?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 5 May 2010
Adrenergic modulation of focal
adhesion kinase protects human
ovarian cancer cells from anoikis
Anil K. Sood,1,2,3 Guillermo N. Armaiz-Pena,1 Jyotsnabaran Halder,1 Alpa M. Nick,1
Rebecca L. Stone,1 Wei Hu,1 Amy R. Carroll,1 Whitney A. Spannuth,1
Michael T. Deavers,4 Julie K. Allen,1 Liz Y. Han,1 Aparna A. Kamat,1 Mian M.K. Shahzad,1
Bradley W. McIntyre,5 Claudia M. Diaz-Montero,5 Nicholas B. Jennings,1 Yvonne G. Lin,1
William M. Merritt,1 Koen DeGeest,6 Pablo E. Vivas-Mejia,7 Gabriel Lopez-Berestein,2,3,7
Michael D. Schaller,8 Steven W. Cole,9 and Susan K. Lutgendorf6,10
1Department of Gynecologic Oncology, 2Department of Cancer Biology, 3Center for RNA Interference and Non-Coding RNA,
4Department of Pathology, 5Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
6Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, University of Iowa, Iowa City, Iowa, USA.
7Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, Texas, USA. 8Department of Biochemistry,
West Virginia University, Morgantown, West Virginia, USA. 9Department of Medical Oncology Hematology, UCLA, Los Angeles, California, USA.
10Departments of Psychology and Urology and Holden Comprehensive Cancer Center, University of Iowa, Iowa City, Iowa, USA.
There is growing evidence supporting the role of stress biology in
cardiovascular (1), cancer (2, 3), and other disease states. Several
epidemiological and experimental studies have suggested that
behavioral stress factors may accelerate growth of existing tumors
(4); however, the underlying mechanisms are not fully understood.
Much research has suggested that neuroendocrine stress media-
tors might enhance cancer pathogenesis by inhibiting antitumor
immune responses (5), and we recently demonstrated that sym-
pathetic nervous system (SNS) activity can also directly enhance
the pathogenesis of ovarian carcinoma by upregulating angiogenic
pathways in the tumor microenvironment (6). The latter effects
were mediated through activation of tumor cell β2-adrenergic
receptors (ADRB2) and the associated cAMP/PKA signaling path-
way. Additional signaling and pathogenetic pathways may also be
involved but are not fully understood at present.
Based on the effects of some neuropeptides, such as bombesin,
on focal adhesion kinase (FAK) (7), we considered whether FAK
might be involved in the tumor-promoting effects of chronic
stress. FAK is a non–receptor protein tyrosine kinase that local-
izes to focal adhesions (8) and mediates physical attachment of
cells to their ECM (9). It is not known whether stress hormones
such as norepinephrine and epinephrine can activate FAK or what
functional significance those dynamics might have for tumor cell
survival. Normal cells enter apoptosis when separated from ECM
and neighboring cells (a process known as anoikis) (10). In the
present study, we sought to determine whether chronic stress and
associated neuroendocrine dynamics could affect anoikis in ovar-
ian cancer cells, and if so, what signaling pathways might mediate
these effects. Analysis of cellular models and an orthotopic mouse
model of human ovarian cancer showed that catecholamines can
protect ovarian cancer cells from anoikis and these effects are
mediated by FAK phosphorylation through ADRB2-dependent
activation of Src. Parallel results were observed in human ovar-
ian cancer in vivo, linking increased levels of stress/depression to
increased FAK activation and showing accelerated disease progres-
sion in patients with high levels of FAK activity.
Catecholamines protect tumor cells from anoikis. Tumor cells develop
resistance to anoikis, which allows for their survival during the
process of metastasis. FAK is known to promote tumor cell surviv-
al and may play a significant role in avoidance of anoikis (11–13).
We have previously demonstrated that norepinephrine promotes
ovarian cancer growth via stimulation of angiogenic pathways.
To determine whether norepinephrine and epinephrine might
also inhibit anoikis via FAK activation, we analyzed ovarian can-
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article:?J Clin Invest. 2010;120(5):1515–1523. doi:10.1172/JCI40802.
1516?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 5 May 2010
cer cells maintained in poly-HEMA–coated tissue culture plates,
which allows for anchorage-independent growth. These cells grew
predominantly as a single-cell suspension, with 48-hour anoikis
rates of 23.0% for SKOV3 cells and 51.3% for EG cells and 72-hour
anoikis rates of 62.0% and 72.6%, respectively (Figure 1A). How-
ever, exposure to either epinephrine or norepinephrine resulted in
significant inhibition of anoikis (Figure 1, A and B).
