recombinant arabidopsis hsp70 sustains cell survival and ... · idase-coupled secondary antibodies...

Cancer Biology and Signal Transduction

Recombinant Arabidopsis HSP70 SustainsCell Survival and Metastatic Potential of BreastCancer CellsAlessandra Nigro1, Loredana Mauro2, Francesca Giordano2, Salvatore Panza3,Rina Iannacone4, Grazia Maria Liuzzi5, Saveria Aquila2,3, Francesca De Amicis2,3,Francesco Cellini4, Cesare Indiveri1, and Maria Luisa Panno2

Abstract

The chaperone HSP70 protein is widely present in manydifferent tumors and its expression correlates with an increasedcell survival, lowdifferentiation, and poor therapeutic outcome inhuman breast cancer. The intracellular protein has prevalently acytoprotective function, while the extracellular HSP70 mediatesimmunologic responses. Evolutionarily, HSPs are well conservedfrom prokaryotes to eukaryotes, and human HSP70 shows astrong similarity to that of plant origin. In the current article, wehave tested the potential effect of recombinant HSP70, fromArabidopsis thaliana, on cell survival and metastatic properties ofbreast cancer cells. Our data show that HSP70 sustains cellviability in MCF-7 and MDA-MB-231 breast tumoral cells andincreases Cyclin D1 and Survivin expression. The extracellularHSP70 triggers cell migration and the activation of MMPs par-

ticularly in MDA-MB-231 cells. Furthermore, under UV-inducedstress condition, the low levels of phospho-AKTwere increased byexogenous HSP70, together with the upregulation of Cyclin D1,particularly in the tumoral cell phenotype. On the other hand, UVincreased TP53 expression, and the coincubation ofHSP70 lowersthe TP53 levels similar to the control. Thesefindings correlatewiththe cytoprotective and antiapoptotic role of HSPs, as reported indifferent cellular contexts. This is the first study onmammary cellsthat highlights how the heterologous HSP70 from Arabidopsisthaliana sustains cell survival prevalently in breast cancer celltypes, thus maintaining their metastatic potential. Therefore,targeting HSP70 would be of clinical importance since HSP70blocking selectively targets tumor cells, in which it supports cellgrowth and survival. Mol Cancer Ther; 15(5); 1063–73. �2016 AACR.

IntroductionThe expression of HSPs is a basic and well-conserved cellular

response to a wide variety of stress conditions. In fact, althoughthese proteins are produced at low levels in basal conditions,their expression increases during cellular damage in order totrigger the repair, necessary for the salutary resolution of theinsult.

However, HSPs play also a key role in the folding and pro-tein metabolism participating in the assembly of multimericprotein complexes as well as in their transport. These actionsinvolve their pivotal role within the cell, even though it has beenrecently highlighted the extracellular function of the HSPs. Inthis compartment, HSPs act as alert stress signals priming othercells, particularly those of the immune system, to avoid thepropagation of the insult and favor resolution (1). Extracellular

HSP70 has also been reported to increase microbicide capacityand chemotaxis of neutrophils (2, 3), to enhance phagocytosis(4), and to modulate the response of monocytes to endo-toxin (5). Overall, extracellular HSP70 has been associated withboth immunostimulatory and immunosuppressive activities,depending on the cell context and on the microenvironmentin which the protein exists (6).

HSPs interact with antigen-presenting cells eliciting a cascade ofevents including representation of HSP-chaperoned peptides byMHC and maturation of dendritic cells. These properties of HSPsalso allow them to be used for immunotherapy of cancers andinfections in novel ways (7).

However, various reports have emphasized the involvement ofHSPs in tumorigenesis, as theymay provide a selection advantageto cancer cells to better support cell growth and survival. Inparticular, HSP70 has been shown to play a negative role in themitochondrial apoptotic pathway by directly binding to Apaf-1,thus avoiding caspase activation (8, 9).

Studies, in this field, have demonstrated that extracellularHSP70 in breast cancer cells, with other assistant proteinsHop, HSP40, and p23, increases the association of HSP90aand MMP-2 to address its activation (10, 11). Accordingly,high expressions of HSP70 are always associated with moreaggressive tumor phenotypes, lymph node metastases, andincreased breast cancer cell survival that correlates with poorprognosis (12–14).

Tumor progression, mediated by exogenous HSP70, is sup-ported bymany signals, such as activation of EGFR, stimulation ofMAPK signaling, secretion of IL8, upregulation of NF-kB/AP-1,

1Department of Biology, Ecology and Earth Sciences, University ofCalabria, Arcavacata di Rende (CS), Italy. 2Department of Pharmacyand Health and Nutrition Sciences, University of Calabria, Arcavacatadi Rende (CS), Italy. 3Health Center, University of Calabria, Arcavacatadi Rende (CS), Italy. 4ALSIA-Research Center Metapontum Agrobios,Metaponto, Matera, Italy. 5Department of Biosciences, Biotechnolo-gies and Biopharmaceutics, Aldo Moro University, Bari, Italy.

