignancy in the United States, accounting for 45,000
molecular mechanisms underlying its etiology and patho-
physiology are poorly understood. Endometrial carcinomas
are derived from glandular epithelium and typically divided
into two subtypes based on clinical, histological, and molec-
ular characteristics [2, 3]. Type I tumors, comprising 80%
of cases, are generally well or moderately differentiated with
endometrioid morphology and are associated with chronic
unopposed estrogen exposure and hyperplasia. By contrast,
type II tumors are more heterogeneous, poorly differentiated
atrophic endometrium [2, 4]. The prevalence of advanced
static disease is increasing [5, 6]. Such cancers typically have
a poorer prognosis and are refractory to current therapeutic
Endometrioid tumors retain expression of estrogen (ER)
regulator of endometrial proliferation [9, 10]. Indeed, the
majority of endometrial cancers are thought to arise due to
nant transformation [2, 11]. However, our understanding of
the molecular mechanisms underlying the pathophysiology
of endometrial cancer lags far behind that of other hormone-
dependent malignancies such as breast, prostate and ovarian
cancer [2, 8, 12, 13].
2Obstetrics and Gynecology International
The protein kinase C (PKC) family has been implicated
in the regulation of numerous signal transduction pathways,
modulating cell growth, differentiation, and survival [14–16].
In endometrial cancer cells and primary endometrial epithe-
lium, expression of PKC훼 is increased in response to treat-
liferative actions of these agents in the endometrium [17, 18].
We have previously shown that PKC훼 is aberrantly expres-
transformation, invasion, and response to chemotherapy [21,
sed in human endometrial tumors [19, 20] and is a critical
regulator of endometrial cancer cell survival, proliferation,
22]. In addition, we demonstrated that knockdown of PKC훼
In this study, we present evidence that, in type I endome-
inhibits growth of estrogen-dependent endometrial cancers
in an in vivo model .
trial cancer cells, PKC훼 induces hormone-independent acti-
vation of ER, potentiates estrogen transcriptional responses,
and regulates estrogen-dependent proliferation and gene ex-
pression. Thus, PKC훼 signaling may be a critical ele-
ment of the supraphysiologic activation of ER thought to
underlie the development of endometrial hyperplasia and
2. Materials and Methods
Ishikawa cells expressing luciferase (luc) or PKC훼 shRNAs
have been described . Unless stated otherwise, all cell
lines were maintained in 5% CO2, phenol red free DMEM,
L-glutamine. Prior to estrogen treatment (100nM Estradiol,
Sigma Aldrich, St. Louis, MO, USA), cells were transferred
to phenol red free DMEM containing 1x SR-1 serum replace-
ment (Sigma Aldrich, St. Louis, MO, USA). Cell lines used
were authenticated by analysis of DNA microsatellite short
tandem repeats (STRS), as described previously .
10units/mL penicillin, 10휇g/mL streptomycin, and 200휇M
2.2. Cell Proliferation. Cell number and viability were deter-
mined from subconfluent cultures using a Vi-Cell Coulter
Counter (Beckman-Coulter, Inc., Fullerton, CA, USA) as
described in .
2.3. Luciferase Reporter Assays. The ERE-luc and pS2-luc
promoter reporter constructs have been described in [24–
26]. Myristoylated PKC훼 vector  was obtained from
using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA)
as per the manufacturers protocol. 0.5휇g pCMV훽, encod-
ciency and cell number. Total DNA was kept constant by
addition of empty vectors. Promoter activity was determined
ing 훽-galactosidase under control of the CMV constitutive
promoter, was included as a control for transfection effi-
by Luciferase and 훽-galactosidase assays, as described in
2.4. RNA Isolation and Quantitative RTPCR. RNA was iso-
lated from 106cells using a Qiagen RNeasy kit (Qiagen, Ger-
mantown, MD, USA) according to the manufacturer’s direc-
tions and quantitated using a NanoDrop ND1000 spectro-
photometer. Aliquots were evaluated by chromatography
nologies, Santa Clara, CA, USA) on an Agilent Bioanalyzer
2100 system. cDNAs were prepared using iScript cDNA
synthesis kit (Bio-Rad, Hercules, CA, USA) as per the man-
ufacturers instructions. The samples were amplified by real-
CA, USA) on a Bio-Rad CFX96 C1000 Thermal Cycler using
15 seconds at 95∘C and 1 minute at 60∘C. Negative control
RNA samples were not reverse transcribed or did not lack
(Bio-Rad Hercules, CA, USA), and changes in expression,
reverse, 5耠to 3耠) were as follows: 훽-Actin: AGCCTCGCC-
TTTCCGTAACCTCAAG; PRKCA: GCTTCCAGTGCC-
AAGTTTGC and GCACCCGGACAAGAAAAAGTAA;
LTF: ATGGTGGTTTCATATACGAGGCA and GCCACG-
AGGGAAA and GTCTGTCTCCGCTTGGAGTGTAT; pS2
(TFF1): AGGCCCAGACAGAGACGTGTAC and CGT-
CGAAACAGCAGCCCTTA. Primers were designed using
Primer3 software (http://primer3.wi.mit.edu) and obtained
from Eurofins MWG Operon (Huntsville, AL, USA) or
Integrated DNA Technologies (Coralville, IA, USA).
