Honokiol suppresses pancreatic tumor growth, metastasis and desmoplasia by interfering with tumor–stromal cross-talk
Honokiol suppresses pancreatic tumor growth, metastasis and desmoplasia by interfering with tumor-stromal cross-talk
Courey Averet 2
Arun Bhardwa 2
Sumit Arora 2
Sanjeev K.Srivastava 2
Mohammad Aslam Khan 2
Aamir Ahmad 2
Seema Singh 1 2
James E.Carter 0
Moh'd Khushman 3
Ajay P.Singh 1 2
0 Department of Pathology, College of Medicine an
1 tDee,partment of Biochemistry and Molecular Biology, College of Medicine
2 Department of Oncologic Sciences , Mitchell Cancer Institu
3 dDepartment of Interdisciplinary Clinical Oncology, Mitchell Cancer Institute, University of South Alabama , 1660 Springhill Avenue, Mobile, AL 36604-1405 , USA
The poor clinical outcome of pancreatic cancer (PC) is largely attributed to its aggressive nature and refractoriness to currently available therapeutic modalities. We previously reported antitumor efficacy of honokiol (HNK), a phytochemical isolated from various parts of Magnolia plant, against PC cells in short-tienrvmitro growth assays. Here, we report that HNK reduces plating efficiency and anchorage-independent growth of PC cells and suppresses their migration and invasiveness. Furthermore, significant inhibition of pancreatic tumor growth by HNK is observed in orthotopic mouse model along with complete-blockage of distant metastases. Histological examination suggests reduced desmoplasia in tumors from HNK-treated mice, later confirmed by immunohistochemical analyses of myofibroblast and extracellular matrix marker proteins (α-SMA and collagen I, respectively). At the molecular level, HNK treatment leads to decreased expression of sonic hedgehog (SHH) and CXCR4, two established mediators of bidirectional tumor-stromal cross-talk, binotvhitro and in vivo. We also show that the conditioned media (CM) from HNK-treated PC cells have little growth-inducing effect on pancreatic stellate cells (PSCs) that could be regained by the addition of exogenous recombinant SHH. Moreover, pretreatment of CM of vehicle-treated PC cells with SHH-neutralizing antibody abolishes their growth-inducing potential on PSCs. Likewise, HNK-treated PC cells respond poorly to CM from PSCs due to decreased CXCR4 expression. Lastly, we show that the transfection of PC cells with constitutively activeβIKmKutant reverses the suppressive effect of HNK on nuclear factorkappaB activation and partially restores CXCR4 and SHH expression. Taken together, these findings suggest that HNK interferes with tumor-stromal cross-talk via downregulation of CXCR4 and SHH and decreases pancreatic tumor growth and metastasis.
Pancreatic cancer (PC) remains a clinical challenge despite as combination therapies; however, none has provided
sigsignificant advancements in our understanding of its mole-cu nificantly superior benefit in patients’ surviva3l,4(). Moreover,
lar pathobiology1(). This year, it is predicted to inflict ~50370 some therapies cause extreme side effects and thus not
recompeople and claim 41 780 lives to become the third leading cause mended to older patients 5(). As a result, 5-year postdiagnosis
of cancer-related deaths in the USA2)(. Over the years, new survival rate of PC patients has remained between 4.0 and 7.0%
treatment options have been tested either as single agents or for the past three decades2(). Clearly, there remains a dire need
Maximum sensitivity substrate kit (Thermo Scientific, Logan, UT);
immunohistochemical (IHC) analysis reagent EZ-Dewax (Biogenex,
Fremont, CA); background sniper, polymer and probe (Biocare Medical,
Concord, CA); VivoGlo™ Luciferin (Promega, Madison, WI)T. he following
antibodies were used: CXCR4 (1:1000; rabbit monoclonal), SHH,
alphasmooth muscle actin (α-SMA) (1:100, rabbit monoclonal) (Epitomics,
Burlingame, CA), collagen I (1:100, rabbit polyclonal) (Abcam, Cambridge,
MA), SHH-neutralizing antibody 5E1 [Developmental Studies Hybridoma
Bank (DSHB), University of Iowa, Iowa City, IA (deposited by T.M.Jessell/S.
Brenner-Morton)], mouse biotinylated antβi--actin (1:20000; Sigma–
Aldrich) and horseradish peroxidase labeled secondary antibodies
(1:2000; Santa Cruz Biotechnology, Dallas, TX).
for novel agents that are more effective, yet relatively safer, in Cell culture and treatment
curbing the aggressive growth of PC. PC cells, MiaPaCa and Colo-357, were procured and maintained in culture
Natural compounds have made a significant impact on the as adherent monolayer as described earlier18(). Cell lines used in this
anticancer drug discovery process6(). One-third of all the drugs study were authenticated by short tandem repeats genotyping (Genetica
approved by the United States Food and Drug Administration DNA Laboratories, Burlington, NC). For HNK treatment, stock solution
(10 mM) of HNK was prepared in dimethyl sulfoxide, stored at −20°C and
(USFDA) for cancer treatment are either natural compounds or diluted at desired concentration with fresh complete medium immed-i
their derivatives 7(,8). Honokiol (HNK), a small biphenolic li-g ately before use. An equal volume of dimethyl sulfoxide (<0.1%) was added
nan consisting of a bioactive para-allyl and ortho-allyl phenols, to the control.