CorrespondingAuthor:Maria Luisa Panno, Department of Pharmacy andHealthand Nutrition Sciences, University of Calabria, via Pietro Bucci, cubo 4C,Arcavacata di Rende, Cosenza 87036, Italy. Phone: 399-8449-2927; Fax:399-8449-2929; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0830

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

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on February 4, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst March 3, 2016; DOI: 10.1158/1535-7163.MCT-15-0830

http://mct.aacrjournals.org/

and subsequent activation of MMPs (15–18). In addition, extra-cellular HSP70 acts as a risk signal on mononuclear cells toprovide an inflammatory tumor microenvironment. By acting onthe immune system, it can trigger signaling in tumor cells in anautocrine or paracrine fashion via binding to Toll-like receptors,thus promoting invasion and angiogenesis (13).

Evolutionarily, HSPs are well conserved from prokaryotes toeukaryotes, such as in plants and humans (19).The utilization ofHSPsderived fromplants canbe exploited to assess their actiononmammalian immune system or to evaluate their action on tumorcells.

HSPs purified from plants represent a powerful option sincethey have several advantages including low costs, absence ofmammalian pathogens, and no endotoxic side effect.

Recently, it has been reported how recombinant HSP70(r-AtHSP70) of plant origin, purified from bacterial cultures, wasable to protect rat cardiac and hepatic function under ischemicand sepsis conditions (20).

Starting from such evidence, in the current article, we used therecombinant HSP70, obtained from heat stressed Arabidopsisthaliana cDNA, to evaluate, for the first time, its potential effecton the survival response in the normal MCF-10A human mam-mary epithelia cells and in bothMCF-7 andMDA-MB-231 humanbreast-derived cells with low and high tumorigenic potential,respectively.

Materials and MethodsMaterials

DMEM/F12, DMEM, L-glutamine, penicillin, streptomycin,FBS, horse serum, and PBS were purchased from Gibco. Insulin,hydrocortisone, cholera toxin, EGF acetone, Tris-HCl, SDS, bro-mophenol, glycerol, gelatin, Commassie Brilliant Blue R250 werefrom Sigma. Acrylamide/bis acrylamide, 40% solution was fromApplichem. Anti-antibodies HSP70, Cyclin D1, TP53, Survivin,phosphoERK1/2, ERK1/2,b actin, and endostatinwere purchasedfrom Santa Cruz Biotechnology, while anti-antibodies phospho-Akt, Akt, phospho-p38MAPK, p38MAPK from Epitomics. Anti-antibody phospho-TP53 (Ser-15) was from Cell Signaling Tech-nology. The His Tag antibody was purchased from GenScript.

Cell culturesMCF-7, MDA-MB-231, MCF-10A, and BJhTERT cells were

obtained from ATCC where they were authenticated, storedaccording to the supplier's instructions, and used within 4months after frozen aliquot resuscitations. Every 4 months,cells were authenticated by single tandem repeat analysis at ourSequencing Core; morphology, doubling times, estrogen sen-sitivity, and mycoplasma negativity were tested (Myco Alert;Lonza). ER-positive MCF-7 human breast cancer cells werecultured in DMEM/Nutrient Mixture F-12Ham (DMEM/F-12)supplemented with 5% FBS; L-glutamine (1%) and penicillin/streptomycin (1%).

MDA-MB-231 is a metastatic human breast cancer cell linetriple-negative for ERa, PR, and HER-2. MDA-MB-231 cells werecultured inDMEM supplemented with 5% FBS; L-glutamine (1%)and penicillin/streptomycin (1%).

The MCF-10A cell line is a nontumorigenic human epithelialbreast cell line responsive to insulin, glucocorticoids, choleratoxin, and EGF. The MCF-10A cells were cultured in DMEM/F-12 supplemented with 5% horse serum (HS); L-glutamine

(1%), penicillin/streptomycin (1%), 100 ng/mL cholera toxin;hydrocortisone (0.5 mg/mL); insulin (10 mg/mL) and EGF(20 ng/mL).

The BJhTERT human fibroblast cell line was cultured in min-imum essential medium (MEM) with 10% FBS, L-glutamine(1%), and penicillin/streptomycin (1%). All cells were grown at37�C under 5% CO2 humidified incubator.

Normal lymphocytesPeripheral blood was obtained from healthy volunteers.