2.5. Statistical Analysis. Data were expressed as mean ±
the following conditions: 10 minutes at 95∘C and 40 cycles of
relative to 훽-actin and rpl13a controls, were estimated using
TTTGCCGA and GCGCGGCGATATCATCATC; RPL13A:
TACCAGAAAGTTTGCTTACGTGGG and TGCCTG-
the ΔCT method . Primer pair sequences (forward and
standard deviation or standard error of the mean and ana-
lyzed using Student’s 푡-test. 푃 values <0.05 were considered
[21, 27]. As shown in Figure 1, expression of myrPKC훼, in
of a canonical estrogen response element (ERE) fused to
in the regulation of ER-dependent transcription, Ishikawa
endometrial cancer cells were transiently transfected with a
myristoylated PKC훼 construct (myrPKC훼) that is targeted
the absence of estrogen, resulted in a dose-dependent acti-
vation of transcription from a promoter containing 3 copies
to membranes and thereby rendered constitutively active
of an ERE and potentiated the effect of estrogen. Similar
results were obtained using the pS2 (TFF1) promoter, an
stimulated ERE promoter activity was further increased over
endogenous E2 regulated gene  (Figure 2(b)). myrPKC훼
expression induced a marked increase in basal pS2 promoter
Obstetrics and Gynecology International3
are mean ± s.d. (푛 = 3).
activityand enhanced the stimulatoryeffect of E2. Treatment
cells were transiently transfected with 0.5휇g pEREluc, 0.3휇g
and promoter activity expressed as fold increase over control. Data
(pCDNA3). Luciferase activity was normalized to 훽-galactosidase
with E2 had no effect on the level of myrPKC훼 expression in
receptor (ER) , activity of the ERE and pS2 promoters
Ishikawa cells (not shown).
In HEC-50 endometrial cancer cells, which lack estrogen
ment with E2 (in the presence or absence of myrPKC훼) had
(Figure 3). Accordingly, transfection of HEC-50 cells with
naling induces ligand-independent activation of ER-depen-
dent transcription and thereby potentiates responses to E2.
Activation of the phosphoinositide 3-kinase (PI3K)/Akt
pathway is one of the most critical steps in endometrial
carcinogenesis  and has been shown to mediate ligand-
naling in PKC훼 regulation of transcription, we treated
on the ERE promoter (Figure 5). Treatment of Ishikawa cells
with LY29004 or Akt-I-1/2 significantly inhibited the ability
transfected with ER훼 (Figure 5(b)). LY29004 and Akt-I-1/2
was minimal (Figure 3). Expression of active PKC훼 or treat-
pHEGO encoding ER훼 reconstituted ERE and pS2 tran-
no effect on pS2 or ERE promoter activity, indicating that
the effects of PKC훼 and E2 are dependent on ER expression
scriptional responses to both E2 and myrPKC훼 (Figure 4).
Together, these results (Figures 1–4) indicate that PKC훼 sig-
Ishikawa cells with pharmacological inhibitors of PI3K (LY
29004) or Akt (Akt-I-1/2) [35, 36] and examined their effects
of myrPKC훼 to enhance E2 activation of the ERE promoter
treatment resulted in the expected decrease in phosphoryla-
tion of Akt and GSK3, respectively, and did not impact
trial cancer cells . To investigate the role of PI3K/Akt sig-
(Figure 5(a)). Similar results were obtained in HEC-50 cells
expression of myrPKC훼 (not shown). Thus, the effects of
To confirm the results, using the ERE and pS2 promoter
constructs, we examined expression of a panel of estrogen-
dependent genes implicated in endometrial neoplastic trans-
formation [33, 34]. Levels of pS2 (TFF1), lactotransferrin
(Ltf), and c-fos mRNA were determined by real-time reverse
knockdown of PKC훼 in Ishikawa cells significantly reduced
[9, 37–39]. We therefore determined the effect of PKC훼
control shRNA targeting luciferase, reflected by an increase
in the number of viable cells (Figure 7). Knockdown of
PKC훼 on E2- and ER-dependent transcription are mediated,
in part, by the PI3K/Akt pathway.
