Raf Kinase Inhibitor Protein (RKIP) Blocks Signal Transducer and ...€¦ · Raf Kinase Inhibitor...

Raf Kinase Inhibitor Protein (RKIP) Blocks SignalTransducer and Activator of Transcription 3 (STAT3)Activation in Breast and Prostate CancerSaad Yousuf1., MeiLi Duan1,2., Erika L. Moen1, Sam Cross-Knorr1, Kate Brilliant1, Benjamin Bonavida3,

Theresa LaValle4, Kam C. Yeung5, Fahd Al-Mulla6, Eugene Chin7, Devasis Chatterjee1*

1 Department of Medicine, Rhode Island Hospital and The Alpert Medical School of Brown University, Providence, Rhode Island, United States of America, 2 Department of

Critical Care Medicine, Beijing Friendship Hospital, Capital Medical University, Beijing, China, 3 Department of Microbiology, Immunology and Molecular Genetics,

University of California Los Angeles, Los Angeles, California, United States of America, 4 Kolltan Pharmaceuticals, Inc., New Haven, Connecticut, United States of America,

5 Department of Biochemistry and Cancer Biology, University of Toledo, College of Medicine, Toledo, Ohio, United States of America, 6 Department of Pathology, Faculty

of Medicine, Kuwait University, Safat, Kuwait, 7 Department of Surgical Research, Rhode Island Hospital and The Alpert Medical School of Brown University, Providence,

Rhode Island, United States of America

Abstract

Raf kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine-binding-protein (PEBP) family thatmodulates the action of many kinases involved in cellular growth, apoptosis, epithelial to mesenchymal transition, motility,invasion and metastasis. Previously, we described an inverse association between RKIP and signal transducers and activatorsof transcription 3 (STAT3) expression in gastric adenocarcinoma patients. In this study, we elucidated the mechanism bywhich RKIP regulates STAT3 activity in breast and prostate cancer cell lines. RKIP over expression inhibited c-Src auto-phosphorylation and activation, as well as IL-6-, JAK1 and 2-, and activated Raf-mediated STAT3 tyrosine and serinephosphorylation and subsequent activation. In MDA-231 breast cancer cells that stably over express RKIP, IL-6 treatmentblocked STAT3 phosphorylation and transcriptional activation. Conversely, in RKIP knockdown MDA-231 cells: STAT3phosphorylation and activation increased in comparison to parental MDA-231 cells. RKIP over expression resulted inconstitutive physical interaction with STAT3 and blocked c-Src and STAT3 association. The treatment of DU145 prostate, butnot PC3 prostate or MDA-231 breast, cancer cell lines with ENMD-1198 or MKC-1 dramatically increased expression of RKIP.Overexpression of RKIP sensitized PC3 and MDA-231 cells to MTI-induced apoptosis. Moreover, MTI treatment resulted in adecrease in Src-mediated STAT3 tyrosine phosphorylation and activation, an effect that was significantly enhanced by RKIPover expression. In stable RKIP over expressing MDA-231 cells, tumor xenograft growth induced by activated STAT3 isinhibited. RKIP synergizes with MTIs to induce apoptosis and inhibit STAT3 activation of breast and prostate cancer cells.RKIP plays a critical role in opposing the effects of pro-oncogenic STAT3 activation.

Citation: Yousuf S, Duan M, Moen EL, Cross-Knorr S, Brilliant K, et al. (2014) Raf Kinase Inhibitor Protein (RKIP) Blocks Signal Transducer and Activator ofTranscription 3 (STAT3) Activation in Breast and Prostate Cancer. PLoS ONE 9(3): e92478. doi:10.1371/journal.pone.0092478

Editor: Lucia R. Languino, Thomas Jefferson University, United States of America

Received May 20, 2013; Accepted February 23, 2014; Published March 21, 2014

Copyright: � 2014 Yousuf et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the U.S. Army Medical Breast Cancer Research Program under Materiel Command W81XWH-07-1-0556 and W81XWH-08-1-0516 (DC). Research reported in this publication was also supported by the National Institute of General Medical Sciences of the National Institutes of Healthunder Award Number P20GM103421. The previous segment of this project was supported by the National Center for Research Resources (NCRR) under P20 RR017695 (DC) and 1R01 CA102128-01A2 (EC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of themanuscript.

Competing Interests: The authors declare that previous affiliation to MedImmune and current affiliation to Kolltan, Inc. does not alter their adherence to all thePLOS ONE policies on sharing data and materials.

* E-mail: [email protected]

. These authors contributed equally to this work.

Introduction

Members of the signal transducer and activator of transcription

(STAT) family are transcription factors located in the cytoplasm

that, upon activation and nuclear translocation, regulate the

expression of genes involved in cell growth, apoptosis, survival,

and differentiation [1,2]. Upon activation, STAT3 undergoes

multiple posttranslational modifications, including phosphoryla-

tion and acetylation of STAT-family-conserved tyrosine, serine,

and lysine residues in the carboxy-terminal region [3–6]. These

specific modification events can be induced by treatment of cells

with cytokines, growth factors, and hormones. Both Janus kinase

(JAK) family and Src family tyrosine kinases can be recruited by

cytokines or growth factor receptors to catalyze STAT3 tyrosine

phosphorylation [7–10].

Cytokine/growth factor-activated STAT3 transcribes numerous

genes that inhibit apoptosis and promote cell survival and

neoplastic progression, including bcl-XL, bcl-2, and mcl-1 [11,12];

it also stimulates metastasis and invasion [13,14]. Moreover,

STAT3 is required for microtubule stabilization and stimulation of

cell migration via inhibition of the microtubule-destabilizing

Stathmin [15]. Aberrant JAK or Src protein kinase activity and

constitutive STAT3 activation have been associated with the

development of a tumorigenic phenotype in several cancers [1,16–

PLOS ONE | www.plosone.org 1 March 2014 | Volume 9 | Issue 3 | e92478

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24]. Hence, the STAT3 pathway is a popular target for

chemotherapeutic agents.

RKIP is a member of the phosphatidylethanolamine-binding

protein family. In contrast to STAT3, RKIP antagonizes multiple

cell-survival pathways, including the Ras-Raf-1 pathway [25], NF-

kB (Nuclear Factor kappa Beta) [26], and GRK2 (G Protein-

Coupled Receptor Kinase 2) [27] and activates others, like

GSK3b [28]. RKIP is required for some human cancer cells to

undergo drug-induced apoptosis [29]. RKIP is a metastasis

suppressor in colon, breast, melanoma and prostate cancer and

its expression is predictive of clinical outcome: better outcome with

higher expression [30–35]. In addition, there is an inverse

relationship between clinical outcome for gastric cancer patients

and expression of RKIP and STAT3 [36]. RKIP expression levels

vary in prostate and breast cancer cell lines depending on their

metastatic capacity; as the levels of RKIP expression decreases,

metastatic-potential of the cancer increases [37]. The lower levels

of RKIP in these cells are caused by the direct inhibition of RKIP

transcription by the zinc-transcriptional repressor protein Snail

[37], which has also been found to down-regulate the tumor

metastasis suppressor protein E-cadherin [38].

The anti-proliferative effects of microtubule inhibitors (MTIs),

such as taxol, have been exploited for quite some time as potential

chemotherapeutic agents preventing microvasculature prolifera-

tion and stimulating apoptosis of various tumor cell lines including

breast and prostate cancer cells [39,40]. 2-Methoxyestradiol (2-

ME2) is an estrogen-derived mammalian metabolite that has

antitumor and antiangiogenic effects [41]; it causes apoptosis by

inhibiting oncogenic proteins such as HIF-1a and disrupting

microtubule polymerization via weak competitive binding to

colchicine-binding sites [42]. ENMD-1198, an analog of 2-ME2

designed to retain the same mechanisms of action as 2-ME2

without undergoing the rapid in vivo metabolism of 2-ME2, was

tested in a Phase I clinical trial. Not only does ENMD-1198 inhibit

HIF1-a, but it also decreases STAT3 and NF-kB levels [43].

MKC-1 is a cell-cycle inhibitor that prevents mitotic spindle

formation by interacting at the colchicine-binding site of

microtubules [44]. MKC-1 also antagonizes the Akt-mTOR

signaling pathway, the most frequently mutated pathway in

human tumors with mutations that promote tumor progression

and decrease survival among cancer patients [45].