Peripheral blood mononuclear cells (PBMC) were isolated byFicoll–Paque Plus (GE Healthcare) gradient centrifugation from10 mL of blood and then washed once with cold PBS. Totalprotein extracts fromPBMCwere lysed inRIPAbuffer andused forWestern blot analysis.

HSP-70 His-tag ArabidopsisThe recombinant HSP70 protein, tested in our experiments,

derives from Arabidopsis thaliana plants. The coding region of thegene HSP70-4 was amplified by PCR from cDNA coming fromleaf tissues exposed to heat stress. The amplified sequence wasconfirmed by sequencing and cloned in the E. coli expressionvector pET27þ resulting in pETAtHSP70. Competent BL21 cellswere transformed with the pETAtHSP70 plasmid. The recombi-nant AtHSP70 protein was extracted and purified as reportedpreviously (20). Briefly, the protein was purified by affinitychromatography and then dialyzed. The purity of r-AtHSP70 waschecked by SDS-PAGE and Western blot analysis as describedpreviously (20).

These experiments were performed at the Research Center"Metapontum Agrobios," which supports research and develop-ment of the agri-food system through plant biotechnology. In allexperiments, purified r-AtHSP70 was solubilized in Tris-HCl, pH7.5, phosphate buffer. The same buffer alone was added to thecontrol samples.

Western blot analysisMCF-7, MDA-MB-231, MCF-10A, and BJhTERT cells were

grown in 10-cm dishes to 70% to 80% confluence, treated ornot with r-AtHSP70 for 72 hours and lysed. Total cellular extractswere subject to SDS-PAGE and transferred to a nitrocellulosemembrane by electroblotting as previously described (21, 22).Finally, the membranes were incubated with horseradish perox-idase-coupled secondary antibodies for 1 hour at room temper-ature. The membranes were washed with TBST 1� (TBS andTween 20) after each antibody binding reaction. Detection ofeach protein was performed using an ECL kit (Santa CruzBiotechnology).

ImmunofluorescenceAs to immunofluorescence analysis, the cells were fixed with

4% paraformaldehyde, permeabilized with 0.2% Triton X-100,followed by blocking with BSA (3%, 30 minutes), and thenincubatedwith anti-r-AtHSP70His-tag antibody (4�C, overnight)and with fluorescein-conjugated secondary antibody (30 min-utes, room temperature). IgG fluorescein-conjugated secondaryantibody was used as negative control (NCtrl). DAPI staining wasused for nuclei detection (Sigma). Protein cellular localizationwas observed under a fluorescence microscope (Olympus BX51fluorescence microscope; �100 objective).

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MTT assayFor viability assay, MCF-7, MDA-MB-231, and MCF-10A cells

were seeded (1 � 104) in 96-well plates and serum-starvedfor 24 hours before the addition of treatment. On the

second day, the cells were treated with r-AtHSP70 at 5, 10,and 20 mg/mL and incubates at 37�C for 72 hours. At the endof treatment, the medium was removed and replaced withMTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium

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Figure 1.Expression of HSP70 in normal and breast cancer cells. A, endogenous protein levels of HSP70 were assessed in normal cells: BJhTERT human fibroblast cell line,human peripheral lymphocytes and MCF-10A cell line, as well as in MCF-7 and MDA-MB-231 breast tumor cells. Protein extracts were resolved by SDS-PAGE andsubjected to immunoblot analysis with rabbit antiserum against human HSP70. The histograms represent the mean � SD of three separate experimentsin which band intensities were evaluated in terms of optical density arbitrary units. B, 40 mg extracts of MCF-7 cells untreated (Ctrl) or treated (r-AtHSP70) for 72hourswith His-tailed r-AtHSP70 and immunoblotted with a-His-tag r-AtHSP70 antibody (top). Middle, the filter was reprobed with anti-human HSP70 (a-hHSP70).Bottom, b-actinwas used as control for protein loading. (Ctrlþ) correspond to 40 mg of pure r-AtHSP70 loaded onto the gel. C, immunofluorescence of recombinant-AtHSP70 through a-His-tag r-AtHSP70 (FITC-conjugated) in MCF-7 and MCF-10A cells treated for 48 hours with vehicle (Ctrl) or 10 mg of r-AtHSP70.DAPI staining was used to visualize the cell nucleus. NCtrl, negative control, coincides to the sample processed without primary antibody. Representative resultsare shown.

Effects of r-AtHSP70 on Breast Cancer Cells

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bromide (2 mg/mL)] and incubated at 37�C for 3 to 4 hours(22). During this incubation, viable cells with active metabo-lism convert MTT into a purple colored formazan product withan absorbance maximum near 570 nm. Finally, the absorbancewas measured on each well in a multiplate reader (Multiscan exThermo Scientific).