to knockdown PKC훼. Control cells were transduced with
expression of the estrogen-dependent genes pS2, ltf, and c-
shRNA targeting luciferase . As shown in Figure 6,
cer growth and stimulates proliferation of Ishikawa cells
decrease in PKC훼 mRNA levels (Figure 6).
Estrogen is a critical regulator of type I endometrial can-
knockdown on estrogen-dependent proliferation. E2 treat-
ment stimulated proliferation of Ishikawa cells expressing a
cantly different between cell lines and was not affected by E2
number at 72h and essentially abrogated the E2 proliferative
response at 144h. Cell viability (89%–96%) was not signifi-
Together, these results indicate that PKC훼 is a critical
positive endometrial cancer cells.
regulator of ER-dependent gene expression and modulates
both E2-stimulated transcription and cell proliferation in ER
Estrogen, acting through ER, is a major contributor to endo-
metrial proliferation. Indeed, hormone-dependent, type
I endometrial cancers are thought to arise due to excess
mitogenesis, atypical hyperplasia, and the transition to
malignant adenocarcinoma [4, 8, 11]. In this study, we have
the effects of estrogen on both gene expression and endo-
estrogenic environment, resulting in estrogen-independent
activation of ER-dependent transcription and potentiating
metrial cancer cell proliferation. The primary effect of PKC훼
enhancing levels of genes linked to endometrial hyperplasia
is to stimulate basal, unliganded ER transactivation, thereby
pS2/TFF1, and c-fos) implicated in proliferation of normal
and transformed endometrial cells and linked to the devel-
opment of endometrial carcinoma [11, 33, 34, 40, 41]. Knock-
by both ER and PKC훼. Accordingly, treatment of breast
of a subset of estrogen responsive genes (lactotransferrin,
down of PKC훼 in endometrial cancer cells reduced expres-
4Obstetrics and Gynecology International
Figure 2: PKC훼 enhances ER-dependent promoter activity. Ishikawa cells were transiently transfected with (a) 0.5휇g pEREluc or (b) 0.5휇g
pPS2luc and 0.3휇g pCMV훽 in the presence or absence of 0.5휇g pmyrPKC훼 or vector control (pCDNA3). Cells were treated with ±100nM
Data are mean ± s.e.m of 6 experiments conducted in triplicate.
Fold activation (RLU)
normalized to 훽-galactosidase and promoter activity expressed as
cells, lacking ER, were transiently transfected with 0.5휇g pEREluc
Relative Light Units (RLU). Data are mean ± s.e.m of 4 experiments
or 0.5휇g pPS2luc and 0.3휇g pCMV훽 in the presence or absence of
0.5휇g pmyrPKC훼 or vector control (pCDNA3). Cells were treated
with ±100nM estradiol (E2), as indicated. Luciferase activity was
conducted in triplicate.
and endometrial cancer cells with phorbol esters, to activate
PKC, has been shown to induce expression of pS2 and c-fos
and augment their increased levels observed in response to
estrogen treatment [41–43].
Cyclin D1 is also an important mediator of estrogen-
dependent endometrial cell proliferation and is over
expressed in endometrioid tumors [9, 37]. Consistent with
interaction of E2 and PKC훼 mitogenic signaling pathways,
expression of the cyclin-dependent kinase (CDK) inhibitor
p21 is decreased in endometrial cancers, correlating with
poorer prognosis [44, 45]. Estrogen-induced Ishikawa cell
proliferation paralleled a decline in p21 protein expression
, whilst progesterone mediated growth inhibition was
linked to elevated p21 levels . Expression of p21 was
we previously demonstrated that PKC훼 activates the cyclin
D1 promoter in endometrial cancer cells . In addition,
also upregulated in response to knockdown of PKC훼 ,
cancer cell proliferation.
The PI3K/Akt pathway is commonly dysregulated in
type I endometrial cancers. More than 80% of endometrioid
activating mutations in PI3K [47–49]. PTEN heterozygous
mice develop endometrial hyperplasia and adenocarcinoma,
characteristic of human endometrioid tumors [11, 33, 34].