In this study we examined the role of RKIP in the apoptotic

inducing effects of MTIs and whether RKIP modulates MTI-

mediated STAT3 activation in multiple experimental models

[43,44]. Through our experiments, we gained additional under-

standing of the multifunctional role and mechanisms by which

RKIP inhibits cell survival and promotes apoptosis.

Materials and Methods

Ethics StatementThe animal care facilities at Rhode Island Hospital operate in

full compliance with the OLAW/PHS policy on the Humane

Care and use of Laboratory Animals and the USDA Animal

Welfare act. The Hospital’s NIH Assurance number is A-3922-01

and the USDA Registration number is 15-R-002. This study was

performed with approval from Rhode Island Hospital IAUCUC

CMT #0169-08. Any animal that exhibited anorexia or decreased

water intake for 24 hours or decreased activity, hunched posture,

excessive grooming or any other overt sign of distress was

euthanized to limit further suffering. A 15% reduction in body

weight compared to cage mates was also considered significant and

affected animals were euthanized by CO2 asphyxiation.

Materials2-ME2, ENMD-1198 and MKC-1 were provided by Entremed

(Rockville, MD). All reagents and chemicals were purchased from

Sigma Chemical Co. (St. Louis, MO) unless otherwise noted.

Protein quantification reagents were obtained from Bio-Rad

Laboratories, Inc. (Hercules, CA). Enhanced chemiluminescence

reagents and secondary mouse and rabbit horseradish peroxidase-

conjugated antibodies for Western blot analysis were ordered from

GE Healthcare (Arlington Heights, IL). The antibodies to Src

pY416 (2101S), STAT3 pY705 (9131S) and PARP (9542S) were

purchased from Cell Signaling Technology (Beverly, MA); the

RKIP (07-137), VEGF (06-585), Muc1 (05-652) and Cxcr4

(PC389) were purchased from Millipore (Hopkington, MA); the

actin-HRP (sc-1616), c-Src (sc-19) and STAT3 (sc-482) antibodies

were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Cells and PlasmidsThe human prostate carcinoma cell lines PC3 and DU145,

human breast carcinoma MDA-MB231 (MDA) and human normal

renal epithelial cell lines 293T cells were purchased from ATCC

(Rockville, MD). The cell lines in our lab were used between passage

numbers 35-45. The prostate and breast cancer cells were grown in

RPMI 1640 medium and 293T cells were grown in Dulbecco’s

modified Eagles medium. All cell lines were supplemented with 10%

fetal calf serum, glutamine, non-essential amino acids, 100 units/ml

penicillin and 50 units/ml streptomycin and cultured in a

humidified incubator at 37uC containing 5% CO2. The expression

plasmids for pcDNA3 c-myc STAT3, HA-STAT3, CMV-HA-

RKIP, CMV-FLAG-RKIP and c-Src have been described [6,29].

For RNAi-based silencing of expression of RKIP in the MDA

cells, we prepared knockdown cells expressing the small interfering

RNAs (siRNAs) specific for human RKIP or a firefly luciferase-

specific siRNA as control from the short hairpin RNA (shRNA)

expression vectors pSUPER.retro-puro (Oligoengene). The target

sequences were: 59GATTCAGGGAAGCTCTACA-39 (RKIP-1/

PEBP-1) and 59 CGTACGCGGAATACTTCGA-39 (firefly lucif-

erase control). RKIP was over expressed in MDA cells by

retroviral-mediated gene transfer. Retroviral constructs encoding

N-terminally FLAG-tagged rat RKIP in the pQCXIP (Clontech)

vector were used to generate RKIP-over expressing MDA cells as

previously described [29]. Expression of the tagged RKIP fusions

was confirmed by Western blot analysis.

Cell TransfectionCells were transiently transfected using the Lipofectamine

transfection reagent (Invitrogen) according to the manufacturer’s

protocol. Transfection conditions were assessed and optimized by

FACS analysis of cells transfected with a Green Fluorescent

Protein (GFP)-expressing plasmid. Transfection efficiencies were

consistently high (approximately 88–93% of cells). Total DNA

quantities between 1 and 3 mg were transfected per sample.

Western Blot AnalysisTotal cell extracts were prepared as previously described [46].

Protein concentrations of lysates were determined using the

Bradford assay kit (BioRad). Proteins were separated by SDS-

PAGE and electrophoretically transferred from the gel to nitrocel-

lulose membranes (GE Healthcare). Proteins recognized by the

antibodies were detected by enhanced chemilluminescence reagents

(GE Healthcare). Densitometry of Western blots were performed

according to the protocol listed at the following site: http://

lukemiller.org/journal/2007/. Actin was used for normalization of

loading of protein, and band densities were determined using

RKIP Inhibits STAT3 Activation in Prostate and Breast Cancer


Photoshop CS6. The relative intensity was determined by dividing

the absolute intensity of each sample band by the absolute intensity

of the Actin loading control.

Flow Cytometry AnalysisThe percentage of apoptotic cells (sub-GO) was determined by

flow cytometric analysis of propidium iodide stained cells. The

analysis was performed using a dual laser FACSCalibur flow

cytometer (Becton Dickinson) with cell cycle modeling and analysis

software from Modfit LT (Verity Software House, Inc., Topsham,

MN) as previously described [46]. All experiments were repeated

at least 3 times.

Luciferase Reporter AssayCells (5 6 105 cells/60 mm dish) were transiently transfected

with 0.5 mg of a reporter plasmid containing STAT3 binding SIE-

fragment of the promoter region of mouse IRF1 gene (p2xSIE-

Luc) or the RKIP promoter using lipofectamine in serum-free

medium [6,37]. After 4 h OptiMEM, containing 20% FBS (final

concentration), was added to the cells. To normalize transfection

efficiencies, approximately 12 h after batch transfection of tissue

culture dishes with the appropriate plasmids, cells were trypsin-

ized, harvested and re-seeded into the appropriate number of

dishes required for the individual experiment. After 12 h the cells

were left untreated or treated with the indicated drug for the

indicated time. Cells were harvested by scrapping, washing twice

with PBS and lysing in passive lysis buffer (Promega, Madison,

WI). The luciferase activity in the cytosolic supernatant was

evaluated using the Dual Luciferase Reporter Assay (Promega)

and measured using a luminometer to estimate transcriptional

activity.

Application of Small Interfering RNA (siRNA) against RKIPDU145 cells were plated in a 6-well plate 24 h prior to

transfection in an antibiotic-free growth medium. RKIP siRNA

(Santa Cruz Biotechnology Inc.), or a relevant amount of a control

siRNA solution was mixed with transfection reagent in OptiMEM.

Complex formation was allowed for 30 min at room temperature.

The mix was added to OptiMEM-washed cells together with

OptiMEM and incubated for 6 h. At 6 h 1 ml of medium

supplemented with 20% FBS was added to the cells and

incubation was continued for a total of 48 h before treatment

and cell harvesting. RKIP inhibition at the protein level was

confirmed using Western blot analysis.

Electrophoretic Gel Mobility Shift Assays (EMSA)Cells (106) transfected with EV and HA-RKIP then treated with

40 ng/ml IL-6 for 12 h were harvested after treatment and

washed twice with cold Dulbecco’s PBS (Cellgro). After washing,

the cells were lysed in 1 ml of NP40 lysis buffer (10 mM Tris-HCl

pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% NP40 and 0.1 mM

EDTA) on ice for 5 min. Samples were centrifuged at 300 6 g at

4uC for 5 min. The pellet was washed twice in NP40 buffer.

Nuclei were then lysed in nuclear extraction buffer (20 mM

HEPES pH 7.9, 25% glycerol, 0.42 mM NaCl, 1.5 mM MgCl2,

0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and

0.5 mM DTT) and sonicated for 10 s at 4uC. The protein

concentration was determined using the Bio-Rad protein assay.

The nuclear proteins were frozen at 280uC. Both buffers

contained the complete protease inhibitor cocktail tablets from

Roche. Nuclear protein (10 mg) was mixed for 30 min at room

temperature with Biotin-labeled oligonucleotide probe STAT3,

using the EMSA Kit purchased from PanomicsTM (Panomics, Inc.

Redwood City CA), following the manufacturer’s instructions.