Clonogenic assayClonogenic assay was performed by plating MCF-7, MDA-MB-

231, andMCF-10A cells in 6-well plates at a density of 3,000 cells/well. The day after, the cells were starved and then r-AtHSP70(10mg/mLand20mg/mL)was added to cultures. Themediumwasreplaced with fresh medium containing r-AtHSP70 every 3 days.On the tenthday, themediumwas removed and cell colonieswerestained with crystal violet (0.1% in 20% methanol). Coloniescontaining >50 normal-appearing cells were counted and photo-graphed through a digital camera. The experiment was repeatedthree times in triplicates.

Detection of MMPs by zymographyThe MCF-7, MDA-MB-231, and MCF-10A cells were treated

or untreated with r-AtHSP70 at final concentration of 10 and20 mg/mL for 24 and 48 hours at 37�C and, at the end of

incubation, the conditioned medium was collected and sub-jected to zymographic analysis (23). Briefly, 40 mL of eachsupernatant were precipitated with cold acetone at 20�C for1 hour, centrifuged and the pellets were solubilized with aloading buffer (Tris-HCl, pH 6.8, 125 mol/L, SDS 4%, bromo-phenol 0.10%, Glycerol 0.25%). The obtained samples wereseparated in 7.5% polyacrylamide gels copolymerized with0.1% (w/v) gelatin. Stacking gels contained 5.4% polyacryl-amide. After electrophoresis, the gels were washed in 2.5%(w/v) Triton X-100, 10 mmol/L CaCl2, 50 mmol/L Tris-HCl,pH 7.4, and then incubated overnight at 37�C in 1% (w/v)Triton X-100, 50 mmol/L Tris-HCl, 10 mmol/L CaCl2, pH 7.4(developing buffer). After staining and de-staining of the gels,gelatinase activity was detected as a lysis area on the bluebackground of the gel fractionated by SDS-PAGE on a 7,5%gel polyacrylamide 40% (Acrylamide/bis acrylamide, 40%solution, Applichem) containing 1.0 mg/mL gelatin (Sigma)under nonreducing conditions.

After washing in Tris buffer, the gel was stainedwith CoomassieBrilliant Blue R-250, and subsequently destained with 10% (v/v)acetic acid. The non-staining bands resulting from digestion ofthe substrate by gelatinase enzymes were then visualized.

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Figure 2.Effects of r-AtHSP70 on the proliferation and survival of breast cells. A, MTT growth assays were performed in MCF-7, MDA-MB-231, and MCF-10A cells treated withvehicle (Ctrl) or increasing doses of r-AtHSP70 as indicated for 72 hours. Cell proliferation is expressed as fold change � SD relative to vehicle-treated cells ofthree different experiments eachperformed in triplicate. � , P < 0.05 comparedwith vehicle. B, clonogenic assay inMCF-7, MDA-MB-231 andMCF-10A cells. The cellsweregrown in the absence (Ctrl) or presence of r-AtHSP70 (10 and 20 mg/mL) for 10 days. The results shown are representative of three independent experimentsperformed in triplicates. C, the histogram is the number of colonies expressed as percentage relative to control. � , P < 0.01, compared with untreated cells (Ctrl).

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Cell migration assayCell migration was assessed in scratch assays. Briefly, confluent

MCF-7,MDA-MB-231, andMCF-10A cells plated on tissue culturedishes were manually scratched with a 10-mL pipette tip, washedwith PBS, to remove cells in suspension, and then incubated at37�C in the absence or presence of r-AtHSP70 (10 mg/mL). Phasecontrast images at specific scratch sites were captured at 0, 12, 24,and 48 hours.

Transwell migration assayCell migration was performed using Transwell chambers

(Corning) with a pore size of 8 mm. For the migration assay, atotal of 2 � 104 cells/mL were placed into the top chamber inserum-free medium, and in the bottom chamber r-AtHSP70(10 mg/mL) was added. After 12 hours of incubation at 37�Cwith 5% CO2, the cells on the top surface of the chamber wereremoved using cotton swabs, and then, the migrated cells on thebottom surface were fixed in 4% paraformaldehyde (Sigma-Aldrich), stained with 0.1% crystal violet (Sigma-Aldrich), andcounted under a light microscope in five random fields. Data areexpressed as number of cell migrating standardized to the respec-tive untreated control set to 100%. The experiment was repeatedthree times in triplicates.

UV treatmentMCF-7 andMCF-10A cells were subjected toUVB (l¼ 365 nm)

for a cycle of 30 minutes and then treated with vehicle or with ofr-AtHSP70 (10 mg/mL). After that, cell cultures were stowed in theincubator at 37�C for the following 72 hours. At the end of thisperiod, the cells were lysed and used for theWestern blot analysis.