Endometrial tumorigenesis in this model is associated with
upregulation of estrogen-stimulated gene expression and
ligand-independent activation of ER , mediated by Akt
suggesting that the CDK inhibitor is a target of both PKC훼
and estrogen signaling pathways, regulating endometrial
. Consistentwiththeseresults,we haveshown thatPKC훼
Obstetrics and Gynecology International5
Figure 4: Reconstitution of PKC훼 regulated, ER-dependent transcription in HEC-50 cells. Cells were transiently transfected with 0.5휇g
pHEGO (ER훼) and (a) 0.5휇g pEREluc or (b) 0.5휇g pPS2luc and 0.3휇g pCMV훽 in the presence or absence of 0.5휇g pmyrPKC훼 or vector
control (pCDNA3). Cells were treated with ±100nM estradiol (E2), as indicated. Promoter activity was determined as in Figure 2. Data are
mean ± s.e.m of 6 experiments conducted in triplicate.
E2 + PKC훼
E2 + PKC훼
Figure 5: PKC훼 effects on ER-dependent transcription are mediated by the PI3-kinase/Akt pathway. (a) Ishikawa cells were transiently
∗푃 < 0.05.
is required to maintain Akt activity in endometrial cancer
cells  and that amplification of estrogen/ER mediated
transfected with 0.5휇g pEREluc and 0.3휇g pCMV훽, in the presence or absence of 0.5휇g pmyrPKC훼. (b) HEC-50 cells were transiently
wasdeterminedasinFigure 2andexpressedasfoldincreaseovertheappropriateinhibitorordiluentcontrol.Resultsaremean±s.d.(푛 = 6).
way (Figure 5).
Phosphorylation of ER has been implicated in regulation
phorylation of serine 167, by Akt, induces activation of ER
, and phosphorylation of serines 104, 106, and 118
6Obstetrics and Gynecology International
Figure 6: Knockdown of PKC훼 reduces ER-dependent gene expression. Ishikawa cells were stably transduced with shRNAs targeting PKC훼
or luciferase (luc). RNA was isolated and analyzed by real time reverse transcription PCR, using primers specific for the indicated gene, as
(푛 = 6).
described in Section 2. ΔCt values were calculated relative to a control gene (rp13) and relative levels expressed as 2Δct. Data are mean ± s.e.m
Fold E2 stimulation
of Ishikawa cells. Cells were stably transfected with shRNAs tar-
lines were treated with ±100nM estrogen (E2) and harvested at the
as the fold increase in cell number induced by estrogen treatment.
modulates ER interaction with co activators . PKC훼-
geting PKC훼 or luciferase. Control (luc) or PKC훼 knockdown cell
using a Beckmann Coulter Vi-CELL analyzer. Results are expressed
Data are mean ± s.e.m (푛 = 6).∗푃 ≤ 0.05.
dependent ER phosphorylation and its functional role in
endometrial cancer cells remain to be established; however,
indicated time points. Cell number and viability were determined
these latter sites match the consensus substrate sequence for
the effects of PKC훼 may be mediated by direct or indirect
phosphorylation of ER.
In summary, we have shown that activation of PKC훼 induces
Thus, PKC훼-dependent signal transduction is a critical com-
development of endometrial hyperplasia and endometrioid
adenocarcinoma. Furthermore, estrogen exposure may
estrogen-independent activation of ER-dependent gene
expression and potentiates the effects of estrogen on trans-
cription. Evidence also implicates PKC훼 in the regulation of
ponent of the environment of excessive estrogen and supra-
increase PKC훼 expression and/or activity in endometrial
The incidence of endometrial cancer continues to rise,
survival has not significantly improved [54–56]. Thus, there
estrogen-dependent endometrial cancer cell proliferation.
cancer cells [17, 18, 53], providing a potential positive feed-
back loop to amplify estrogen and ER-dependent responses.
progression of endometrial carcinogenesis. PKC훼 specific
growth factor/ERK-dependent signal transduction pathways
regulating the growth of type I tumors [20–22]. Hence,
inhibition of PKC훼-dependent signaling would enable the
simultaneous targeting of multiple estrogen-dependent and
-independent pathways implicated in the development and
Obstetrics and Gynecology International7
Conflict of Interests
The authors have no conflict of interests.
This paper is funded by the Cancer League of Colorado, Uni-
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