FAK activation is known to play a role in protecting cells from
anoikis, and we have previously demonstrated that ovarian cancer
cells express higher levels of FAK compared with nontransformed
epithelial ovarian cells (14). To determine whether norepineph-
rine-mediated inhibition of anoikis might be mediated by FAK
activation, we examined phosphorylation of all FAK tyrosine sites
after treating SKOV3 cells with norepinephrine. Increased phos-
phorylation at pFAKY397 was noted in response to norepinephrine
treatment, but no significant changes were noted at the other
sites (Supplemental Figure 1; available online with this article;
doi:10.1172/JCI40802DS1). Similar effects were observed in EG
cells (Figure 2A) and after epinephrine treatment (Figure 2A), and
subsequent experiments therefore focused on pFAKY397. Norepi-
nephrine-induced FAK tyrosine phosphorylation was detectable
within 10 minutes and peaked at 30 minutes (Supplemental
Figure 2, A and B). Similar effects occurred after the loss of cell
attachment, with both SKOV3ip1 and EG cells showing norepi-
nephrine-induced FAKY397 phosphorylation even when cells were
maintained in suspension (Figure 2B). Immunofluorescence
analyses verified that norepinephrine induced a dose-dependent
increase in pFAKY397, localized specifically to focal adhesions in
SKOV3ip1 cells (Figure 2C).
To confirm that FAK mediated the effects of norepinephrine
on anoikis, we used siRNA to suppress FAK protein levels by 80%
(Supplemental Figure 3). Norepinephrine continued to inhibit
anoikis in ovarian cancer cells treated with a control siRNA, but
FAK siRNA completely abrogated the effect of norepinephrine
on ovarian cancer cell survival under anchorage-independent
conditions (Figure 2D).
FAK activation by catecholamines is dependent on ADRB and Src. Our
previous studies have shown that norepinephrine’s effects on
ovarian cancer cell production of angiogenic factors are medi-
ated specifically by tumor cell β-adrenergic receptors (6, 15). To
identify the receptor subtype mediating norepinephrine effects
on FAK activation and anoikis, we examined the effects of both
α- and β-antagonists. The nonspecific beta blocker propranolol
(1 μM) blocked norepinephrine-induced FAK activation in both
SKOV3ip1 (Figure 3A) and EG (Figure 3B) cells. Propranolol also
blocked norepinephrine’s effects on anchorage-independent apop-
tosis (Figure 3C), which were similar to the effects observed with
FAK siRNA. The ADRB1-specific inhibitor atenolol had minimal
effects on norepinephrine-induced pFAKY397, whereas the ADRB2-
specific blocker butoxamine (1 μM) abrogated the effects of nor-
epinephrine (Figure 3, A and B). The beta blockers had no effect
on FAK or pFAKY397 levels in the absence of norepinephrine (Sup-
plemental Figure 4). Similar effects were observed using siRNA to
inhibit expression and downstream signaling of specific β-adren-
ergic receptors (Supplemental Figure 5, A and B). ADRB2-targeted
siRNA blocked the effects of norepinephrine on FAK activation,
but ADRB1-targeted siRNA had no effect (Figure 3D). α-Adrener-
gic blockers had no effect on norepinephrine-mediated FAK activa-
tion (Supplemental Figure 6). Consistent with the effects on FAK
activation, propranolol and ADRB2-siRNA reversed the protective
effects of norepinephrine on anoikis (Figure 3C), but inhibition
of other adrenergic receptor subtypes had no effect (Supplemen-
tal Figures 7 and 8). Consistent with the role of ADRB2, norepi-
nephrine-mediated protection against anoikis was not observed
in the ADRB2-null A2780-PAR and RMG-II cells (Supplemental
Figure 9A). To further support the role of ADRB2 in mediating the
effects of norepinephrine on anoikis, we transfected either ADRB2
(RMG-II-ADRB2) or empty vector (RMG-II-neo) into the RMG-II
cells. The RMG-II-ADRB2 cells had significantly lower rates of
anoikis compared with the controls upon stimulation with iso-
proterenol (Supplemental Figure 9B).