Statistical analysisEach datum point represents the mean � SD of at least three

independent experiments. Data were analyzed by Student t testusing the GraphPad Prism 4 software program (GraphPad Soft-ware). P < 0.05 was considered as statistically significant.

ResultsHeterologous HSP70 crosses the cells and positively regulatesthe viability

Thebasal levels of endogenousHSP70proteinwere analyzed inMCF-7 andMDA-MB-231breast tumoral cells aswell as innormalMCF-10Amammary cells, BJhTERThumanfibroblast cell line andhuman lymphocytes. The neoplastic cells, especially the moreaggressive tumoral MDA-MB-231 cell line, showed a slightlyincreased expression of the endogenous HSP70 compared withnormal cells (Fig. 1A) in line with previous data showing HSPsincrease in mammary tumors (24). To evaluate whether r-AtHSP70used in the treatment, could enter the cells or, otherwise,could affect the expression level of endogenous protein, we usedthe specific anti-polyHistidine (His-tag) antibody that recognizesthe polyHistidine tail linked to the protein. As shown in Fig. 1B,the MCF-7 cells incubated for 72 hours with r-AtHSP70 resultpositive to the anti His-tag antibody, confirming that the heter-ologous HSP70 protein does cross the membrane. To test thespecificity of this reaction, we reprobed the filter with the anti-humanHSP70. In untreatedMCF-7 cells, the endogenous HSP70is well expressed. A slight increase of this protein level is obtainedby the treatment with r-AtHSP70, indicating that the treatmentmight also affect the endogenous protein expression. Immuno-fluorescence analysis shows ahighpositivity to anti-polyHistidine

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Figure 3.r-AtHSP70 influences the expression of proteins involved in cell survival. A, immunoblotting for Cyclin D1, TP53, and survivin expression in MCF-7, MDA-MB-231, andMCF-10A cells treated for 72 hours with vehicle (Ctrl) or increasing doses of r-AtHSP70 (5, 10, and 20 mg/mL). b-Actin was used as control of equal loadingand transfer. B, histograms represent the average fold change in Cyclin D1, TP53 and survivin relative to Ctrl. � , P < 0.05 compared with Ctrl.






r-HSP70-FITC in treated MCF-7 and MCF-10A cells, with respectto the empty vehicle. The image reveals a broad immunoreactivityof the r-AtHSP70 at nuclear level as well around the cytoplasm,confirming the entrance into the cells. No reaction was noticed inthe nuclei and in the cells processed without primary antibody(NCtrl; Fig. 1C).

Because exogenous r-AtHSP70 was able to migrate within thecell, we investigated the effects of this protein on cell growth.Withthis aim, we tested different concentrations of the molecule after72 hours of incubation in MTT assay. As shown in Fig. 2A,treatment with r-AtHSP70 increased cell proliferation of bothbreast cancer cell lines, while no effect was revealed in normalMCF-10A cells. Analogously, the clonogenic assay, performedafter 10 days, has confirmed a better survival of breast tumorcells under r-AtHSP70 treatment (Fig. 2B and C).

Effects of r-AtHSP70 on breast cancer progression and invasionNext, we evaluated the expression levels of Cyclin D1, Survivin,

and TP53 as they are important regulators of cell growth andapoptosis.

Western blot analysis revealed that r-AtHSP70, in the examinedcell lines, induces an upregulation of both CyclinD1 and Survivinproteins, with prevalent effects in breast cancer cell phenotypes(Fig. 3). On the other hand, a lowering of TP53 expression isevidenced inMDA-MB-231 cells and under the highest doses of r-AtHSP70 also in MCF-7 cell line. Then, we evaluated the func-

tional status of the MAPKs (ERK1/2 and p38), as they play acentral role in proliferative responses, differentiation, and stress.The phosphorylation of ERK1/2 tends to increase by the exoge-nous r-AtHSP70, particularly in MDA-MB-231 and MCF-10Acells, while the same treatment decreases the basal level ofphospho-p38 (Fig. 4). Overall, these findings, together with MTTand clonogenic assays, support the assumption that extracellularr-AtHSP70 signal gives an important contribution to breast cancerproliferation and survival.

MMPs play a critical role in the multiple steps of tumor pro-gression, including angiogenesis, invasion, andmetastases.On thebasis of these concepts, we wanted to investigate whether extra-cellular AtHSP70 can trigger the activity of MMP-2 and MMP-9.The zymography results, as reported in Fig. 5A, show a differentbasal level of metalloproteases among the three cell lines, withbarely detectable levels of MMP-2 andMMP-9 in MCF-7 cells. Onthe contrary, the MDA-MB-231 cell line expresses constitutivelyhigh levels of MMP-9, which result to be clearly activated byextracellular r-AtHSP70 already after the first 24 hours. In thesame cells, the MMP-2, which is less expressed than MMP-9, isweakly induced by 24 hours of treatment (Fig. 5A). Surprisingly,even in the normal MCF-10A cell line, the exogenous r-AtHSP70induces the upregulation of MMP-9 and MMP-2. With the pro-longation of the incubation up to 48 hours, in MCF-7 cells, theMMP-9 is slightly induced by the highest concentration of exog-enous r-AtHSP70, whereas the MMP-2 tends to decrease.