is derived from various parts of the plants oMfagnolia species
(9). Lately, it has attracted a great deal of attention in cancerPlating efficiency assay
research due to its antitumor efficacy along with a desirable Cells (1× 103 cells/well) were seeded in six-well plates and allowed to
spectrum of bioavailability after intravenous administration in adhere and establish for 2h4. Subsequently, cells were treated with ve-hi
animal models (9–11). We also reported previously that it s u-p cle or various doses of HNK (0–5 μM). Fresh media containing HNK or veh-i
pressed growth of PC cells by inducing 1G/S cell-cycle arrest and cle was replaced after every third day. After 2 weeks, colonies were fixed
apoptosis (12). However, a more comprehensive examination of with methanol, stained with crystal violet, photographed and counted
its anticancer efficacy remained to be explored. using image analysis software (Gene Tools, Syngene, Frederick, MD).
In the present study, we evaluated the efficacy of HNK
against long-term growth and malignant phenotypes of PC cells
in vitro and in an orthotopic mouse model of PC, and delineated
underlying molecular mechanisms. HNK reduced the plating
efficiency, anchorage-independent growth, and migratory and
invasive potential of PC cells. In addition, HNK treatment -sig
Soft-agar assay was performed as described previously by us1(9). Briefly,
equal volumes of agarose (1.6%) and regular growth medium were mixed
and plated to form bottom layer (0.8% agar growth medium) in six-well
plates. Cells (2.5× 103 cells/ml) were suspended in regular culture media,
mixed with equal volume of 0.6% agarose, and cell suspension-agar mix
nificantly inhibited the growth of orthotopic pancreatic tumors (2 ml) was seeded as top layer in each well and replenished with tre-at
in nude mice. Moreover, no visible metastases were detected ment media (vehicle or HNK), and incubated for 3 weeks. Fresh media
in any of the HNK-treated mice on bioluminescence and hist-o containing HNK or vehicle was replaced every third day. Colonies were
logical examinations. In addition, pancreatic tumors from HNK- stained with 0.005% crystal violet in phosphate-buffered saline, observed
treated group exhibited decreased desmoplasia as confirmed using Nikon Eclipse microscope (Nikon Instruments) and counted in 10
by immunostaining of extracellular matrix and myofibroblast randomly selected fields (×100 magnification).
marker proteins. Expression of CXCR4 and sonic hedgehog
(SHH), two known promoters of tumor growth, metastasis and Migration and invasion assays
desmoplasia (13–17), was reduced in HNK-treated pancreatic
tumor xenografts and in cancer cell lineins vitro. Further
biochemical studies suggested the role of nuclear factor-kappaB treatment, cells were trypsinized, counted and plated at equal density in
(NF-κB) in suppression of CXCR4 and SHH expression. Together, the upper chamber of non-coated polyethylene teraphthalate membrane
(Boyden Chamber, six-well insert, 8.0 µm; BD Biosciences) (for migration)
these findings lend additional experimental and strong precl-ini or Matrigel-coated polycarbonate membrane (24-well insert, 8.0 µm, BD
cal support for the candidacy of HNK as a novel and effective Biosciences) (for invasion) in serum-free medium. Media supplemented
agent for PC therapy and prevention, either alone or in combi-na with 10% fetal bovine serum was used as chemoattractant in the lower
tion with other treatment modalities. chamber. Cells were allowed to migrate/invade for h1,6 and then cells
remaining in the upper portion were removed. Cells that had migrated/
Materials and methods invaded were fixed, stained with Diff-Quick cell staining kit (Dade Behring,
Newark, DE), mounted on slides and counted in 10 random fields under
Reagents, plasmids and antibodies microscope.
PC cells were grown in six-well plates to subconfluence level, treated
with vehicle or varying concentrations of HNK (0–5 µM) for
h4.8PostThe following reagents were used in this study: Roswell Park Memorial
Institute medium (RPMI-1640); Dulbecco’s modified Eagle medium; Orthotopic xenograft study
penicillin and streptomycin (Invitrogen, Carlsbad, CA); fetal bovine All animal experiments were performed in compliance with Institutional
serum (Atlanta Biologicals, Lawrenceville, GA); HNK (Cayman Chemical Animal Care and Use Committee (IACUC) guidelines.Immunocompromised
Company, Ann Arbor, MI); AMD3100 and Cremophor EL (Sigma–Aldrich, female mice (4–6 weeks old; Harlan Laboratories, Prattville, AL) were a-nes
St Louis, MO); recombinant human SHH (R & D Systems, Minneapolis, thetized with intraperitoneal (i.p.) injection of ketamine (1m00g/kg) and
MN); pGL4.32 (luc2P/NF-B-RE/Hygro) and pRL-TK plasmids (Promega, xylazine (15mg/kg). Luciferase-tagged MiaPaCa cells (×1106/50 μl) were
Madison, WI). pCMV-IKKβ S177E S181E (plasmid number 11105) (A. injected into the pancreas of immunocompromised mice as described
Rao Laboratory; procured through Addgene, Cambridge, MA); pCMV previously (19). Once tumor became palpable (~7 days after injection),
(Origene, Rockville, MD); X-tremeGENE HP DNA Transfection Reagent the animals were randomly divided into two groups (six mice per group).