10 ml was subjected to denaturing 5% polyacrylamide gel

electrophoresis and transferred to Nylon transfer membrane

Hybond-N+ (Amersham) using the Trans-BlotH SD semi-dry

Transfer cell System (Bio-Rad). The membranes were transferred

to a UV Cross-linker FB-UVXL-1000 Fisher technology (Fisher

Scientific) for 3 min. The detection was made as per the

manufacturer’s instructions, after the membranes were exposed

using Hyperfilm ECL (GE Healthcare). The oligonucleotide

sequences used for STAT 3 were: 59-GATCCTTCTGG-

GAATTCC TAGATC-39 as described [18,47].

STAT3-DNA BindingA standard transcription factor enrichment assay was modified

to assess STAT3 dimer binding to DNA. MDA cells were

transiently transfected with an empty vector or an expression

plasmid for RKIP. After 48 h, some cells were treated with IL-6

(40 ng/ml) for 12 h. Cells were harvested, and cell lysates were

generated as previously described [18]. Four mg of total protein/

condition was incubated overnight at 4uC in 3 ml of 100 mM

HEPES-KOH (pH 7.9), 25 mM MgCl2, 300 mM KCl, 50 mM

dithiothreitol, 0.5% Nonidet P-40, and 50% glycerol, containing

50 mg of the STAT3-DNA binding site oligonucleotide (5’-

GATCCTTCTGGGAATTCCTAGATC-3’) [47] agarose conju-

gate. Agarose beads were then removed by centrifugation, washed,

and incubated in 50 ml of Laemmli buffer for 10 min at 100uC.

Samples were then analyzed by Western blot analysis for STAT3

content.

Immunoprecipitation AssayCells were transfected with EV or the indicated plasmids for

48 h. In the experiment investigating IL-6 stimulation, cells were

treated with 40 ng/ml IL-6 for 2 h. The cells were lysed in RIPA

buffer. Five percent of the sample was removed to verify and

analyze proteins present for the immunoprecipitation input.

Approximately 1.5 mg of protein was incubated with 3 mg of

monoclonal c-Myc antibody for 4 hr at 4uC. Immunocomplexes

were collected on protein G-agarose beads separated 10% SDS-

PAGE and electroblotted to nitrocellulose membrane. Proteins

were detected after incubation with specific antibodies described

and identified using the ECL detection system.

Identification of Proteins Interacting with RKIP via LiquidChromatography-Mass Spectrometry (LC-MS/MS)

Cells were transfected with HA-RKIP for 48 h. Lysed cells were

immunoprecipitated with an antibody to HA and proteins were

separated via 10% SDS-PAGE. The gel was stained with coomasie

and the gel bands were excised by extracting gel particles with

glass Pasteur pipettes. Sample preparation, including protein

alkylation before proteolytic digestion, was performed as previ-

ously described [48]. After alkylation, the solution was removed

and the gel pieces washed and dried. After rehydratation for 15

minutes at 4uC in digestion buffer consisting of 0.05 M

ammonium bicarbonate, 5 mM CaCl2 and 12.5 mg/mL trypsin

(porcine, sequence grade, Promega, Madison, WI, USA), the

proteins were digested. After allowing the gel plugs to swell for 15

minutes, an additional volume of digestion buffer was added to

cover the gel plugs that had completely adsorbed all initially added

buffer, and the samples were then placed for 16 h in an incubator

set at 37uC. After digestion, the peptides were recovered from the

mixture by centrifugation (Eppendorf centrifuge). Peptides re-

maining in the gel were extracted with a solution of 50% (v/v)

acetonitrile containing 1% (v/v) TFA (Pierce) in 25 mM



ammonium bicarbonate for 10 min with shaking and subsequently

pooled with the first fraction. The tryptic digest was held at 280uCuntil ready for LC-MS/MS analysis.

Tryptic ‘‘in-gel’’ or ‘‘in-solution’’ digests were separated with an

RP column (C-18 PepMap 100, LC Packings/Dionex, Sunnyvale,

CA, USA) [49]. The gradient was starting with 5% (v/v)

acetonitrile in 0.1% (v/v) formic acid to 35% acetonitrile in

formic acid (solvent B) for 75 minutes. The column eluate was

introduced directly onto a QSTAR XL mass spectrometer

(SCIEX and Applied Biosystems, Concord, Ontario, Canada)

via ESI. Candidate ion selection, fragmentation and data

collection were performed as described previously [50]. Protein

identifications were performed with ProteinPilot software (Sciex

and Applied Biosystems), using a human ‘‘RefSeq’’ databases from

NCBI (http://www.ncbi.nim.nih.gov/RefSeq/). This software is

the successor of ProID and ProGroup, and uses the same scoring

method. Briefly, given a protein score S, the likelihood that the

protein assignment is incorrect is 10-S, and the scores above 2.0

require that at least two sequence-independent peptides be

identified. Protein identification was performed in at least two

independent experiments.

Wound Healing/Scratch AssayMDA MB-231 cells were seeded in 60 mm dishes containing

glass coverslips at a density of 26105 cells/dish in RPMI 1640

medium containing 10% FBS [51] The cells were transiently

transfected the following day with the indicated plasmids (see

results). The confluent cells (,85%) were starved in starvation

medium for 18 h. and a wound line was drawn across the adherent

cells on the glass coverslip. Non adherent cells were removed by

washing the dishes with PBS. The cells were incubated in medium

containing 1% FBS and after an additional 24 h, the cells were

washed, fixed and stained with crystal violet and the cells

migrating into the wound space counted.

Growth of Tumor XenograftsMDA cells (107) that stably express: 1). empty vector, 2).

activated and over expressed STAT3, 3). over expressed RKIP, 4).

RKIP knockout and 5). activated and over expressed STAT3 and

over expressed RKIP were injected into the two rear dorsal flanks

of 6 week old female NU/J mice (Jackson Labs). After tumor

implantation, mice were weighed and had tumor measurements

taken twice weekly, starting on day 1. These tumor measurements

were converted to tumor weight (mg) using an established formula:

Weight (mg) = length 6 width (mm2)/2. Experiments were

terminated when tumors in control animals reached a size of

approximately 0.5 g. At termination, all of the mice were weighed

and sacrificed, and their tumors excised.

Statistical methodsAll cell culture experiments were repeated at least 3 times,

unless indicated otherwise, and paired t-tests were performed to

determine statistical significance.

Results

RKIP over expression results in the reduction of c-Src-

mediated STAT3 activation. C-Src-triggered STAT3 activa-

tion enhances breast cancer progression and prevents depolymer-

ization of microtubules [15,52]. Phosphorylated STAT3 partici-

pates in c-Src- and cytokine-mediated signal transduction [53].

Therefore, to explore the role of RKIP on c-Src-mediated STAT3

activation, MDA cells were transiently transfected with c-Src,

RKIP, or the combination in the presence or absence of ENMD.

Our results indicate a 2.9 fold increase (relative intensity 0.41 vs.

1.3) of STAT3 pY705 phosphorylation after transient transfection

with c-Src when compared to untreated empty vector cells

(Fig. 1A). In cells transiently transfected with c-Src and: a) co-

transfected with HA-RKIP, STAT3 pY705 phosphorylation was

reduced 38% (relative intensity 1.3 vs. 0.81); b) treated with

ENMD treatment of c-Src transfected cells lowered STAT3 pY705

phosphorylation 43% (relative intensity 1.3 vs. 0.74); and c) co-

transfected with HA-RKIP and treated with ENMD, STAT3

pY705 phosphorylation was reduced 47% (relative intensity 1.3 vs.

0.7).

C-Src transient transfection results indicate a 5.3 fold increase

(relative intensity 0.22 vs. 1.15) of c-Src pY416 phosphorylation

when compared to empty vector transfected MDA cells (Fig. 1A).

In cells transiently transfected with c-Src and: a) co-transfected with

HA-RKIP, c-Src pY416 phosphorylation was reduced 34%

(relative intensity 1.15 vs. 0.75); b) treated with ENMD, c-Src

pY416 phosphorylation was reduced 40% (relative intensity 1.15 vs.

0.72); and c) co-transfected with HA-RKIP and treated with

ENMD, c-Src pY416 phosphorylation was reduced 70% (relative

intensity 1.15 vs. 0.29). Collectively these results indicate that HA-

RKIP overexpression and ENMD treatment can, independently

and concomitantly, inhibit c-Src-mediated STAT3 pY705 and c-

Src pY416 phosphorylation.