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Figure 4.Role of r-AtHSP70 on MAPK signal. A, Western blot analysis on protein lysates fromMCF-7, MDA-MB-231, and MCF-10A cells treated for 72 hours with vehicle (Ctrl)or increasing doses of r-AtHSP70 (5, 10, and 20 mg/mL) showing ERK and p38 activation. The immunoblot analyses were stripped and reprobed with totalERK and total p38. b-Actin was used as loading control. The results are the mean � SD of three separate experiments in which the band intensities wereevaluated in terms of optical density arbitrary units and expressed as the percentage of the control assumed to be 100%. B, histograms represent the averagefold change in pERK/total ERK and pp38/total p38 ratio relative to Ctrl. � , P < 0.05 compared with Ctrl.

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In themost aggressiveMDA-MB-231 cell phenotype, theMMP-9 at 48 hours results as the highest activated, in order to reach thesaturation level, regardless of the experimental conditions.

In untransformedMCF-10A cells, at the same time, the MMP-9is no longer upregulated by exogenous r-AtHSP70, while theweakactivation of the MMP-2 persists (Fig. 5A).

However, the unexpected activation of MMPs also in nor-mal breast cells has led us to examine the metalloprotease-related endostatin, an antiangiogenetic protein. As presumed,in MCF-10A cells, and, in a lower extent, in MCF-7 cells, theextracellular r-AtHSP70 induces an upregulation of endostatin,whereas inMDA-MB-231 cells, the protein expression is reducedand maintained at a lower level under treatment (Fig. 5B).

Next, we performed scratch assay and here we reported theimages captured at the most significant time points of r-AtHSPtreatment (T0, 12, 24, and 48 hours). The data, analyzed with theprogress of time, validate that r-AtHSP70 promoted net move-ment of MCF-7 and especially of MDA-MB-231 cells, to almostclose the gap created (Fig. 6A). Themigratory potential of the cellswas also determined through the Boyden chamber assay (Fig. 6B).The r-AtHSP70placed below the cell permeablemembrane is ableto attract MCF-7 and, in a greater extent, MDA-MB-231 cells.However, it is worth highlighting how MCF-10A cells, understimulation, show no migratory potential to both tests used(Fig. 6A and B).

In UV-treated cells, HSP70 preserves cell survivalBecause elevated HSP70 expression has been shown to pro-

vide a selection advantage to cancer cells, also preventing celldeath under stressful conditions, we wanted to study whether inUV-treated cells the extracellular r-AtHSP70might better preservecell survival. To this aim, MCF-7 and MCF-10A cells were subjectto UV ray (l ¼ 365 nm) for 30 minutes and then treated oruntreated with HSP70 for the following 72 hours. As presumed,the survival factor phospho-Akt results to be downregulatedunder UV ray, whereas on the same irradiated cells, the coincuba-tion with HSP70 retrieves the phosphorylation levels of theprotein than the previous condition (Fig. 7). Moreover, CyclinD1 follows the same pattern of expression of phospho-Akt. Suchstressful condition increases TP53 and its phosphorylation (Ser15) in MCF-7 cells; however, the addition of HSP70 to cellspreviously undergone to UV reduces the oncosuppressor proteinat levels similar to the controls. All these events are emphasized incancer cells compared with normal MCF-10A cell line (Fig. 7).

DiscussionElevated expression levels of HSPs in tumoral cells play a

cytoprotective role by preventing apoptosis and by restoringprotein homeostasis to promote survival of several client proteins(25, 26). Indeed, high levels of HSPs are closely associated with

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Figure 5.Effect of r-AtHSP70 on the activity of matrixmetalloproteinase and endostatin expression intransformed and untransformed breast cells.A, the cells were treated with r-AtHSP70(10 and 20 mg/mL) for the indicated time.Conditioned media were collected and analyzedby gelatin zymography. Representativezymogram of three separate experiments isshown. B, Western blotting determination ofendostatin levels at 48 hours. b-Actin was usedas loading control. C, histograms represent themean � SD of three separate experiments inwhich band intensitieswere evaluated in termsofoptical density arbitrary units and expressed asthe percentage of the control assumed as 100%.� , P < 0.05 compared with Ctrl.






invasive potential of breast cancer andwith a poor prognosis (27).Among different HSPs members, in our study we focused onHSP70, which results to be more expressed in breast cancer cells,and particular in the more aggressive MDA-MB-231 phenotype,compared with noncancer cell types.