The major target of norepinephrine-induced FAK phosphoryla-
tion, Y397, is a high-affinity binding site for the SH2 domain of
Src (16). To determine whether Src could directly induce FAKY397
phosphorylation, we performed in vitro kinase assays. In these
experiments, Src induced phosphorylation of either FAK or a
kinase-dead FAK (i.e., mutation at K454M, resulting in alteration
of the ATP-binding site; Supplemental Figure 10). Moreover, the
Src inhibitor AP23846 prevented Src-induced FAKY397 phosphory-
lation, further demonstrating the effect of Src. β-Adrenergic acti-
vation of G proteins can directly stimulate Src activation (17), so
we pretreated ovarian cancer cells with the Src family kinase inhib-
itor PP2 (18) (or its inactive congener PP3) for 30 minutes prior
to norepinephrine exposure. PP2 (10 μM) completely blocked
the norepinephrine-mediated increases in pFAKY397 (Figure 4A),
whereas pretreatment with PP3 had no effect (Figure 4A). Similar
experiments were also performed with Src siRNA and demonstrat-
ed abrogation of norepinephrine-mediated FAKY397 phosphoryla-
tion after Src gene silencing (Figure 4A). We also examined cells
null for Src, Yes, and Fyn (SYF), and norepinephrine failed to
stimulate FAK phosphorylation in these cells at Y397 (Figure 4B)
or any other site (Supplemental Figure 11). Moreover, when Src
was transiently introduced, norepinephrine treatment resulted in
FAKY397 phosphorylation (Figure 4B).
To determine whether norepinephrine enhanced direct inter-
actions between Src and FAK, we performed coimmunoprecipi-
tation assays of SKOV3ip1 cell lysates obtained 10 minutes after
norepinephrine exposure. Immunoprecipitation of Src followed
by anti-FAK Western blot analysis revealed a direct association
Effect of (A) 10 μM norepinephrine or (B) 10 μM epinephrine on anoi-
kis in ovarian cancer cells. Gray bars, vehicle treatment; black bars,
norepinephrine (A) or epinephrine (B) treatment. Results represent the
mean ± SEM. *P < 0.05.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 5 May 2010
between the 2 molecules that was significantly increased by nor-
epinephrine (Figure 4C). Parallel effects were observed when FAK
immunoprecipitates were assayed for Src (Figure 4C).
Norepinephrine is also known to increase intracellular calcium
levels (19, 20), which can lead to phosphorylation of tyrosine
kinases. To determine whether the stimulation of FAK phosphor-
ylation by norepinephrine was mediated by intracellular Ca2+, we
treated cells with the Ca2+-ATPase inhibitor thapsigargin for 30
minutes prior to norepinephrine exposure. Thapsigargin abol-
ished norepinephrine-induced calcium flux (data not shown), but
it did not block norepinephrine’s effects on FAK-Src complex for-
mation or FAKY397 phosphorylation (Figure 4D). Similar effects
were observed when extracellular calcium was chelated with EGTA
(Figure 4D). To determine whether norepinephrine-induced FAK
activation required an intact actin skeleton, we exposed ovarian
cancer cells to the actin polymerization inhibitor cytochalasin D
for 2 hours and then stimulated them with norepinephrine (21).
Cytochalasin D substantially diminished the norepinephrine-
induced increase in pFAKY397 (Figure 4E).
Effect of FAK silencing on norepinephrine-mediated tumor growth. To
assess the effects of catecholamine-induced FAK activation on in
vivo tumor growth, we analyzed the effects of restraint stress on
the growth and anoikis of ascites-producing 2774 ovarian cancer
cells implanted into the peritoneal cavity of nude mice. Animals
exposed to 2 hours of daily restraint stress had a significantly lower
rate of tumor cell apoptosis in ascites (P ≤ 0.01; Figure 5A), indi-
cating lower levels of anoikis. Similar effects were observed with
the β-agonist isoproterenol (P ≤ 0.01; Figure 5B), and those effects
were blocked by propranolol. Similar findings were noted with
the SKOV3ip1 model (data not shown). Both chronic stress and
isoproterenol also increased phosphorylation of FAKY397 in tumor
cells, and these effects were blocked by propranolol (Supplemental
Effect of catecholamines on FAK activation. (A) SKOV3ip1 or EG cells were plated and treated with either norepinephrine (NE) or epinephrine
(Epi) and then subjected to Western blot analysis for FAK or pFAKY397. (B) SKOV3ip1 cells kept in suspension were treated with norepinephrine,
followed by Western blot for FAK and pFAKY397. (C) SKOV3ip1 cells were treated with norepinephrine, then subjected to immunofluorescence
analysis for pFAKY397 and actin. Quantification of pFAKY397 staining is shown in the graph. Scale bars: 10 μm. (D) Effect of 10 μM norepinephrine
with or without FAK silencing with siRNA on anoikis. Results represent the mean ± SEM. *P ≤ 0.01.