The results of the Western blot analysis were confirmed by a

luciferase reporter assay in MDA cells. MDA cells were transiently

transfected with a reporter plasmid containing a STAT3-binding

SIE-fragment of the promoter region of mouse IRF1 gene

(p2xSIE-Luc) in serum-free medium [6]. Transient transfection

of MDA cells with c-Src resulted in a significant (7-fold) increase in

STAT3 luciferase reporter activity (p,0.00000001) when com-

pared to the empty vector control (Fig. 1B). There was a greater

than 4-fold (p,0.00000005) decrease in transcriptional activity in

cells transfected with c-Src and RKIP when compared to c-Src

alone (Fig. 1B).

RKIP over expression results in the reduction of IL-6-mediated STAT3 activation

To determine if the inhibition of STAT3 phosphorylation and

transcriptional activation by RKIP was only c-Src-specific, we

examined the ability of RKIP to inhibit IL-6-mediated STAT3

activation. MDA cells were transiently transfected with CMV

empty vector or HA-RKIP for 24 h and then treated with IL-6

(40 ng/ml) for 2 h. As shown in Figure 1C, treatment of CMV

empty vector-transfected cells with IL-6 resulted in significant

STAT3 pY705 transcriptional activation, as measured by a STAT3

luciferase reporter assay (p,0.000003). Transient transfection of

HA-RKIP inhibited STAT3 phosphorylation and IL-6 mediated

transcriptional activation (p,0.00004) (Fig. 1C).

In MDA cells treated with IL-6, we observed a 3.5-fold increase

(relative intensity 0.64 vs. 2.82) of STAT3 pY705 phosphorylation

(Fig. 1C). Transient transfection of HA-RKIP inhibited IL-6

mediated STAT3 pY705 phosphorylation 50% (relative intensity

2.82 vs. 1.4). These results demonstrate that HA-RKIP is a potent

inhibitor of IL-6-mediated STAT3 pY705 phosphorylation.

To determine if the inhibition of STAT3 phosphorylation and

transcriptional activation was specific to breast cancer cells, we

performed the same experiment described in Figure 1C, with

DU145 prostate cancer cells. Our results indicate that transfection

of RKIP inhibited IL-6-mediated STAT3 transcriptional activa-

tion (p,0.0002) and a 76% inhibition of STAT3 pY705

phosphorylation (relative intensity 0.78 vs. 0.18) (Fig. 1D). These

results demonstrate that RKIP can also inhibit IL-6-mediated

STAT3 activation and phosphorylation in prostate cancer cells.



Figure 1. ENMD-1198 inhibits c-Src-mediated STAT3 activation. (A)MDA-231 cells were transiently transfected with expression plasmids forc-Src and HA-RKIP for 48 h. Following this, the cells were treated with ENMD-1198 for 24 h and samples prepared for Western blot analysis andexamined for the indicated proteins. Densitometry was performed and the relative intensity when compared to Actin for lanes 1-8, respectively, were:RKIP (top band): 0.225, 0.32, 0.81, 0.22, 0.85, 0.22, 1.02, 0.76; RKIP (bottom band): 0.42, 0.5, 0.4, 0.73, 0.4, 0.59, 0.68, 0.6; STAT3 pY705: 0.41, 1.3, 0.31,0.10, 0.81, 0.74, 0.20, 0.70; STAT3: 0.075, 0.048, 0.059, 0.049, 0.042, 0.044, 0.033, 0.071; c-Src pY416: 0.22, 1.15, 0.59, 0.34, 0.75, 0.72, 0.16, 0.29; c-Src: 0.24,0.37, 0.24, 0.31, 0.55, 0.31, 0.49, 0.5. (B) A STAT3 luciferase reporter assay was performed under the experimental conditions described in A. Cells weretransiently transfected with a luciferase plasmid containing the consensus DNA STAT3 binding sequence for the SIE fragment of the promoter regionof mouse IRF1 gene for 48 h. Some cells were treated with ENMD-1198 for an additional 24 h. Luciferase activity was measured in cytosol extracts andcompared with activity observed in non-treated (control) cells, in which the observed activity was set at arbitrary units. A paired t-test was performedto analyze the increase or decrease in STAT3 reporter activity: *(p,0.00000001) in c-Src transfected cells when compared to the empty vector;**(p,0.00000005) decrease in transcriptional activity in cells transfected with c-Src and RKIP and ***(p,0.00005) in cells transfected with c-Src andtreated with ENMD-1198 and ****(p,0.000005) in cells transfected with c-Src, RKIP and treated with ENMD-1198, when compared to c-Src.EV = empty vector. (bars = mean 6 S.D., of 2 experiments performed in triplicate). (C) Top, Western blot analysis was performed in cells transientlytransfected with RKIP or EV for 48 h. Some cells were treated for 2 h with 40 ng/ml IL-6, whole cell extracts prepared and analyzed for the expressionof the indicated proteins. Densitometry was performed and the relative intensity when compared to Actin for lanes 1-4, respectively were: STAT3pY705: 0.64, 2.82, 1.3, 1.4; STAT3: 1.1, 1.2, 1.8, 1.3. Bottom, STAT3 luciferase assay was performed in MDA cells transiently transfected with STAT3luciferase reporter construct or an expression vector for RKIP. After 48 h, some of the cells were treated with 40 ng/ml of IL-6 for 6 h. Cells wereharvested and luciferase reporter activity measured. A paired t-test was performed demonstrating the increase in transcriptional activity of cellstreated with *(p,0.000003) IL-6 when compared to EV. A decrease in transcriptional activity was observed in cells **(p,0.00004) transfected withRKIP and treated with IL-6 when compared to cells treated with IL-6 (bars = mean 6 S.D., of 2 experiments performed in triplicate). (D) The sameexperiment was performed in DU145 prostate cancer cells as described in (C). Top: Western blot analysis of the inhibition of IL-6-mediated STAT3phosphorylation by RKIP overexpression under the same experimental conditions described in (C). Densitometry was performed and the relativeintensity (fold increase) when compared to Actin for lanes 1–4, respectively were: STAT3 pY705: 022, 0.78, 0.24, 0.18; STAT3: 0.75, 0.61, 1.3, 0.67.Bottom: STAT3 luciferase assay was performed in DU145 cells transiently transfected with STAT3 luciferase reporter construct or an expression vectorfor RKIP. A paired t-test was performed demonstrating the increase in transcriptional activity of cells treated with *(p,0.0004) IL-6 when compared toEV. A decrease in transcriptional activity was observed in cells **(p,0.0002) transfected with RKIP and treated with IL-6 when compared to cellstreated with IL-6 (bars = mean 6 S.D., of 2 experiments performed in triplicate). (E) STAT3 DNA binding activity was examined using the STAT3 EMSAkit purchased from Panamoics according to the manufacturer’s instructions. Cells (106) were transfected with EV or RKIP for 48 h. Some cells weretreated with 40 ng/ml IL-6 for 6 h. Cells were harvested, washed and lysed in NP-40 lysis buffer. Nuclei were isolated and lysed according to themanufacturer’s instructions. Nuclear protein (10 mg) was mixed for 30 min at room temperature with Biotin-labeled oligonucleotide probe STAT3,using the Panaomics EMSA Kit. The detection was made as per the manufactory’s instructions. (F) Western blot analysis of STAT3-DNA binding as afunction of RKIP overexpression and IL-6 treatment was determined. Cell lysates were generated, particulate material then was removed bycentrifugation, and the resulting supernatant (1.5 mg of total protein/condition) was incubated overnight at 4uC in binding buffer containing 50 mg



We examined the ability of RKIP to inhibit IL-6-dependent

STAT3 DNA binding. Extracts were generated from cells

transfected with RKIP or EV for 48 h and then exposed for

12 h to 40 ng/ml IL-6. In initial experiments, STAT3 binding to

DNA was assessed by an electrophoretic mobility shift assay

(EMSA) using the Panomics STAT3 EMSA kit. Assessment of

STAT3 DNA binding by EMSA revealed that IL-6 promoted

STAT3 DNA binding, the level of which was significantly reduced

by RKIP over expression. The results of this analysis revealed that

IL-6 promoted STAT3 DNA binding. The consequence of this

reduction in the cellular content of STAT3 pY705 by RKIP over

expression was a significant reduction in STAT3 dimer binding to

DNA (Fig. 1E). To confirm the specificity of this effect, STAT3

binding to DNA also was assessed in total cell extracts by exposure

to the STAT3 DNA binding site consensus sequence immobilized

on agarose beads. Western blot analysis revealed an increase in

binding after IL-6 treatment and again a dramatic reduction in

STAT3 dimer recovery in cells transfected with RKIP (Fig. 1F).