(Roche, Indianapolis, IN); western blotting SuperSignal West Femto One group received i.p. injection of HNK (150mg/kg body weight, once
daily), whereas the other group received vehicle (Cremophor EL) only. Results
Tumor growth was monitored weekly by bioluminescence imaging using
Xenogen-IVIS-cooled CCD optical system (IVIS Spectrum), following i.p. HNK suppresses the plating efficiency,
anchorageinjection ofd-luciferin (150mg/kg). At the end point (28 days after trea-t independent clonogenic growth and malignant
Tmheenrteianfitteiar,tpiornim),afrinyatluimmaogrsinwgewraesrpeesrefcotremde,dwaenigdhaendi,mmaelasswuerreedsaancdrifmiciceed. phenotypes of PC cells
imaged for detection of near and distant metastases. Tumor volume was In our earlier study, we demonstrated the growth inhibitory
calculated by the following formulaA: ×( B2)/2, where A is the larger andB potential of HNK in PC 1(2). Here, we extended our findings
is the smaller of the two dimensions. In addition, the liver, lung and spleen by examining the effect of HNK on the long-term growth, c-lo
were excised and imaged separately, and then fixed in Bouin’s solution. nogenic potential and malignant properties of two aggressive
Histological and IHC analyses PC cell lines (MiaPaCa and Colo-357). We first performed pl-at
ing efficiency assay, which is an ideal test to monitor the
longIHC analysis was performed on deparaffinized and rehydrated tissue se-c term growth of tumor cells21(). MiaPaCa and Colo-357 cells
tions from formalin-fixed, paraffin-embedded blocks of orthotopically were seeded at low density (500 cells/well), treated with HNK
dweevreeloupseedd paatnc1r:1e0a0ticditluutmioornss.aFsodreshcisrtiboeldogeiacrallieer2x0a()m.Ainlalttiohne,atnutmiboordsieasnd (0.625–5 µM) or vehicle (dimethyl sulfoxide) and incubated
metastatic lesions were stained with hematoxylin and eosin (H&E) and for 2 weeks. Our data demonstrate that the plating efficiency
visualized under microscope (×100 and ×400), and photographed. of MiaPaCa and Colo-357 cells was significantly and gradually
decreased with the increasing concentrations of HNK. As shown
Protein isolation and subcellular fractionation in Figure 1A, we observed that MiaPaCa cells exhibited 1.7-, 3.8-,
Total proteins from vehicle- or HNK-treated PC cells and tumor tissues 8.21- and 51.1-folds, whereas Colo-357 exhibited 1.98-, 3.9-,
7.4were prepared in Nonidet P-40 (NP40) buffer supplemented with protease and 34.1-folds decrease in plating efficiency at 0.625, 1.25, 2.5
and phosphatase inhibitors. Cytoplasmic and nuclear protein fractions of and 5.0 μM HNK treatment doses, respectively, as compared
PC cells were isolated using the Nuclear Extract Kit, as per manufacturer’s with the vehicle-treated controls. Further, we examined the
instructions. effect of HNK on the anchorage-independent growth of PC cells
by performing soft-agar-based clonogenic assay. Similar to the
Immunoblot assay plating efficiency data, the clonogenic potential of HNK-treated
Total protein was resolved on 10% polyacrylamide gels and transferred to PC cells was also reduced by 1.9-, 2.9- and 8.5-folds (in MiaPaCa)
polyvinylidene fluoride membranes. Blots were subjected to a standard and ~1.8-, 5.2- and 17.3-folds (in Colo-357) at 0.625, 1.25 and
immunodetection procedure using specific antibodies against and vis-u 2.5 μM of HNK, respectively. Notably, at 5 µM of HNK treatment,
alized using SuperSignal West Femto Maximum sensitivity substrate kit no to very less visible colonies were observed in both MiaPaCa
with a LAS-3000 image analyzer.
and Colo-357 cellsF(igure 1B).