Therefore, inhibitionof these classes of proteins is an importantstrategy of anticancer therapy. In addition to the role of intracel-lular HSPs, even those present in the extracellular compartmentprofoundly affect the tumor aggressiveness. The results obtainedin this investigation are heading in this direction, because theexogenous r-AtHSP70 protein, in a singular manner in trans-

formed breast cells, is able to keep the proliferative and survivalsignals, both in basal and stress-induced conditions. It is worth tokeep also in mind as the neoplastic cells, compared with normalcells, show an overexpression of proteins belonging to the familyofHSPs that boosts cell survival (11). The recombinantArabidopsisthaliana protein, used in this study, is able to cross the membraneof both normal and tumor cell types and to localize in the cytosoland around the nucleus affecting cell behavior, particularly inneoplastic cells. In this respect, it was proposed as the HSP70crosses the membrane, through its ability to bind to phosphati-dylserine and formation of pores, as shown in artificial lipid

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Figure 6.Cell migration induced by r-AtHSP70. A, cell migration was assessed by Scratch assay for control and 10 mg/mL r-AtHSP70–treated cells. The filling of scratch bymigrated cells at time 0, 12, 24, and 48 hours was imaged. MDA-MB-231 cells have a more motile appearance to migrate than MCF-7 and MCF-10A. Theseimages are representative of three independent experiments. B, transmigration assay through Boyden chambers in all three cell lines treated with 10 mg/mLr-AtHSP70 for 12 hours. White histogram, untreated cells; gray histogram, treated cells. � , P < 0.05, compared with Ctrl.

Nigro et al.





membranes (28, 29). The endolysosomal and exosomal transporthas been implicated in HSP70 trafficking in various cell types,thereby influencingmany cellular activities and signaling cascades(30–38). Here, we have shown that recombinant plant HSP70induces an increase of cell viability in breast tumoral MCF-7 andMDA-MB-231 cells, together with upregulation of Cyclin D1 andSurvivin. In contrast, the expression of TP53 is downregulated.

It is well known in literature that Survivin and Cyclin D1, bothtransducers of proliferative and growth signals, are moreexpressed in neoplastic cells and closely related with the increaseof chemoresistance (39, 40). Our results confirm an overexpres-sion of these proteins in the neoplastic clones compared withuntransformed MCF-10A cells. Moreover, increased levels ofSurvivin have been found in the more aggressive MDA-MB-231cell phenotype. In addition to upregulating these proteins,r-AtHSP70 also leads to activation of ERK1/2. This kinase playsa central role in cell proliferation control; in fact, it is necessary forG1 to S-phase progression and for the induction of positiveregulators of the cell cycle (41). One key target of the pathwayis D-type Cyclins and our results are in line with this, as bothfunctional signals are upregulated under HSP stimulus. On thecontrary, p38 MAPK, being most frequently associated with atumor-suppressor function, results decreased following treatmentwith the highest concentrations of r-AtHSP70, coincidently whencell survival is more preserved.

Owing to the ability ofHSP70 to protect cells fromawide rangeof apoptotic signals and to drive tumorigenesis, we wanted toinvestigate whether the protein was able to influence the meta-static and proinvasive properties of the neoplastic breast cells. To

this,wehavedetermined the activity ofmetalloproteinases,MMP-2 and MMP-9, which, as widely known, are the main markers ofcell invasion and migration (11, 42). The zymographic analysisperformed on the conditionedmediumof the cells, collected aftertreatment with AtHSP70 showed a strong induction of metallo-proteases,most evident at 24hours of treatment. At this time, onlyin the high tumorigenic and metastatic MDA-MB-231 cells, thereis a consistent upregulation of MM-9. The unexpected activa-tion of MMP-2 and MMP-9 in the wild-type MCF-10A cellscorrelates well with the sustained expression of the antiangio-genic protein, endostatin. In fact, data from the literature havedocumented as metalloproteinases can act as negative regula-tors of angiogenesis by releasing antiangiogenic proteins endo-statin, angiostatin, and tumostatin (43, 44). As we demonstrat-ed, endostatin is already more expressed in MCF-10A cells bothin basal condition as well as under HSP70 stimulation. There-fore, the activation of MMP-2 and MMP-9 found in these cells isnot correlated to metastatic reaction, but rather to antiangio-genic response. In contrast, in the two tumoral phenotypes, asexpected, the endostatin is barely detectable, especially in themore aggressive MDA-MB-231 cells, in which, however, theMMPs are significantly active.