These results suggest that one mechanism of RKIP-mediated

inhibition of STAT3 activation occurs by preventing STAT3

DNA binding via inhibition of phosphorylation.

MTIs induce RKIP expression in a prostate cancer cell lineMicrotubule inhibitors such as taxol are important agents that

have been used extensively and investigated for the treatment of

breast and prostate cancer for many years [54-56]. We hypoth-

esized that the anti-tumor effects of MTIs are not only mediated

by a direct effect on microtubules, but also by the induction of

tumor suppressor RKIP. Many naturally occurring or synthetic

compounds are known to induce the expression of RKIP [57].

Human prostate and breast carcinoma cells undergo apoptosis

when treated with various chemotherapeutic compounds, includ-

ing camptothecins (CPT) [46] cisplatin [58] and taxol [39,40] In

addition, we have shown that RKIP is required for chemotherapy-

triggered apoptosis [29].

Therefore, we investigated the effects of MTIs on prostate and

breast cancer cell apoptosis and RKIP protein expression. DU145,

PC3 and MDA cells were treated with ENMD-1198 and MKC-1

at IC50 concentration (200 nM) for 6 or 12 h and were then

analyzed via Western blot analysis. As shown in Fig. 2A, both

MTIs induced the expression of RKIP in DU145 but not PC3 cells

or MDA cells. We examined the time course for RKIP induction

in DU145 cells. EMND-1198-treated DU145 cells showed a more

rapid increase of RKIP expression when compared to cells treated

with MKC-1 (Fig. 2B).

RKIP overexpression restores sensitivity to MTI-associatedapoptosis in resistant cancer cells

We determined the apoptotic effects of MTIs in prostate and

breast cancer cell lines using flow cytometry analysis. Cells were

treated with ENMD-1198 or MKC-1 at the indicated concentra-

tions for 24 h and were then collected and analyzed for apoptosis

via flow cytometry (Fig. 2C). We found that the percentage of

apoptosis substantially increased in a dose-dependent manner in

DU145 prostate cancer cell lines treated with ENMD-1198 and

MKC-1 (Fig. 2C). However, PC3 prostate and MDA breast

cancer cells were resistant to MTI-induced apoptosis, (Fig. 2C).

These results suggest that the resistance of PC3 and MDA cells to

MTI-mediated apoptosis may be due to the lack of RKIP

induction.

RKIP over expression sensitizes breast and prostate cancer cells

to drug-induced apoptosis [35]. Therefore, we sought to determine

whether over expression of RKIP could sensitize ENMD-1198-

resistant PC3 prostate and MDA breast cancer cells to apoptosis.

Both cell lines were transiently transfected with HA-RKIP

expression plasmid for 24 h and were then treated with ENMD-

1198 for 12 h. Our results indicate that MDA and PC3 cells

underwent substantial cell death, as measured by Poly (ADP-

ribose) polymerase (PARP) cleavage upon transfection with RKIP

and treatment with ENMD-1198 (Fig. 2D). These results were

reproduced in both cell lines after treatment with MKC-1 (data

not shown). We also measured PARP cleavage upon treatment

with ENMD-1198 and MKC-1 in DU145 cells. Both MTIs

induced RKIP expression and PARP cleavage, as shown in

Fig. 2D, right panel.

To confirm that RKIP is required during MTI-triggered

apoptosis, DU145 cells were transiently transfected with RKIP

expression plasmid, RKIP siRNA, or scramble siRNA for 48 h

and then treated with ENMD-1198 for 24 h. Our results indicate

that there was a greater than 3-fold increase in apoptosis in

DU145 cells transiently transfected with RKIP (p,0.001) or

treated with ENMD-1198 (p,0.00007) and greater than 5-fold

with the combination (p,0.0002) (Fig. 2D). In the presence of

RKIP siRNA, apoptosis due to ENMD-1198 treatment was

reduced greater than 3.5-fold (p,0.000012) when compared to

the combination of RKIP transfection and ENMD-1198 treat-

ment (Fig. 2E, top). The results from the siRNA experiment were

substantiated by Western blot analysis (Fig. 2E, bottom). The

expression of RKIP was reduced greater than 50% in cells

transfected with RKIP siRNA (Fig. 2E, bottom). Collectively, our

results indicate that the increase in endogenous RKIP is required

for enhanced MTI-triggered apoptosis.

RKIP inhibits JAK- and Raf-mediated STAT3 activationHaving determined that RKIP and ENMD-1198 were effective

in inhibiting STAT3 activation and phosphorylation, we next

focused on the ability of RKIP and ENMD-1198 to inhibit JAK-

mediated activation of STAT3 using the luciferase reporter assay.

We chose to use 293T renal epithelial cells to gain further

understanding of RKIP function for three main reasons. First, it

has been previously established that STAT3 is activated in DU145

and PC3 cells by more than one JAK kinase [59]. Second, we have

previously shown that STAT3 expression was not changed by

modulating RKIP expression in 293T cells [60] and third, because

293T cells have a higher transfection efficiency with the plasmids

we were using, especially under the serum free conditions used in

the experiments described below. 293T cells were transfected with

the IRF-1 plasmid along with cDNA encoding either JAK1, 2, or 3

or TYK2 in the presence or absence of ENMD-1198 or transiently

expressed HA-RKIP. Our data demonstrate that JAK1, 2, 3 and

TYK2 were able to significantly stimulate STAT3 luciferase

reporter activity (Fig. 3A). Ectopic RKIP expression significantly

inhibited STAT3 luciferase reporter activity in cells transfected

with JAK1 and 2, but not JAK3 or TYK2 (Fig 3A). Treatment of

IRF-1 and JAK1, 2, 3 or TYK2 transfected cells with ENMD-

1198 for 24 h did not affect STAT3 transcriptional reporter

activity. The induction of RKIP occurs after 6 h of exposure to

ENMD-1198 in some cell lines (Fig. 2B), which indicates that

of STAT3-DNA binding site oligonucleotide-agarose conjugate. The resulting supernatant was analyzed by Western blot methods to assess STAT3content [18].doi:10.1371/journal.pone.0092478.g001



Figure 2. Microtubule inhibitors cause cell cycle arrest and induce RKIP expression. (A) DU145 and PC3 cells were treated with ENMD-1198or MKC-1. Cells were harvested and washed twice with PBS and whole cell lysates were prepared for Western blot analysis as we have previouslyreported [28]. Proteins (unless indicated 50 mg/sample was used for Western blot analysis) were separated by 10% SDS-PAGE, transferred tonitrocellulose and analyzed with antibodies to the indicated proteins. Note: for all Western blots described in this Figure legend and for all othersubsequent Figure legends, the exposure time used to identify the various proteins was variable. (B) DU145 cells were treated with 200 nM ENMD-1198 orMKC-1 for 6 or 12 h. Cells were prepared for Western blot analysis for the indicated proteins. (C) DU145, PC3 prostate and MDA breast cancer cells (16105) were used for FACS analysis after treatment with the indicated concentrations of ENMD-1198 or MKC-1. Cells were harvested, washed twicewith phosphate buffered saline (PBS) and prepared for flow cytometry as previously described (43). Analysis for apoptosis was performed using aFACSCalibur flow cytometer (Data represents the mean 6 S.D., of 2 independent experiments performed in duplicate). (D) (Left panel) PC3 and MDAcells were transiently transfected with HA-RKIP expression plasmid for 48 h. Whole cell lysates were prepared and Western blot analysis wasperformed as described in A for the indicated proteins. Note for this and all other subsequent experiments described where RKIP was transfected intocells, the lower RKIP band represents endogenous RKIP, while the upper RKIP band represents ectopic RKIP (HA-RKIP). (Right panel), DU145 cells weretreated with ENMD-1198 or MKC-1 for 24 h. Whole cell lysates were prepared and Western blot analysis was performed for the indicated proteins. (E,top) DU145 cells were transiently transfected with various combinations with expression plasmids for RKIP, RKIP siRNA, scramble siRNA or emptyvector (EV) for 48 h. The cells were treated with 200 nM ENMD-1198 for 24 h. Cells were harvested and analyzed for apoptosis as described in (C). Apaired t-test was performed to analyze the increase in apoptosis in cells when compared to empty vector (EV): *(p,0.001) transiently transfected withRKIP; **(p,0.00007) treated with ENMD-1198; ***(p,0.0002) with the combination of RKIP transfection and ENMD-1198 treatment.



inhibition of JAK activity is ENMD-1198-independent. The

inhibition of STAT3 luciferase reporter assay by HA-RKIP was

comparable to the JAK inhibitors tyrphostin and AG490 (data not

shown). We also examined the ability of RKIP to inhibit JAK1-, 2-

mediated inhibition of STAT3 pY705 phosphorylation. Western

blot analysis demonstrated that transiently expressed RKIP

(0.5 mg) effectively inhibited JAK1- and 2-mediated phosphoryla-

tion of STAT3 (Fig. 3B).