Collection of conditioned media We next determined the effect of HNK on the aggressive
malignant phenotypes of PC cells. For this, PC cells were treated
PC cells were grown in 10m0m Petri dishes up to 65–70% confluency
and treated with vehicle or HNK (10 µM) for 1h2in regular media. Post- with increasing doses of HNK for 48h, and then trypsinized and
treatment, cells were washed with phosphate-buffered saline and c-ul used for the assessment of migration and invasion ability. We
tured in low serum supplemented regular media for 4h8. Thereafter, observed that the motility of PC was drastically decreased on
conditioned media (CM) was collected, centrifuged at 3g00for 10min to HNK treatment. These data show that in comparison with ve-hi
remove cell debris and designated as CM-Veh (from vehicle-treated cells) cle controls, the number of migratory cells were decreased ~2.2-,
and CM-HNK (from HNK-treated cells). To obtain CM, pancreatic stellate 3.2-, 6.4- and 13.2-folds (in MiaPaCa) and ~1.2-, 2.8-, 7.2- and
11.3cells (PSCs) were grown in low serum supplemented media for 48h, super- folds (in Colo-357) at 0.625, 1.25, 2.5 and 5.0 μM of HNK, respe-c
natant was collected, centrifuged and used in subsequent experiments. tively F(igure 1C). Similarly, invasive potential of MiaPaCa and
WST-1 assay Colo-357 cells was also suppressed by ~1.64- to 12.9-folds and
2.4- to 11.2-folds, respectively, on HNK treatment (0.625–5 µM)
PSCs were seeded in 96-well plate (3000 cells/well), grown forh2u4nder as compared with vehicle-treated controlFsi g(ure 1D). Together,
regular culture conditions and treated with Veh-CM or HNK-CM collectedthese findings indicate that HNK effectively inhibits plating-effi
fcreolmlsvweehricelteroeartHeNdKw-ittrheavteehdicPleCocrelHlNs,Kre(1s0p eμcMti)vfeolryh,4,f8coorlh7l.2eIcntpeadrbayllterly,pPs-Ci ciency, clonogenic potential and malignant phenotypes of PC
nization, counted and equally seeded (3000 cells/well) in 96-well plate. cells.
After overnight incubation, PC cells were treated fohr 7w2ith CM collected
form PSCs (PSCs-CM). Subsequently, viability of PSCs or PC cells was mea-s HNK inhibits pancreatic tumor growth and
ured by WST-1 assay, and percent viability was calculated as described metastasis in an orthotopic mouse model
earlier 1(4,15). To examine the role of SHH, PSCs were treated with either
SHH-neutralizing antibody (in case of Veh-CM) or recombinant SHH (in
case of HNK-CM), and effect on cell viability was examined by WST-1 assay.
Next, we evaluated the antitumor efficacy of HNiKn vivo using
an orthotopic xenograft mouse model of PC. For this, we chose
MiaPaCa cells, which are shown to be highly tumorigenic and
metastatic in mice (19). These cells were luciferase-tagged to
enable non-invasive real-time monitoring of their growth.
Cells were implanted directly into the mouse pancreas and
tumor growth examined on alternate days by palpation. After
7 days of implantation, when tumors became palpable, mice
were divided into two groups. One group of mice received a
daily i.p. injection of HNK (150mg/kg), where the other was
administered only the vehicleF(igure 2A). Tumor growth was
monitored once a week using IVIS imaging system following
i.p. injection of d-luciferin. At the end point (28 days after
treatment initiation), mice were imaged one final time and
To understand the role of NFκ-B/p65, PC cells were transiently tra-ns
fected with constitutively active IKβKmutant (pCMV-IKKβ S177E S181E) or
with its control vector (pCMV) using X-tremeGENE HP DNA Transfection
Reagent as per the instructions by the manufacture.
All the experiments were performed at least three times, independently,
and all data are expressed as mean ± SD. Wherever appropriate, the data
were also subjected to unpaired two-tailed Studentt-’tsest or analysis of
variance.P < 0.05 was considered statistically significant.
then sacrificed. Non-invasive imaging analysis showed that (Figure 2B and C). Moreover, our end-point measurements
tumor growth in HNK-treated group of mice was significantly revealed smaller tumors with average weight of 0.g77and size
decreased in comparison with the vehicle-treated mice group of 99.6 mm 3 in HNK-treated mice, as compared with average
weight of 2.88g and size of 1361.0mm 3 in vehicle-treated mice of desmoplasia, viz. α-SMA and collagen I 1(3). These data
dem(Figure 2D and E). onstrated intense staining oαf-SMA (Figure 4B) and collagen
After removal of primary tumors, mice were imaged again to I (Figure 4C) in tumor tissue sections from vehicle-treated group,
examine the presence of metastatic lesions and secondary me-t whereas weak or no staining was detected in tumor sections of
astatic organs collected. Metastatic dissemination of pancreatic HNK-treated mice (Figure 4B and C). Taken together, these fin-d
tumor cells to various distinct organs [liver (66.6%), lungs (66.6%) ings suggest that HNK inhibits desmoplastic reaction in panc-re
and spleen (83.3%)] of mice was observed in majority of the mice atic tumors.