1518?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 5 May 2010
Figure 12 and data not shown). Next, to confirm the functional
role of FAK activation as a mediator of stress-induced protection
from anoikis, we delivered human FAK-specific siRNA (Supple-
mental Figure 13) in vivo by incorporation into 1,2-dioleoyl-sn-
glycero-3-phosphatidylcholine (DOPC) nanoliposomes (22, 23).
Control siRNA-DOPC had no effect on SKOV3ip1 tumor growth
(Figure 5C), but FAK-specific siRNA-DOPC completely blocked
stress-induced increases in tumor growth. Similar effects of FAK
silencing were observed in the HeyA8 model (Figure 5D). In these
experiments, FAK siRNA-DOPC also blocked the protective effects
of stress on tumor cell apoptosis (Figure 5E). We also performed
similar experiments with the 2774 model to examine effects of
propranolol (Figure 5F) or FAK siRNA-DOPC (Figure 5F) on
tumor cell apoptosis in ascites as a measure of anoikis. Treatment
with either propranolol or FAK siRNA-DOPC completely blocked
the protective effects of daily restraint stress or isoproterenol on
tumor cell apoptosis in ascites. On the basis of our in vitro find-
ings regarding the role of Src in stress-mediated pFAKY397 activa-
tion, we examined the effects of Src silencing using the restraint
stress model. Mice bearing SKOV3ip1 tumors were exposed to
daily restraint stress and treated with either control siRNA-DOPC
or Src siRNA-DOPC. In comparison to the controls, tumors from
Src siRNA–treated animals had significantly lower pFAKY397 levels
(Supplemental Figure 14).
FAK expression in human ovarian carcinoma. To assess the signifi-
cance of stress-induced FAK activation for human clinical cancer,
we examined FAK activity in 80 cases of invasive epithelial ovarian
cancer. Consistent with our previous data (11, 14, 24), increased
FAK expression was noted in 67% of the tumors. Increased levels
of pFAKY397 were observed in 50% of tumors (Figure 6A), and both
increased FAK expression and increased FAKY397 phosphorylation
were associated with poor overall patient survival (Figure 6B).
The pFAKY397 scores were normalized to total FAK scores, and
high expression of normalized pFAKY397 remained significantly
associated with poor overall survival (Supplemental Figure 15).
To assess the role of adrenergic signaling in these relationships,
we grouped patients based on high versus low levels of depres-
sion, which has been linked to differential adrenergic signaling
(25) in ovarian cancer patients. High levels of depression (Center
for Epidemiological Studies Depression [CESD] scale ≥16) were
associated with a marginally significant increase in FAK expres-
sion (P = 0.08) and a highly significant increase in the level of
phosphorylated FAKY397 (P = 0.003; Figure 6C). We also measured
norepinephrine levels in 51 tumors and found increased FAKY397
phosphorylation (P < 0.04; Figure 6D) and marginally increased
levels of total FAK (P = 0.06; Figure 6D) in tumors with above-
median norepinephrine content (>0.84 pg/mg). The box plots of
FAK and pFAKY397 scores based on CESD and norepinephrine lev-
els are shown in Supplemental Figure 16.
The key findings from our study are that norepinephrine and
epinephrine protect ovarian cancer cells from anoikis via a FAK-
mediated signaling pathway that is initiated by ADRB2 and
involves subsequent Src-associated phosphorylation of FAKY397.
Norepinephrine-induced FAK activation was also found to play a
significant role in the effects of chronic stress on ovarian cancer
growth in vivo in an orthotopic mouse model. Additional stud-
ies of human clinical tumors showed that both depression and
tumor norepinephrine content were associated with increased
Effect of propranolol (nonspecific β-antago-
nist), atenolol (β1 antagonist), butoxamine
(β2 antagonist), or SR59230A (β3-antago-
nist) on FAK and pFAKY397 in (A) SKOV3ip1
and (B) EG cells. (C) Effect of 10 μM nor-
epinephrine with or without propranolol
or ADRB1- or ADRB2-targeted (β1 or β2)
siRNA in SKOV3ip1 cells on anoikis. (D)
Effect of β1 and β2 targeted siRNA on
pFAKY397 and FAK in SKOV3ip1 cells. In A,
B, and D, the immunoblot is shown at the top,
and quantification of band intensity relative
to total FAK intensity is shown below. For all
panels, results represent the mean ± SEM
of triplicate experiments. *P < 0.01.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 5 May 2010
FAK activation and that increased FAK activation was associated
with substantially accelerated mortality times. Thus, these stud-
ies identify norepinephrine- and epinephrine-induced FAK activa-
tion as a novel mechanism by which stress might accelerate the
pathogenesis of ovarian cancer.