RKIP was originally identified as an inhibitor of Raf [25], which

is the activating kinase for the MAPK cascade. We examined the

ability of Raf-BXB, an untagged constitutively active version of

Raf [25], to phosphorylate STAT3 and the ability of RKIP to

interfere with this process. Serum-starved 293T cells were

transfected with Raf-BXB in the presence or absence of HA-

RKIP expression vector. After 48 h in serum-free medium, cells

were left untreated or exposed to 20 ng/ml of IL-6. As shown in

Fig. 3C, Raf-BXB and IL-6 caused an increase in the STAT3

pY705 levels, an effect that appeared to be additive when cells were

exposed to Raf-BXB and treated with IL-6 (Fig. 3C). This may be

due to the fact that overexpression of BXB bypasses RKIP’s

constitutive role and leads to a regulatory loop that results in the

phosphorylation of RKIP by ERK.

Transient transfection with HA-RKIP blocked Raf-BXB and

IL-6 STAT3 pY705 phosphorylation (Fig. 3C).

We investigated whether RKIP could block Raf-BXB-mediat-

ed-STAT3 activation as measured by a reporter plasmid

containing STAT3-binding SIE-fragment transfected in 293T

cells. Cells were transfected with Raf-BXB or HA-RKIP as

described above and after 24 h were treated with the MAP-kinase

inhibitor PD98059 for an additional 24 h. As shown in Fig. 3D,

Scrmbl = scrambled siRNA. A decrease in apoptosis was observed ****(p,0.000012) in cells treated with ENMD-1198 when compared to cellstransiently transfected with RKIP siRNA and treated with ENMD-1198. (Data represents the mean 6 S.D., of 2 independent experiments performed intriplicate). (E, bottom) Western blot analysis of the same experimental conditions described in Fig. 2E, top. The Western blot analysis was repeated 3times and the Figure is representative of one of the experiments. Densitometry was performed and the relative intensity (fold increase) whencompared to Actin for lanes 1-8, respectively were 0.78, 1.7, 0.19, 0.84, 0.99, 0.67, 0.36 and 0.92.doi:10.1371/journal.pone.0092478.g002

Figure 3. RKIP blocks JAK and Raf-mediated STAT3 activation. (A) RKIP inhibits JAK1, 2-triggered STAT3 activation. 293T cells weretransfected with the Luc reporter construct containing 26 STAT3 binding SIE-fragment and co-transfected with either JAK1, 2, 3 or TYK2 in thepresence or absence HA-RKIP and ENMD-1198. Cells were placed in serum free medium and cell extracts were subjected to luciferase assay toestimate transcriptional activity. Averages and standard deviations (error bars) of the values of luciferase activities from triplicates of 3 representativeexperiments are shown. (B) Western blot analysis demonstrating inhibition of STAT3 pY705 phosphorylation in HA-RKIP and JAK1 or 2 transientlytransfected 293T cells. Raf-mediated STAT3 pY705 phosphorylation is inhibited by RKIP: (C) 293T cells were transfected with Raf-BXB, CMV emptyvector or HA-RKIP. After 48 h cells were left untreated or exposed to IL-6 for 1 h. Cell lysates were analyzed by Western blot. (D) 293T cells weretransfected with the STAT3 luciferase reporter construct and/or with EV, Raf-BXB or HA-RKIP. After 24 h, some cells were treated with PD98059 (PD)and after 48 h from the initial time of transfection, some cells were treated with IL-6 for 6 h. A paired t-test was performed indicating *(p,0.000005)increase in BXB transfected cells when compared to EV; decrease in STAT3 transcriptional activity **(p,0.00006) in PD treated cells transfected withBXB and ***(p,0.00004) in RKIP and BXB transfected cells when compared to the cells transfected with BXB. Luciferase reporter activities fortriplicates of 3 representative experiments +/- s.d. are shown. Western blot analysis of cells transfected with the indicated proteins in the presence orabsence of PD treatment.doi:10.1371/journal.pone.0092478.g003



Raf-BXB caused significant (p,0.000005) induction of STAT3

transcriptional activity as measured by the luciferase reporter

assay. Treatment with PD98059 for 24 h after transfection

(p,0.00006) or transfection of 293T cells with HA-RKIP

significantly (P,0.00004) inhibited Raf-BXB-mediated STAT3

activation (Fig. 3D). Interestingly, PD98059 reduces STAT3

pY705 in combination with BXB (data not shown). Our results

indicate that RKIP can block Raf-dependent STAT3 pY705

phosphorylation and transcriptional activation. Our data also

indicate that RKIP acts as a multiple kinase inhibitor as it inhibits

JAK- and Raf-mediated STAT3 pY705 phosphorylation and

transcriptional activation.

RKIP interacts with, and inhibits STAT3 activationTo delineate the mechanism by which RKIP inhibits c-Src- and

JAK1 and 2-mediated STAT3 activation, a series of RKIP

deletion constructs (Fig. 4A, upper panel) were examined for their

ability to inhibit JAK1-mediated STAT3 activation. As noted in

Fig. 4A, middle panel, full-length and clone N60 RKIP expression

constructs potently inhibited Src- and JAK1-mediated STAT3

phosphorylation. RKIP deletion constructs N93, C134 and C93

did not block c-Src-mediated STAT3 pY705 phosphorylation,

shown with Western blot analysis (Fig. 4A, lower panel).

Although both N60 and C134 bind to Raf, clone N60 has a

higher affinity than C134 for binding to Raf [29], suggesting that

the N60 sequence may also have a greater ability to disrupt c-

Src:STAT3 interactions. To determine whether there is any

interaction among RKIP, c-Src, and STAT3, MDA cells were

transiently transfected with the following series of epitope-tagged

expression plasmids: c-myc empty vector, c-myc-Src and HA-

STAT3, c-myc-Src and Flag-RKIP, HA-STAT3 and Flag-RKIP

and c-myc-Src, HA-STAT3 and Flag-RKIP. We did not use the

MDA RKIP stable cell line for this experiment because we did not

have a stable cell line for c-Src. After 48 h, lysates were prepared

and incubated with an antibody to c-myc as described in the

Experimental Procedures section. Western blot analysis was

performed and the nitrocellulose filter was analyzed by incubation

to an antibody to HA. As shown in Fig. 4B, a constitutive

interaction was noted between Src and STAT3. That interaction

was no longer present in cells that were transfected with Src,

STAT3, and RKIP. LC/MS was performed to determine if RKIP

constitutively associated with c-Src, STAT3 or other proteins in

the JAK/STAT pathway. Cells were transfected with HA-RKIP

plasmid and cell lysates immunoprecipitated with an antibody to

HA. After excision and ‘‘in gel’’ digestion the protein in one of the

bands was identified as STAT3, the isoform 1 of signal transducer

and activator of transcription 3 with a very high confidence (score

62.46). Thirty-four peptides belonging to this protein were

identified, and the sequence coverage was 53.9%. This result

supports our IP result indicating the interruption of Src:STAT3

association after RKIP over expression.

Activated Src (pY416) was observed in the Western blot analysis

of the co-IP input samples transfected with c-myc, Src, and/or

HA-STAT3. When Src was co-transfected with RKIP, however,

Src was no longer in its activated phosphorylated form (Fig. 4B),

indicating that RKIP inhibited Src autophosphorylation. These

results further indicate the versatility of RKIP in blocking STAT3

activation.