Further confirmation of the prometastatic effect of HSP70comes from scratch and Transwell migration assays. In fact, whilenormal cells show no migratory response, tumor cells, and, inparticular, theMDA-MB-231 cells are highlymobile enough to fillthe space created. Besides,many studies onHSPs and the family ofHSP70 reported that these proteins are involved in the formationof metastases and dissemination of tumor cells.

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Figure 7.r-AtHSP70 sustains survival in UV-treated breast cells. A, Western blot analysis on protein lysates from MCF-7 and MCF-10A cells subjected to UVB ( �l ¼ 365 nm)for a cycle of 30 minutes and then treated for 72 hours with vehicle (Ctrl) or r-AtHSP70 (10 mg/mL) showing the phosphorylated form of Akt-Ser473 (pAkt),TP53-Ser15 (pTP53). The immunoblot analyses were stripped and reprobed with Cyclin D1, total Akt, and TP53. b-Actin was used as loading control. B,histograms represent the average fold change in pAkt/total Akt and pTP53/TP53 ratio, Cyclin D1 relative to Ctrl. � , P < 0.05 compared with Control.






After assessing the role of extracellular HSP70 on the growthand on the prometastatic properties of the cells, we studied theeffects of the exogenous r-AtHSP70 in MCF-7 and MCF-10A cellsunderUV-stress condition. As expected, the decreased levels of Aktphosphorylation in irradiatedMCF-7 cells were well restored by r-AtHSP70 and these data correlate with the pattern of Cyclin D1expression.

According to the above data, the increase of TP53 and itsphosphorylation at Ser 15, which have been extensively used asa marker for TP53 stability and function, in UV-induced MCF-7cells, is lost under r-AtHSP70 protein exposure (45, 46). Thisreinforces the concept of the cytoprotective and antiapoptotic roleof HSPs, as reported in different cellular contexts (47–49).

This is the first study on mammary cells, transformed and not,that highlights how the recombinant HSP70 from Arabidopsisthaliana dramatically improves cell survival, particularly in thetransformed clones. Within these clones, the most significanteffects have been documented in the more tumorigenic MDA-MB-231 cells. In doing so, the heterologous r-AtHSP70 potenti-ates the directional movement as well as the metastatic potentialof the tumoral cells. Therefore, the study of these proteins and thecharacterization of their expression in the transformed pheno-types could be useful to identify molecules capable of interferingon their biologic action in order to increase the probability ofsuccess and effectiveness of some antitumor drugs.

Because the protein mainly influences the behavior of cancercells, as here reported, the use of specific inhibitors may be ofclinical importance because of their selectivity of action towardthe transformed cells, thus reducing the side effects in normaltissues.

Although r-AtHSP70 seems to enter cells, it could be used as atool for studying mechanisms of action of this protein on tumorprogression. Indeed, mutants of r-AtHSP70 produced in heterol-ogous systems might help in identifying the critical sequences of

the protein responsible for structure/function relationships withtumor induction and maintenance.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A. Nigro, R. Iannacone, M.L. PannoDevelopment of methodology: A. Nigro, S. Panza, G.M. Liuzzi, F. CelliniAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Nigro, L. Mauro, S. Panza, S. Aquila, F. De AmicisAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Nigro, L. Mauro, F. Giordano, G.M. Liuzzi,S. Aquila, M.L. PannoWriting, review, and/or revision of the manuscript: A. Nigro, L. Mauro,R. Iannacone, G.M. Liuzzi, C. Indiveri, M.L. PannoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Nigro, F. Giordano, S. Panza, F. De Amicis,F. CelliniStudy supervision: A. Nigro, L. Mauro, G.M. Liuzzi, M.L. PannoOther (financial support): C. Indiveri

AcknowledgmentsThe authors thankDr. SturinoD(Professor of English,University ofCalabria,

Cosenza) for reviewing the language of the manuscript.

Grant SupportThis work was supported by grant: Programma Operativo Nazionale

[01_00937] - MIUR "Modelli sperimentali biotecnologici integrati per losviluppo e la selezione di molecole di interesse per la salute dell'uomo" (toA. Nigro, L. Mauro, F. Giordano, S. Panza, R. Iannacone, G. M. Liuzzi, S. Aquila,F. De Amicis, F. Cellini, C. Indiveri, M. L. Panno).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 12, 2015; revised January 26, 2016; accepted February 8,2016; published OnlineFirst March 3, 2016.

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2016;15:1063-1073. Published OnlineFirst March 3, 2016.Mol Cancer Ther Alessandra Nigro, Loredana Mauro, Francesca Giordano, et al. Metastatic Potential of Breast Cancer CellsRecombinant Arabidopsis HSP70 Sustains Cell Survival and

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