of vehicle-treated group, as was evident from the strong lu-mi
nescent signal. No metastases were detected in any of the mice, HNK interferes with tumor–stromal cross-talk by
which received HNK (Figure 3A–C). The presence or absence of downregulating the expression of CXCR4 and SHH
metastatic tumor-cell nests to secondary sites was further-con in PC cells
firmed by microscopic analysis of H&E-stained tissue sections Previous studies from our lab and elsewhere have provided
(Figure 3D). Altogether, our data suggest that HNK suppresses strong support for the role of CXCR4 and SHH in pancreatic
pancreatic tumor growth and eliminates metastatic spread. tumor growth, metastasis and desmoplasia by enabling bidire-c
tional tumor–stromal cross-tal1k5(,17,18,25–27). Therefore, we
Desmoplasia is decreased in pancreatic tumor examined the expression status of CXCR4 and SHH in tumor
xenografts of HNK-treated mice sections by IHC analyses. Data show a decrease in CXCR4 and
Extensive desmoplasia is a fundamental characteristic of pa-n SHH expression in pancreatic tumors of HNK-treated group, as
creatic tumors, which has been suggested to be of significance compared with that of vehicle-treated grouFpi(gure 5A). This is
from the pathobiological and clinical standpoints22–(24). further supported by the immunoblotting data from proteins
Therefore, we next examined the effect of HNK on the desm-o isolated from fresh-frozen tumor xenograftsFi(gure 5B). To
furplastic reaction in orthotopic pancreatic tumors. To accomplish ther confirm these observations, we treated both MiaPaCa and
this, tumor tissue sections were stained with H&E and exa-m Colo-357 cells in culture with various doses of HNK or vehicle
ined under microscope. The presence of excessive dense fibrotic and examined the expression of CXCR4 and SHH by quantitative
area was revealed in the tumors of control group mice, whereas reverse transcription–PCR and immunoblot assays. Treatment
it was only minimal in sections of tumor tissues from HNK- with HNK for 48h resulted in a dose-dependent decrease in the
treated group of mice F(igure 4A). The presence of desmoplasia expression of both CXCR4 and SHH at mRNA (Supplementary
was further confirmed by immunostaining for specific markers Figure 1 is available atCarcinogenesis Online) and protein
levels F(igure 5C). To confirm the role of SHH downregulation in Significant growth inductionP (< 0.01) was observed only in
decreased tumor–stromal cross-talk, we treated PSCs with CM PSCs treated with Veh-CMF(igure 5C). Furthermore, this growth
from either vehicle (Veh-CM) or HNK-treated PC cells (HNK-CM). induction was abrogated when Veh-CM was preincubated
with anti-SHH antibodies, whereas it was induced in HNK- (pCMV) prior to the HNK treatment.The effects of these trans-fec
CM-treated PSCs on addition of exogenous recombinant SHH tions on the transcriptional activity and nuclear localization of
(Figure 5D). In a reverse approach, when we exposed vehicle NF-κB were examined following HNK treatment. Transcriptional
or HNK-pretreated PC cells with CM of PSCs (CM-PSCs), we activity F(igure 6A) and nuclear localization of NκFB- (Figure 6B)
observed significantly less growth induction in case of HNK- was inhibited in control vector-transfected cells on HNK t-reat
pretreated MiaPaCa and Colo-357 (44.1 and 51.5%, respectively) ment; where no inhibitory effects were observed in MiaPaCa and
cells as compared with that pretreated with vehiclFeig(ure 5E). Colo-357 cells transfected with IKβKmutant (Figure 6A and B).
Further, vehicle- and HNK-treated PC cells were incubated with Moreover, when we examined the effect of restored NFκ-B
actiAMD3100, a CXCR4 antagonist, 30min. prior to their exposure vation on the HNK-mediated downregulation of CXCR4 and
to CM-PSCs to ascertain the role of CXCR4 in the CM-PSCs- SHH, we observed that the expression of CXCR4 and SHH was
mediated growth induction. Data demonstrate that CM-PSCs- regained to an appreciable extent, but not completelFyig(ure 6C).
induced growth of PC cells is remarkably abrogated when they These findings suggest that suppression of NFκ-B activation by
are pretreated with AMD3100, whereas no effect of AMD3100 HNK is, at least partly, responsible for its inhibition of CXCR4
is observed in HNK-pretreated PC cells, suggesting that CXCR4 and SHH expression in PC cells.