We explored the consequence of RKIP disrupting Src-STAT3

interaction. Activated Src and STAT3 have been associated with

promoting epithelial-mesenchymal transition (EMT) in tumor cells

[61,62]. We investigated the ability of RKIP over expression to

inhibit c-Src-mediated induction of proteins that are intimately

involved with EMT, migration, and angiogenesis. As shown in

Fig. 4C, c-Src transient transfection of MDA cells resulted in the

induction of Cxcr-4, VEGF and Muc1 protein expression, and

RKIP over expression significantly inhibited the expression of

these EMT-associated proteins (Figure 4C). To complement this

result, we showed that transfection of MDA cells with c-Src

resulted in an approximately 4-fold and significant increase

(p,0.0002) in migration of MDA cells (Fig. 4D). In cells co-

transfected with c-Src and RKIP, there was approximately a 60%

reduction in migration (P,0.005) when compared to cells that had

been transfected with c-Src (Fig. 4D). Transfection of MDA cells

with RKIP siRNA did not effect Src-mediated migration. These

results indicate that RKIP can inhibit c-Src-mediated MDA cell

migration.

RKIP inhibits IL-6-mediated STAT3 activation in stablytransfected cell lines

Our data thus far has indicated that RKIP can inhibit multiple

mechanisms of STAT3 activation (Figs. 1, 3). Our experiments

were performed with transient transfection over expression of

RKIP (Figs. 1-3) and RKIP siRNA (Fig. 2). Therefore, to

accurately assess the role of RKIP in the inhibition of STAT3

activation, stable MDA cell lines were created that over express

(MDA RKIP) and do not express RKIP (knockdown, MDA si175).

We examined the effect of IL-6 on STAT3 luciferase reporter

activity in both models, including the appropriate empty vector

controls. MDA EV and MDA RKIP were transfected with the

STAT3 reporter and treated with IL-6 as previously described in

Fig. 3. As shown in Fig. 5A, treatment of MDA EV cells with IL-6

resulted in a greater than 2.5 fold (p,0.007) increase in STAT3

luciferase reporter activity. In contrast, in MDA RKIP cells that

stably over express RKIP there was a slight decrease (1-fold) in

reporter activity when compared to untreated cells (Fig. 5A). In the

knockdown cells, IL-6 treatment resulted in a greater than 2-fold

increase in reporter activity in the MDA si175 empty vector

control cells (Fig. 5B). In contrast, in the stable MDA si175 RKIP

knockdown cells, IL-6 treatment resulted in a greater than 11-fold

(p,0.005) increase in STAT3 luciferase reporter activity (Fig. 5B).

We examined the effect of IL-6 treatment on STAT3 pY705

phosphorylation in MDA RKIP and MDA si175 cells. In MDA

EV cells, IL-6 treatment resulted in STAT3 pY705 phosphoryla-

tion, but not in MDA RKIP cells (Fig. 5C). In contrast, in MDA

si175 EV cells IL-6 treatment resulted in STAT3 pY705

phosphorylation, which was significantly increased in MDA

si175 RKIP knockdown cells (Fig. 5C).

RKIP blocks STAT3 promotion of tumor xenograft growthSTAT3 has been shown to promote breast cancer tumor growth

[63,64] and our results have shown that RKIP is a potent inhibitor

of STAT3 activation in vitro. Therefore, we examined if RKIP

could inhibit STAT3-mediated tumor xenograft growth. Our

results indicate that mice inoculated with parental MDA EV cells

had an average tumor growth of 215 mg (Fig. 5D). Mice

inoculated with MDA RKIP had a 1.4-fold reduction in tumor

mass (p,0.002), MDA si175 RKIP a 1.2-fold (p,0.008) and

MDA STAT3 a 1.4-fold (p,0.002) increase in tumor mass when

compared to MDA EV (Fig. 5D). MDA cells that over express

RKIP and activated STAT3 had a 1.1-fold and not significant

increase (p,0.13) in tumor growth when compared to MDA EV,

but also a significant 1.3 fold (P,0.009) reduction in tumor mass

when compared to MDA STAT3 (Fig. 5D). Taken together, our

studies indicate that RKIP overexpression inhibits and RKIP

ablation significantly enhances IL-6 mediated STAT3 activation

and that RKIP can inhibit STAT3-mediated tumor growth. Our



data suggest that RKIP is a native negative regulator of STAT3

activity.

Discussion

In this study we show that MTIs ENMD-1198 and MKC-1

induced apoptosis in prostate and breast cancer cell lines is

dependent upon the induction of RKIP. Src is able to induce and

activate STAT3 expression and stabilize microtubules. MTIs

inhibit STAT3 phosphorylation and in MTI-treated MDA breast

cancer cells, where c-Src promotes STAT3 activation, we detected

the inhibition of c-Src- and IL-6-mediated STAT3 phosphoryla-

tion and activation, effects that were greatly enhanced by RKIP

over expression. IL-6-mediated STAT3 activation was RKIP

dependent as demonstrated by the lack of STAT3 activation in

MDA cells that over express RKIP and a significant enhancement

of activation in cells that have knockdown of RKIP. In addition,

RKIP blocked JAK1 and 2- and Raf-mediated STAT3 activation

and STAT3-mediated tumor xenograft growth. Our studies

demonstrate that RKIP: i) is required for MTI-mediated

apoptosis; ii) inhibits JAK and Raf kinase activities; iii) inhibits

Src association with STAT3 and iv) is required to inhibit STAT3

activation and STAT3-mediated tumor formation. Although over

expression systems may be interpreted as artificial, our results with

the knockdown of RKIP, or basically loss of RKIP function,

Figure 4. RKIP inhibits Src-STAT3 interaction. (A), top panel, Cartoon depicting a series of RKIP deletion constructs that were examined for theirability to inhibit c-Src- and JAK1-mediated STAT3 activation. Middle panel, Some of the deletion constructs were transfected in the presence of c-Srcor JAK1 into cells for 48 h. Whole cell protein lysates were examined for the expression of STAT3, STAT3 pY705 and actin. Lower panel, Cells weretransfected with EV, c-Src or RKIP deletion constructs N93 and C93. After 48 h, cells were harvested and whole cell lysates prepared to examine theexpression of the indicated proteins via Western blot analysis. (B) Cells were transfected with c-myc EV, or the following tagged expression plasmids:myc-c-Src (2 mg), HA-STAT3 (2 mg), Flag-RKIP (4 mg) or the combination for 48 h. Cells were lysed in RIPA buffer, cellular debris removed, a portion ofthe sample removed as the IP input and the remaining supernatant incubated and rotated with an antibody to c-myc for 4 h at 4uC. Subsequently,protein G agarose was added and the samples rotated overnight at 4uC. Protein G agarose containing immuno complexes was extensively washed,26 sample buffer added and the samples heated at 95uC for 5 min. Proteins were separated by SDS-PAGE, transferred onto nitrocellulose. Thesamples subjected to IP were divided in half. One filter was examined with an antibody to HA the other with an antibody to c-myc. The HA filter wasstripped and examined for STAT3 levels. The input samples were examined for the expression of the indicated proteins. (C) Western blot analysis ofcells transfected with EV (lane 1), c-Src (lane 2), HA-RKIP (lane 3) and c-Src + HA-RKIP (lane 4) to examine the expression of the indicated proteins thatwere separated on 15% SDS-PAGE. (D) RKIP inhibits c-Src-mediated MDA-231 cell migration. MDA-231 cells were transfected with empty vector, c-Src,c-, HA-RKIP, RKIP siRNA, scrambled siRNA or the combination. The cells were allowed to grow to near confluence. Cells were wounded with a pipetteand after 24 h the migrated cells were fixed then stained with crystal violet and quantified. A paired t-test was performed indicating *(p,0.0002) anincrease in migration in c-Src transfected MDA cells when compared to EV transfected cells and **(P,0.005) inhibition of c-Src-mediated increase inmigration when compared to cells transfected with c-Src and RKIP. The data represents two independent experiments performed in triplicate.doi:10.1371/journal.pone.0092478.g004



indicate its pivotal role in STAT3 and c-Src activation,

phosphorylation and tumor formation.