FAK signaling is critical in many biological pathways, including
embryonic development (26), cell migration and invasion (14, 27,
28), proliferation (27, 29), and apoptosis (11, 24). FAK is a particu-
larly important regulator of signaling processes between the ECM
and tumor cells (30, 31). FAK phosphorylation at Y397 follows
integrin stimulation or ligand binding by growth factor receptors
(32). The present study identified the ADRB2/Src signaling axis
as a molecular pathway for inhibiting ovarian cancer anoikis via
FAKY397 phosphorylation. Avoidance of anoikis provides a selective
advantage for metastatic cancer cells to allow for transit to new
sites for attachment (33). The present studies suggest that inhibi-
tion of the ADRB2/Src/FAK pathway by beta blockade or siRNA
inhibition might provide a novel strategy for suppressing tumor
growth and metastasis in clinical settings.
All 3 major catecholamines (norepinephrine, epinephrine, and
dopamine) are known to be present in the ovary, with norepineph-
rine being the most abundant (34, 35). Ovarian norepinephrine lev-
els are substantially higher than those in circulating blood (36, 37),
and SNS activity can further enhance those levels to induce precystic
follicles (38, 39). The doses of norepinephrine used for our study
were selected to reflect the physiologic conditions of this hormone
at the level of the tissue microenvironment. Studies suggest that
within the parenchyma of the ovary, concentrations may reach as
high as 10 μmol/l (39). Although the physiological role of norepi-
nephrine and epinephrine in the ovarian tissue environment is not
yet fully known, the present data imply that these signaling mol-
ecules might contribute to disease pathogenesis in the context of an
emerging tumor. Further analysis of the roles of stress hormones in
normal ovarian physiology would provide valuable insights into the
basis for the presently observed effects in tumor cells. Particularly
important in these future studies will be defining the physiologi-
cal role of norepinephrine and epinephrine in modulating cellular
dependence on neighboring cells and ECM. It is possible that stress
hormone–mediated activation of FAK-dependent resistance to anoi-
kis might play a role in normal tissue remodeling or development.
The present study expands the scope of molecular pathways
involved in effects of stress on cancer growth and progression (4). In
addition to potential effects of stress on immune response in can-
cer (4, 5), we and others have demonstrated that stress mediators
such as norepinephrine and epinephrine from the SNS and gluco-
corticoids from the hypothalamic-pituitary-adrenal axis can directly
regulate the function of human cancer cells in ways that promote
their survival and metastasis (4, 6, 40). In the case of the SNS, these
effects are mediated by ADRBs expressed on ovarian, mammary, and
other cancer cells (15, 41, 42). Norepinephrine activation of ADRB2
has been shown to enhance tumor growth in part via induction of
VEGF-dependent angiogenesis (6). Dopamine, an important mem-
ber of the catecholamine family, appears to play an opposing role
against the angiogenic effects of VEGF but is present at reduced
levels under chronic stress conditions (43, 44). Other studies have
identified additional mechanisms by which stress might contribute
to cancer pathogenesis, including impaired DNA repair (45, 46)
and modulation of matrix metalloproteinases (47–49). The current
Mechanism of norepinephrine-mediated (NE-mediated) FAK activation. (A) SKOV3ip1 cells stimulated with 10 μM norepinephrine were treated
with either the Src inhibitor PP2 or its inactive counterpart PP3 followed by immunoblotting for FAK and pFAKY397. In addition, the effect of Src
silencing with siRNA was examined on norepinephrine-mediated FAK activation. (B) SYF-null cells transfected with either empty vector (EV) or
Src (WT Src) were stimulated with 10 μM norepinephrine, followed by immunoblotting for FAK and pFAKY397. (C) SKOV3ip1 cells treated with
10 μM norepinephrine were subjected to immunoprecipitation for Src (left) or FAK (right), followed by immunoblotting for FAK and Src. (D) Cells
stimulated with 10 μM norepinephrine were treated with thapsigargin followed by Western blot for pFAKY397 and FAK (left). Cells stimulated
with 10 μM norepinephrine were treated with EGTA followed by immunoprecipitation for FAK, then immunoblotting for FAK and Src (right). (E)
SKOV3ip1 cells stimulated with 10 μM norepinephrine were treated with cytochalasin D (Cyt D), followed by Western blot for pFAKY397 and FAK.
In A, D, and E, the immunoblot is shown at the top, and quantification of band intensity relative to total FAK intensity is shown below.