downregulation is involved in the poor response of HNK-treated
PC cells to CM-PSCs F(igure 5E). Together, these findings esta-b
lish the role of HNK in interfering with tumor–stromal cross-talk Discussion
via CXCR4 and SHH downregulation. PC is a difficult cancer to manage and treat. The outcomes of
patients with this deadly cancer have not improved much
HNK-induced inhibition of NF-κB is responsible, in over last several decades, with most chemotherapies affo-rd
part, for downregulation of CXCR4 and SHH ing only minor improvements in overall survival 1(). In an
We previously reported HNK as a potent inhibitor of NκFB- acti- earlier study, we observed an anticancer activity of HNK in PC
vation in PC cells1(2). Since NF-κB is also a transcriptional reg-u cells through induction of cell-cycle arrest and apoptos1i2s).(
lator for both CXCR4 and SHH1(5,18), we examined its role in Moreover, our findings established, for the first time, NκFB-as
their observed downregulation in HNK-treated PC cells. For this, a molecular target for HNK, which was also suggested to m-edi
PC cells were transfected with plasmids either expressing c-on ate HNK-potentiated chemosensitization. Since then, HNK has
stitutively active mutant of IKβK(IKKβ-SSEE) or a control vector been shown to affect NFκ-B signaling in lung2(8) and colon (10)
cancer cells as well. The present study further affirms the-sig cells 1(6). In the current study, we noted an inhibitory effect of
nificance of NF-κB targeting by HNK that leads to downreg-ula HNK on this signaling and associated functional consequences.
tion of molecules involved in tumor–stromal cross-talk and thus Not only did we observe downregulation of CXCR4 by HNK, we
suggest wider implications for antitumor efficacy of HNK. also found significant disruption of tumor–stromal interactions.
Pancreatic tumors are highly aggressive in nature and, in most CM from HNK-treated tumor cells failed to stimulate growth of
cases, have already metastasized at the time of its diagnosi2s9(). PSCs and vice versa. Inhibition of CXCR4 and the tumor–str-o
Metastasis is the major cause of cancer-related deaths, and this mal cross-talk by HNK can have big implications because of the
is true for PC as well3(0). In fact, most pancreatic tumors, if not documented role of CXCR4/CXCR12 signaling in cancer meta-s
metastasized, are so genetically advanced that their resection tasis (33) and drug resistance1(
). Activation of this signaling
is feared to cause metastases3(1). Clearly, we need approaches axis induces diverse signaling pathways that act independently
that could target the aggressive nature of pancreatic tumors. Inand cross-talk with each other and/or other active signaling
this regard, our data demonstrating the suppressive effect of pathways to promote a variety of cancer-relevant cellular and
HNK not only on tumor growth but also on malignant phe-no molecular responses 1(
). Besides, we also observed an inh-i
types are highly significant and could have implications for both bition of SHH expression by HNK, which is highly relevant to
PC therapy and prevention. Another unique characteristic of PC tumor–stromal interactions13(,15,25). Tumor–stromal cross-talk
is existence of high desmoplasia, which is suggested to promote was significantly affected when SHH was downregulated, either
lymphangiogenesis, metastasis and chemoresistance 2(2,24). It as a consequence of HNK treatment or its functional inhibition
is also being explored as a factor that influences the balance by the use of SHH antibody as a proof of principal. Abrogation
between immune-dependent and immune-independent regula- of HNK activity by recombinant SHH further supported the
tion of tumor growth2(3). In our study, we observed an inhib-i mechanistic importance of SHH in the disruption of tumor–
tory effect of HNK on desmoplasia, as characterized by reduced stromal interactions by HNK. It thus appears that HNK impacts
secretion of extracellular matrix protein (collagen I) and di-min both tumor and stromal compartments by inhibiting the
crossished staining for myofibroblast marker (a-SMA). Myofibroblasts talk between tumor and stroma cells through its modulation of
are a major component of desmoplastic pancreas and originate CXCR4/CXCR12 axis and downregulation of SHH.
from activated PSCs 3(2). Thus, our data provided direct evidence The role of NF-κB, an oncogenic transcription factor, in p-ro
for a dual impact of HNK on pancreatic tumors through targ-et moting several biological processes of cancer significance, such
ing its tumor and stromal compartments. as proliferation, survival, invasion and metastasis, and th-er
CXCR4 is a chemokine receptor for CXCR12 (also called st-ro apy resistance has been very well documented3(6). Emerging
mal-derive factor 1). CXCR12/CXCR4 signaling is shown to play evidence confirms the constitutive activation of NκFB- in
important roles in tumor–stromal cross-talks in several tumors, tumors of several types including PC3(7,38), wherein its
aberwherein CXCR12 secreted by stromal cells in the tumor mic-ro rant activation enhances the transcription of proinflammatory
environment stimulates the growth of CXCR4-expressing tumor and protumorigenic genes. Similar to our earlier finding1s2(),
we found that HNK inhibited NFκ-B in pancreatic tumor cells. In 8. Dias, D.A. et al. (2012) A historical overview of natural products in drug
addition, our mechanistic studies revealed the partial invo-lve discovery. Metabolites, 2, 303–336.
ment of NF-κB in HNK-mediated downregulation of CXCR4 and 9. Arora, S. et al. (2012) Honokiol: a novel natural agent for cancer pre-ven
SHH in PC cells. We have earlier established the role of NκBF-in tion and therapy. Curr. Mol. Med., 12, 1244–1252.