Numerous chemotherapeutic agents act on the instability of MT

polymerization to exert their cytotoxic effects, leading to cellular

senescence, cell-cycle arrest, and apoptosis. For instance, taxane

drugs such as Paclitaxel stabilize polymeric MTs in their GDP-

bound state, preventing depolymerization [65]. MTIs like

colchicine have the opposite effect: they bind to monomeric

tubulin, preventing MT polymerization and thus causing poly-

merized MTs to destabilize and depolymerize. The MTIs used in

this study bind to the colchicine-binding sites of tubulin inhibiting

spindle formation, but also cause different downstream effects than

original congeners, including the inhibition of the oncogene HIF1-

a. Previous studies have shown the requirement of RKIP for

chemotherapy-triggered apoptosis [29] and inhibition of metasta-

sis in prostate cancer [31]. RKIP’s role in mediating MT dynamics

in prostate or breast cancer has not been given much emphasis

despite recent findings implicating RKIP in the regulation of

mitotic spindle checkpoints via association with centrosomes and

kinetochores [66]. Specifically, RKIP induction activates Aurora B

kinase, a spindle checkpoint regulator, via inhibition of Raf-1 [66].

RKIP also regulates MT dynamics by potently regulating STAT3

levels. Based on the results obtained in this study, we hypothesize

that RKIP’s ability to potentiate the apoptotic effects of the MTIs

used in this study results from its multiple ways of regulating MT

dynamics. It is has been suggested that MTIs indirectly cause MT

instability by decreasing STAT3 levels. STAT3 inhibits the MT-

destabilizing protein Stathmin, allowing MT polymerization to

occur [15]. Therefore, identifying factors or compounds that

negatively regulate MT polymerization-activators and further

cause cell cycle arrest and apoptosis would be an important step in

developing therapies. As we describe here, this can occur via

RKIP over expression, which inhibits constitutive c-Src-STAT3

interaction and activation (Fig. 4).

How exactly the c-Src-STAT3 axis promotes breast cancer is

not clear but may involve an array of cellular processes including

proliferation, survival, epithelial mesenchymal transition (EMT)

and tumor formation [47,67]. Muc1 and VEGF are two proteins

regulated by STAT3 which promote invasion and angiogenesis

[68]. We have found that transfection of c-Src results in the

activation of STAT3 and subsequent induction of Cxcr4, Muc1

and VEGF protein expression and increased breast cancer cell

migration, events, which are negated by RKIP over expression

(Fig. 4). These results demonstrate that RKIP over expression can

Figure 5. RKIP is required to inhibit STAT3 activation and breast tumor xenograft growth. (A) STAT3 luciferase reporter activity wasperformed as described in Figure 2 in cells treated with 40 ng/ml IL-6 for 6 h. A, represents MDA RKIP cells and (B) MDA si175 RKIP knockdown cells.Paired t-test was performed; *(p,0.007) in IL-6 treated cells when compared to EV control. Luciferase reporter activities for duplicate of 2representative experiments +/- s.d. are shown. (C) Western blot analysis was performed in MDA RKIP and MDA si175 RKIP treated with 40 ng/ml IL-6for 2 h. Whole cell lysates were examined for the expression of the indicated proteins. (D) MDA cells were implanted s.c. in the dorsal flanks of Nu/Jfemale mice. Tumors were measured using calipers twice weekly, and volumes were calculated using the formula: weight (mg) = [width (mm)2 6length (mm)]/2. A paired t-test was performed indicating the decrease of RKIP *(p,0.002) or increase of **(p,0.008) si175RKIP, ***(p,0.002) STAT3of tumor growth when compared to EV. A decrease in tumor growth was detected between STAT3 and RKIP/STAT3 ****(p,0.009). Mean tumor sizefor each treatment group is plotted (n = 6) +/2 s.d.doi:10.1371/journal.pone.0092478.g005



inhibit the expression of proteins that are associated with cancer

cell invasion and angiogenesis that are regulated by c-Src as well as

Src-mediated breast cancer cell migration.

The critical roles played by c-Src and STAT3 in cancer

progression make them attractive targets for cancer therapies.

RKIP can simultaneously induce apoptosis and migration and

inhibit cell-survival pathways, including c-Src-STAT3, in human

cancer cells (Figs. 1,3 and 4). We present evidence here that RKIP

can suppress signaling pathways leading to cell proliferation which

suggests that RKIP may be able to inhibit angiogenesis and

metastasis of invasive tumors. There is a clear association between

poor prognosis for patients with tumors and low expression levels

of RKIP. Treatment with chemotherapeutic agents that induce

RKIP expression [29] and/or increase RKIP levels may improve

therapeutic outcome for patients with some cancers.

STAT3 is activated in numerous human tumors including

breast cancer [7] and has been a target for anticancer therapy

[3,5,69]. Osteopontin (OPN), a chemokine-like extracellular

matrix-associated protein, regulates STAT3 activation that leads

to tumor progression and inhibits apoptosis in breast cancer cells

[70]. In contrast, inhibition of the STAT3 signaling pathway using

JAK-specific inhibitors significantly suppresses the growth of

breast cancer cells [71]. Several small molecules have been

identified that directly block STAT3 signaling, and their potency

and specificity are being investigated [72,73]. Small compounds

that block STAT3 signaling through inhibition of Src activity and

induce apoptosis of human breast cancer cells are also under

investigation [74,75]. STAT3 has been shown to be activated in

over 50% of breast tumors, primarily through the IL-6/gp130/

JAK pathway [63,64,76]. Our results indicate that RKIP inhibits

c-Src and IL-6-mediated STAT3 activation and tumor formation

in breast cancer cells (Figs. 1,4 and 5). Significantly, stable over

expression of RKIP attenuates, while ablation of RKIP enhances,

IL-6 mediated STAT3 activation (Fig. 5). Given that STAT3

activation requires both tyrosine phosphorylation by IL-6 and JAK

and serine phosphorylation by MAPK (Raf-dependent), RKIP is a

logical inhibitor of STAT3 activity. Therefore, our data indicate

that RKIP behaves as an inhibitor of multiple pathways leading to

STAT3 activation. It has been suggested that inhibition of STAT3

activation by interfering with IL-6/gp130/JAK signaling pathway

may be an important therapeutic modality in breast cancer [76].

We have shown that RKIP can significantly inhibit activated

STAT3-mediated MDA breast cancer cell tumor xenograft

growth (Fig. 5). Given the realization of the critical roles played

by c-Src and STAT3 in cancer progression, it is imperative to

identify agents to antagonize their function. Clearly, RKIP

represents a protein that can simultaneously induce apoptosis,

inhibit cell survival pathways and tumor growth, including

STAT3, in human breast cancer cells.

Conclusions

RKIP plays a critical role in opposing the effects of pro-

oncogenic STAT3 activation. It is not surprising that expression of

RKIP may be down regulated during tumor progression. There is

a clear association between poor prognosis of a tumor and low

expression levels of RKIP [30–36]. Suppression of prostate and

breast cell survival pathways by RKIP, as demonstrated in this

study, indicates the potential ability of enhanced levels of RKIP to

inhibit angiogenesis and the metastatic capabilities of invasive

tumors. Also, loss of sensitivity to the apoptotic effects of MTIs can

be restored by increased RKIP expression. Thus, it may be

important to consider the use of chemotherapeutic agents, which

induce RKIP expression [29] and/or identify novel compounds to

increase RKIP levels in order to improve therapeutic outcome.

Identifying molecular mechanisms that antagonize STAT3

activation (in this case, via RKIP) will be very informative for

breast and prostate cancer therapy. Further evaluation of RKIP/

STAT3 axis in breast/prostate cancer progression is warranted

and may provide insight into a novel and important therapeutic

approach for the treatment of these diseases.

Acknowledgments

We thank Dr. Djuro Josic of the COBRE Proteomics Core Facility at

Rhode Island Hospital for liquid chromatography/mass spectrometry

analysis and Virginia Hovanesian of the Rhode Island Hospital Core

Laboratories for assistance with figure preparation.

Author Contributions

Conceived and designed the experiments: DC EC KCY. Performed the

experiments: DC SY MD ELM SC-K KB. Analyzed the data: DC SY

ELM SK-C KB KCY EC. Contributed reagents/materials/analysis tools:

KY BB TL EC. Wrote the paper: DC EC BB FA.

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