1520?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 5 May 2010
study identifies a new pathway by which stress biology can impact
tumor growth and progression via ADRB2-dependent activation of
FAK and the resulting protection of cells from anoikis. Resistance to
anoikis is a hallmark of malignant transformation, affording tumor
cells increased survival times in the absence of matrix attachment
and facilitating migration, reattachment, and colonization of sec-
ondary sites (50, 51). Overexpression of oncogenes such as ras, raf,
and src as well as the downregulation of tumor suppressor genes
such as PTEN and p53 (TP53) contribute to protection from anoikis
(52). This study identifies a novel neuroendocrine pathway by which
behavioral stress factors can exert similar effects. These findings also
imply that the neuroendocrine “macroenvironment” may play a sig-
nificant role in shaping cellular activity in the tumor microenviron-
ment in ways that ultimately facilitate cancer progression. Thus,
protective interventions targeting the neuroendocrine system might
simultaneously modulate multiple molecular pathways involved in
tumor metastasis (e.g., anoikis, angiogenesis, and invasion).
Reagents. Norepinephrine, epinephrine, propranolol, butoxamine hydro-
chloride, dobutamine, leupeptin, aprotinin, poly-HEMA, thapsigargin,
EGTA, ATP, and cytochalasin D were obtained from Sigma-Aldrich.
Effect of (A) chronic stress or (B) isoproterenol on tumor cell apoptosis in ascites as a reflection of in vivo anoikis using the 2774 model. Effects
of control siRNA-DOPC or FAK siRNA-DOPC on stress-induced in vivo (C) SKOV3ip1 or (D) HeyA8 tumor growth. (E) Effect of stress on tumor
cell apoptosis in the SKOV3ip1 model. (F) Effect of propranolol or FAK siRNA-DOPC on tumor cell apoptosis in ascites as a measure of anoikis
using the 2774 model. Results represent the mean ± SEM; n = 10 mice per group. *P < 0.01.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 5 May 2010
(RS)-Atenolol and SR59230A were purchased from Tocris Cookson Inc.
PP2, PP3, forskolin, and KT5720 were acquired from Calbiochem. Rabbit
(polyclonal) anti-FAK (pY397) phosphospecific antibody, unconjugated, was
obtained from Biosource International Inc.
Cell culture. The derivation and source of the established ovarian cancer
cell lines SKOV3ip1, HeyA8, 2774, A2780-PAR, RMG-II, and EG have been
reported previously (53). A2780 and RMG-II cells are known to be negative
for ADRB2 expression based on mRNA and protein analysis and lack of
intracellular cAMP response to norepinephrine or isoproterenol (6). These
cells were maintained and propagated in vitro by serial passage in RPMI
1640 supplemented with 15% FBS and 0.1% gentamicin sulfate (Gemini
Bioproducts). The SYF-null cells were maintained in DMEM with 10% FBS
and 0.1% gentamycin sulfate. All of the cell lines are routinely screened for
Mycoplasma species (GenProbe detection kit; Fisher). All experiments were
performed with 70%–80% confluent cultures.
Immunoprecipitation and Western blot analysis. Cells were lysed with modi-
fied 1× RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, and 0.5%
deoxycholate) containing 25 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM
sodium orthovanadate, and 2 mM EDTA. Samples were removed from cul-
ture dishes by cell scraping and centrifuged at 15,000 g for 30 minutes. The
protein concentration of the samples was determined using a bicinchoninic
acid Protein Assay Reagent kit (Pierce), and whole cell lysates were analyzed
by 7.5% SDS-PAGE and stained with Coomassie BBR-250 to ensure equal
loading (data not shown). Samples were transferred to nitrocellulose. Blots
were blocked with 5% nonfat milk at room temperature for 1 hour. Blots
were incubated with the polyclonal antibody (1:1,000 dilution; Biosource)
at room temperature, with agitation for 1 hour, followed by incubation
with a horseradish peroxidase–conjugated anti-mouse secondary antibody
(1:6,000; The Jackson Laboratory). Blots were developed using an enhanced
chemiluminescence detection kit (ECL; Pierce).
For immunoprecipitation experiments, 300 μg of cell lysate was incu-
bated with the Src antibody at 4°C for 1 hour. Protein-antibody complexes
were incubated for at 4°C for 1 hour with protein A/G plus-agarose–con-
jugated beads (Upstate). Protein-antibody complexes were collected by
centrifugation, washed 3 times with the modified RIPA buffer, and boiled
in Laemmli sample buffer. Protein was resolved in SDS-PAGE gels, and
immunoblotting was performed as described above.