direct transcriptional regulation of CXCR4 and SHH in PC cells 10. Hua, H. et al. (2013) Honokiol augments the anti-cancer effects of ox-ali
platin in colon cancer cells. Acta Biochim. Biophys. Sin. (Shanghai), 45,
(15,18). Since NF-κB acts as a downstream and upstream effector 773–779.
of CXCL12/CXCR4 signaling in a positive feedback mechanism, 11. Leeman-Neill, R.J. et al. (2010) Honokiol inhibits epidermal growth f-ac
it not only sustains the increasing activation of this signaling tor receptor signaling and enhances the antitumor effects of epi-der
loop (15,39) but also amplifies the impact on tumor phenotypes mal growth factor receptor inhibitors. Clin. Cancer Res., 16, 2571–2579.
through activation of several other CXCR4-downstream sig-nal 12. Arora, S. et al. (2011) Honokiol arrests cell cycle, induces apoptosis, and
ing pathways. More importantly, through regulation of SHH, it potentiates the cytotoxic effect of gemcitabine in human pancreatic
can further diversify impact by involving stromal cells and -ini cancer cells. PLoS One, 6, e21573.
tiating a bidirectional tumor–stromal cross-ta1l5k).(Therefore, 13. Bailey, J.M. et al. (2008) Sonic hedgehog promotes desmoplasia in
panthe concurrent activation of several cancer-relevant pathways creatic cancer. Clin. Cancer Res., 14, 5995–6004.
14. Bhardwaj,A. et al. (2014) CXCL12/CXCR4 signaling counteracts
docetaxelpromotes the proliferation, survival and metastasis of tumor induced microtubule stabilization via p21-activated kinase 4-dependent
cells 2(0,40,41). In accordance with this, we observed that HNK activation of LIM domain kinase 1. Oncotarget, 5, 11490–11500.
treatment inhibited the growth and metastasis of pancreatic 15. Singh, A.P. et al. (2012) CXCL12/CXCR4 protein signaling axis induces
tumor cells in orthotopic mouse model. These findings are of sonic hedgehog expression in pancreatic cancer cells via extracellular
high clinical importance from the standpoints of develo-p regulated kinase- and Akt kinase-mediated activation of nuclear-fac
ing novel HNK-based therapeutic and prevention strategies. torκB: implications for bidirectional tumor-stromal interactions. J. Biol.
Moreover, these findings establish the mechanistic bases for the Chem., 287, 39115–39124.
inhibitory effects of HNK against PC. 16. Sun, X. et al. (2010) CXCL12/CXCR4/CXCR7 chemokine axis and cancer
Taken together, our study is indicative of a multifaceted a-nti progression. Cancer Metastasis Rev., 29, 709–722.
tumor efficacy of HNK against PC cells, botihn vitro and in vivo. 17. Xu, X. et al. (2014) Sonic hedgehog-Gli1 signaling pathway regulates the
epithelial mesenchymal transition (EMT) by mediating a new target
Our results support the role of HNK in suppressing metastatic gene, S100A4, in pancreatic cancer cells. PLoS One, 9, e96441.
machinery through inhibition of NFκ-B and its downstream 18. Arora, S. et al. (2013) An undesired effect of chemotherapy: gemcitabine
targets CXCR4 and SHH, resulting not only in suppression of promotes pancreatic cancer cell invasiveness through reactive oxygen
tumor growth but also in a complete remission of metastasis species-dependent, nuclear factor kappaB- and hypoxia-inducible factor
in an orthotopic mouse model. This is also accompanied by an 1alpha-mediated up-regulation of CXCR4. J. Biol. Chem., 288; 21197–21207.
inhibitory effect of HNK on desmoplasia through inhibition of 19. Srivastava, S.K. et al. (2015) MYB is a novel regulator of pancreatic
tumor–stromal interactions and could be of significance from tumour growth and metastasis. Br. J. Cancer, 113, 1694–1703.
therapeutic standpoints. Thus, our results hold a lot of promise 20. Bhardwaj,A. et al. (2014) Restoration of PPP2CA expression reverses epit-he
for the eventual goal of developing HNK as an effective th-era lial-to-mesenchymal transition and suppresses prostate tumour growth
and metastasis in an orthotopic mouse model. Br. J. Cancer, 110, 2000–2010.
peutic or preventive agent through its targeting of signaling 21. Munshi, A. et al. (2005) Clonogenic cell survival assay. Methods Mol.
pathways of high significance in PC. Med., 110, 21–28.
22. Erkan, M. et al. (2012) The role of stroma in pancreatic cancer: diag n-os
Supplementary material tic and therapeutic implications. Nat. Rev. Gastroenterol. Hepatol., 9,
Supplementary Figure 1 can be found at http://carcin.oxford- 23. Puré, E. et al. (2016) Can targeting stroma pave the way to enhanced
journals.org/ antitumor immunity and immunotherapy of solid tumors? Cancer
Immunol. Res., 4, 269–278.
