Honokiol suppresses pancreatic tumor growth, metastasis and desmoplasia by interfering with tumor–stromal cross-talk

Carcinogenesis, Nov 2016

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-term in vitro 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, both in vitro 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 active IKKβ mutant reverses the suppressive effect of HNK on nuclear factor-kappaB 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.

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Honokiol suppresses pancreatic tumor growth, metastasis and desmoplasia by interfering with tumor–stromal cross-talk

Carcinogenesis 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. - Introduction 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 Abbreviations 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 Soft-agar assay 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 Transfection 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. Statistical analysis 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( 4,34 ). 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( 6,35 ). 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. 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Averett, Courey, Bhardwaj, Arun, Arora, Sumit, Srivastava, Sanjeev K., Khan, Mohammad Aslam, Ahmad, Aamir, Singh, Seema, Carter, James E., Khushman, Moh’d, Singh, Ajay P.. Honokiol suppresses pancreatic tumor growth, metastasis and desmoplasia by interfering with tumor–stromal cross-talk, Carcinogenesis, 2016, 1052-1061, DOI: 10.1093/carcin/bgw096