Anoikis assays. Cells (5 × 105 per well) were cultured on either plastic or
poly-HEMA–treated 6-well tissue culture plates for 24–72 hours at 37°C
in a 5% CO2 atmosphere. After incubation, adherent cells were detached
with 0.5% trypsin/0.1% EDTA in PBS. Detached and suspended cells
were harvested in complete RPMI 1640 medium and centrifuged at 500 g
for 10 minutes. Pellets were washed with PBS and fixed with ice-cold
75% ethanol (v/v) overnight at 4°C. After fixation, cells were washed
Clinical significance of FAK activation in ovarian carcinoma. (A) Representative images of human ovarian tumors with low or high
immunohistochemical staining for FAK and pFAKY397. Original magnification, ×200. (B) Kaplan-Meier curves of disease-specific mortality for
patients with epithelial ovarian carcinoma based on FAK or pFAKY397 expression. The log-rank test (2-sided) was used to compare differences
between groups. Percentage of ovarian cancers with high FAK or pFAKY397 expression based on CESD scores of at least 16 (C) or tumoral
norepinephrine (NE) levels (greater versus less than median value of 0.84 pg/mg) (D).
1522?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 5 May 2010
with PBS and stained with 500 μl propidium iodide (PI) solution
(50 μg/ml in PBS) containing 25 μg/ml RNase A. Cells were incubated
at 37°C for 30 minutes and analyzed by flow cytometry on an Epics XL
flow cytometer (Coulter).
In vivo anoikis assay. Apoptotic rate of tumor cells from ascites fluid was
determined by dual staining with PI and epithelial surface antigen tagged
with FITC (ESA-FITC). Nude mice inoculated with the 2774 cells were left
undisturbed until they developed detectable ascites. At this point, mice
were divided into 2 groups, receiving either isoproterenol (10 mg/kg) or
PBS. After 5 days of treatment, ascites fluid was drawn out from the peri-
toneal cavity and rapidly centrifuged at 500 g for 10 minutes. Pellets were
washed first with a red blood cell lysis buffer and reconstituted in PBS.
Suspended cells were then incubated with ESA-FITC (1:30 dilution) for
30 minutes at room temperature. After incubation, cells were washed and
fixed overnight at 4°C with ice-cold 75% ethanol. After fixation, cells were
washed and stained with a PI solution (50 μg/ml). Cells were then incu-
bated for 30 minutes at 37°C and analyzed by flow cytometry on an Epics
XL flow cytometer (Coulter).
Kinase assays. HEK 293 cells transiently expressing Flag-tagged FAK, Flag-
tagged kinase-dead FAK, or Flag-tagged Src were individually lysed, and
the protein concentration for each sample was determined. Samples were
incubated overnight with a Flag antibody. Protein-antibody complexes
were precipitated for 1 hour using agarose-conjugated mouse second-
ary antibody. Immune complexes were washed 3 times with RIPA buffer,
1 time with Tris pH 7, and 1 time with kinase buffer with no ATP. The
kinase reaction was carried out by adding 25 μl kinase buffer containing
ATP and incubating for 30 minutes. The reaction was stopped by adding
25 μl of Laemmli sample buffer and boiling samples. Afterward, samples
were subjected to Western blot analysis.
Immunohistochemistry. Formalin-fixed, paraffin-embedded human
tumor samples were stained for total FAK and phospho-FAK (Y397). Slides
were heated overnight at 65°C, deparaffinized, and hydrated with PBS.
Antigen retrieval for total FAK was done with target solution (Dako) in
a vegetable steamer for 40 minutes. Antigen retrieval was not performed
for pFAK staining to maintain adequately low background. After PBS
wash, slides were incubated in 3% H2O2 for 12 minutes to block endog-
enous peroxidase, washed with PBS, and blocked with 5% normal horse
serum (NHS) for 30 minutes at room temperature. Slides were then
incubated with primary antibody at 1:25 overnight at 4°C. After PBS
wash and additional blocking with 5% NHS, slides were incubated with
anti-mouse IgG secondary antibody (MACH4 kit, BioCare Medical) for
20 minutes at room temperature, washed in PBS, and incubated with
polymer-linked horseradish peroxidase (MACH4 kit, BioCare Medical)
for 20 minutes at room temperature. Slides were again washed and incu-
bated with 3,3′-diaminobenzidine (DAB; Phoenix Biotechnologies) for
10 minutes at room temperature to visualize antibody staining. Slides
were counterstained with Gill No. 3 hematoxylin (Sigma-Aldrich) for 15
seconds and washed with distilled water. All samples were reviewed by
a gynecologic pathologist who was blinded to the clinical outcome of
the patients. FAK or pFAKY397 expression was determined by assessing
semiquantitatively the percentage of stained tumor cells and the staining
intensity, as described previously (14). Briefly, the percentage of positive
cells and staining intensity were rated on a scale of 1 to 4; points for
expression and percentage of positive cells were added, and an overall
score (OS) was assigned. Tumors with an OS in the upper tertile were
considered as having protein overexpression. The stained slides were also
scored on the basis of the histochemical score (with a score >100 defined
as high expression and ≤100 as low expression), according to the method
described by McCarty et al., which considers both the intensity of stain-
ing and the percentage of cells stained (54–56).