Funding 24. Olive, K.P. et al. (2009) Inhibition of Hedgehog signaling enhances de-liv
ery of chemotherapy in a mouse model of pancreatic cancer. Science,
NIH/NCI (CA167137 and CA175772 to A.P.S.); USAMCI. 324, 1457–1461.
Conflict of Interest Statement: None declared. 25. Bailey, J.M. et al. (2009) Sonic hedgehog paracrine signaling regulates
metastasis and lymphangiogenesis in pancreatic cancer. Oncogene,
References 28, 3513–3525.
26. Li, X. et al. (2014) Sonic hedgehog paracrine signaling activates stromal
1. Garrido-Laguna, I. et al. (2015) Pancreatic cancer: from state-of-the- cells to promote perineural invasion in pancreatic cancer. Clin. Cancer
art treatments to promising novel therapies. Nat. Rev. Clin. Oncol., 12, Res., 20, 4326–4338.
319–334. 27. Marchesi, F. et al. (2004) Increased survival, proliferation, and mig-ra
2. Siegel, R.L. et al. (2016) Cancer statistics, 2016. CA Cancer J. Clin., 66, tion in metastatic human pancreatic tumor cells expressing functional
7–30. CXCR4. Cancer Res., 64, 8420–8427.
3. Moore, M.J. et al.; National Cancer Institute of Canada Clinical Trials 28. Singh, T. et al. (2013) Honokiol inhibits non-small cell lung cancer cell
Group. (2007) Erlotinib plus gemcitabine compared with gemcitabine migration by targeting PGE(2)-mediated activation of beta-catenin
alone in patients with advanced pancreatic cancer: a phase III trial of signaling. PLoS One, 8, e60749.
the National Cancer Institute of Canada Clinical Trials Group. J. Clin. 29. Vincent, A. et al. (2011) Pancreatic cancer. Lancet, 378, 607–620.
Oncol., 25, 1960–1966. 30. Das, S. et al. (2015) Pancreatic cancer metastasis: are we being
pre4. Von Hoff, D.D. et al. (2013) Increased survival in pancreatic cancer with EMTed? Curr. Pharm. Des., 21, 1249–1255.
nab-paclitaxel plus gemcitabine. N. Engl. J. Med., 369, 1691–1703. 31. Paik, K.Y. et al. (2012) Analysis of liver metastasis after resection for
5. Allison, M. (2012) Hedgehog hopes lifted by approval and stung by fa-il pancreatic ductal adenocarcinoma. World J. Gastrointest. Oncol., 4,
ure. Nat. Biotechnol., 30, 203. 109–114.
6. Newman, D.J. (2008) Natural products as leads to potential drugs: an 32. Omary, M.B. et al. (2007) The pancreatic stellate cell: a star on the rise
old process or the new hope for drug discovery? J. Med. Chem., 51, in pancreatic diseases. J. Clin. Invest., 117, 50–59.
2589–2599. 33. Guo, F. et al. (2016) CXCL12/CXCR4: a symbiotic bridge linking cancer
7. Kneller, R. (2010) The importance of new companies for drug discovery: cells and their stromal neighbors in oncogenic communication n-et
origins of a decade of new drugs. Nat. Rev. Drug Discov., 9, 867–882. works. Oncogene, 35, 816–826.
34. Duda , D.G. et al. ( 2011 ) CXCL12 (SDF1alpha)-CXCR4/CXCR7 pathway 38 . Wharry , C.E. et al. ( 2009 ) Constitutive non-canonical NFkappaB inhibition: an emerging sensitizer for anticancer therapies? Clin. C-an signaling in pancreatic cancer cells . Cancer Biol. Ther. , 8 , 1567 - cer Res., 17 , 2074 - 2080 . 1576.
35. Liu , X. et al. ( 2014 ) Activation of STAT3 is involved in malignancy me-di 39 . Helbig , G. et al. ( 2003 ) NF-kappaB promotes breast cancer cell mig-ra ated by CXCL12-CXCR4 signaling in human breast cancer . Oncol. Rep ., tion and metastasis by inducing the expression of the chemokine 32 , 2760 - 2768 . receptor CXCR4. J. Biol. Chem ., 278 , 21631 - 21638 .
36. Baud , V. et al. ( 2009 ) Is NF-kappaB a good target for cancer therapy? 40 . Fan , Y. et al. ( 2013 ) NF-kappaB and STAT3 signaling pathways collab-o Hopes and pitfalls . Nat. Rev. Drug Discov., 8 , 33 - 40 . ratively link inflammation to cancer . Protein Cell , 4 , 176 - 185 .
37. Lessard , L. et al. ( 2006 ) Nuclear localization of nuclear factor-kappaB 41 . Wen , W. et al. ( 2015 ) Synergistic anti-tumor effect of combined inhi-bi p65 in primary prostate tumors is highly predictive of pelvic lymph tion of EGFR and JAK/STAT3 pathways in human ovarian cancer . Mol. node metastases. Clin. Cancer Res. , 12 , 5741 - 5745 . Cancer, 14 , 100 .