Immunohistochemical staining of orthotopic tumors from therapy
experiments was performed in a similar manner, with the exception of
protein blocking. Nonspecific epitopes were blocked in fragment block
(1:10; Jackson ImmunoResearch Laboratories) in 5% NHS for 5 hours
at room temperature, followed by incubation with primary antibody at
Patient samples. Human epithelial ovarian cancer specimens were obtained
from women enrolled in a prospective study, as described previously
(57). The tumor samples were collected after consent was obtained from
patients. This research was approved by the Institutional Review Boards
of the University of Iowa and University of Texas MD Anderson Cancer
Center. Among the 80 tumor samples, 75 (94%) were of serous histology
and 5 (6%) were endometrioid or mucinous.
Behavioral measures. Patients completed psychosocial questionnaires
between their initial preoperative appointment and surgery. The CESD
scale is a 20-item measure designed to assess depressive symptomatology
and has frequently been used in studies of cancer patients (25). Scores of
16 or higher are associated with clinical depression.
Assessment of tumor norepinephrine levels. Norepinephrine levels in tumor
extracts were determined by high-performance liquid chromatography
with electrochemical detection (HPLC-ED) as previously described (25).
Chromatograph peak areas for norepinephrine were compared with the
average peak areas determined from the injection of 100 pg pure standard
and corrected for extract dilutions and tissue wet weights.
siRNA preparation. We purchased siRNAs targeted against human FAK (tar-
get sequence 5′-AACCACCTGGGCCAGTATTAT-3′) or Src (target sequence
5′-GGCTGAGGAGTGGTATTTT-3′) from QIAGEN and incorporated them
into a neutral liposome (DOPC), as previously described (22, 23).
In vivo model of chronic stress. We obtained female athymic nude mice from
the National Cancer Institute. All experiments were approved by the Insti-
tutional Animal Care and Use Committee of the University of Texas MD
Anderson Cancer Center. For the chronic stress model, we used a physi-
cal restraint system previously used by our group (6). In our model, mice
(n = 10 per group) were injected intraperitoneally with ovarian cancer
cells 7 days after starting stress. Treatment with siRNA designed against
human FAK began 3 days later and continued for 3 weeks. Mice were nec-
ropsied 21 days after tumor cell injection, and tumors were harvested for
immunohistochemistry and Western blot analysis.
Statistics. We compared continuous variables with either 2-tailed Stu-
dent’s t test or ANOVA and compared categorical variables with the χ2 test.
We used nonparametric tests (Mann-Whitney U test), if appropriate, to
compare differences. Kaplan-Meier survival curves and the log-rank test
were used to examine the effects on patient disease-specific survival. A
P value less than 0.05 was considered statistically significant. All statistical
analyses were performed using SPSS version 12 for Windows statistical
software (SPSS Inc.).
The authors thank Gary E. Gallick, Mien-Chie Hung, and Rob-
ert R. Langley for helpful discussions and guidance. We thank
Donna Reynolds for assistance with immunohistochemistry.
G.N. Armaiz-Pena was supported by the NCI F31CA126474 fel-
lowship for minority students. A.M. Nick, A.R. Carroll, R.L. Stone,
W.A. Spannuth, Y.G. Lin, and W.M. Merritt are supported by the
NCI T32 Training Grant (CA101642). This research was funded in
part by support from NIH grants (CA110793 and CA109298), the
University of Texas MD Anderson Cancer Center Ovarian Cancer
Spore (P50 CA083639), the Zarrow Foundation, the EIF Founda-
tion, the Betty Ann Asche Murray Distinguished Professorship, the
Blanton-Davis Ovarian Cancer Research Program, and the Marcus
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 5 May 2010
Foundation to A.K. Sood; NIH grant CA-104825 to S.K. Lutgen-
dorf; and NIH grant A152737 to S.W. Cole.
Received for publication August 12, 2009, and accepted in revised
form February 3, 2010.
Address correspondence to: Anil K. Sood, Departments of Gyne-
cologic Oncology and Cancer Biology, UT MD Anderson Cancer
Center, 1155 Herman Pressler, Unit 1362, Houston, Texas 77030,
USA. Phone: 713.745.5266; Fax: 713.792.7586; E-mail: asood@
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