Recombinant Arabidopsis HSP70 sustains cell survival and … · 2016. 3. 2. · 1 Recombinant...

1

Recombinant Arabidopsis HSP70 sustains cell survival and metastatic potential of breast

cancer cells

Alessandra Nigro1, Loredana Mauro2, Francesca Giordano2, Salvatore Panza3, Rina

Iannacone4, Grazia Maria Liuzzi5, Saveria Aquila2,3, Francesca De Amicis2,3,

Francesco Cellini4, Cesare Indiveri1, Maria Luisa Panno2

1Department of Biology, Ecology and Earth Sciences, 2Department of Pharmacy and Health and

Nutrition Sciences; 3Health Center, University of Calabria, Arcavacata di Rende (CS), Italy

4ALSIA-Research Center Metapontum Agrobios, Metaponto, Matera, Italy

5Department of Biosciences, Biotechnologies and Biopharmaceutics, Aldo Moro University, Bari,

Italy

Running title: Effects of r-AtHSP70 on breast cancer cells

Key words: HSP70, breast cancer cells, cell survival, MMPs, UV ray

Grant support: This research was supported by grant :Programma Operativo Nazionale

[01_00937] - MIUR ‘Modelli sperimentali biotecnologici integrati per lo sviluppo e la selezione di

molecole di interesse per la salute dell’uomo’(to A.Nigro, L. Mauro, F. Giordano, S. Panza, R.

Iannacone, G.M. Liuzzi, S.Aquila, F. De Amicis, F. Cellini, C. Indiveri, M. L. Panno).

Corresponding author: Prof. Maria Luisa Panno, Department of Pharmacy and Health and

Nutrition Sciences, University of Calabria, 87036 Rende, Italy, E-mail: [email protected]

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

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 3, 2016; DOI: 10.1158/1535-7163.MCT-15-0830

http://mct.aacrjournals.org/

2

ABSTRACT

The chaperone HSP70 protein is widely present in many different tumors and its expression

correlates with an increased cell survival, low differentiation and poor therapeutic outcome in

human breast cancer. The intracellular protein has prevalently a cytoprotective function, while the

extracellular HSP70 mediates immunological responses.

Evolutionarily, HSPs are well conserved from prokaryotes to eukaryotes, and human HSP70 shows

a strong similarity to that of plant origin.

In the present paper, we have tested the potential effect of recombinant HSP70, from Arabidopsis

thaliana, on cell survival and metastatic properties of breast cancer cells. Our data show that HSP70

sustains cell viability in MCF-7 and MDA-MB-231 breast tumoral cells and increases Cyclin D1

and Survivin expression. The extracellular HSP70 triggers cell migration and the activation of

MMPs particularly in MDA-MB-231 cells.

Furthermore, under UV-induced stress condition, the low levels of phospho-AKT were increased by

exogenous HSP70, together with the up-regulation of Cyclin D1 particularly in the tumoral cell

phenotype. On the other hand, UV increased TP53 expression and the co-incubation of HSP70

lowers the TP53 levels similar to the control.

These findings correlate with the cytoprotective and anti-apoptotic role of HSPs, as reported in

different cellular contexts. This is the first study on mammary cells that highlights how the

heterologous HSP70 from Arabidopsis thaliana sustains cell survival prevalently in breast cancer

cell types, thus maintaining their metastatic potential. Therefore, targeting HSP70 would be of

clinical importance since HSP70 blocking selectively targets tumor cells, in which it supports cell

growth and survival.




3

INTRODUCTION

The expression of heat shock proteins (HSPs) is a basic and well-conserved cellular response to a

wide variety of stress conditions. In fact, although these proteins are produced at low levels in basal

conditions, their expression increases during cellular damage in order to trigger the repair,

necessary for the salutary resolution of the insult.

However, HSPs play also a key role in the folding and protein metabolism participating in the

assembly of multimeric protein complexes as well as in their transport. These actions involve their

pivotal role within the cell, even though it has been recently highlighted the extracellular function of

the HSPs. In this compartment, HSPs act as alert stress signals priming other cells, particularly

those of the immune system, to avoid the propagation of the insult and favor resolution [1].

Extracellular HSP70 has also been reported to increase microbicide capacity and chemotaxis of

neutrophils [2, 3], to enhance phagocytosis [4], and to modulate the response of monocytes to

endotoxin [5]. Overall, extracellular HSP70 has been associated with both immunostimulatory and

immunosuppressive activities, depending on the cell context and on the microenvironment in which

the protein exists [6].

Heat shock proteins interact with antigen presenting cells eliciting a cascade of events including re-

presentation of heat shock protein-chaperoned peptides by MHC and maturation of dendritic cells.

These properties of heat shock proteins also allow them to be used for immunotherapy of cancers

and infections in novel ways [7].

However, various reports have emphasized the involvement of HSPs in tumorigenesis, as they may

provide a selection advantage to cancer cells to better support cell growth and survival. In

particular, HSP70 has been shown to play a negative role in the mitochondrial apoptotic pathway by

directly binding to Apaf-1, thus avoiding caspase activation [8, 9].

Studies, in this field, have demonstrated that extracellular HSP70 in breast cancer cells, with other

assistant proteins Hop, HSP40 and p23, increases the association of HSP90 alpha and MMP-2 to




4

address its activation [10, 11]. Accordingly, high expressions of HSP70 are always associated with

more aggressive tumor phenotypes, lymph node metastases, and increased breast cancer cell

survival that correlates with poor prognosis [12-14].

Tumor progression, mediated by exogenous HSP70, is supported by many signals, such as

activation of epidermal growth factor receptor, stimulation of MAPK signaling, secretion of

interleukin (IL)-8, up-regulation of NF-kB/AP-1 and subsequent activation of MMPs [15-18].

Additionally, extracellular HSP70 acts as a risk signal on mononuclear cells to provide an

inflammatory tumor microenvironment. By acting on the immune system, it can trigger signaling in

tumor cells in an autocrine or paracrine fashion via binding to Toll-like receptors, thus promoting

invasion and angiogenesis [13].

Evolutionarily, HSPs are well conserved from prokaryotes to eukaryotes, such as in plants and

humans [19].The utilization of HSPs derived from plants can be exploited to assess their action on

mammalian immune system or to evaluate their action on tumor cells.

HSPs purified from plants represent a powerful option since they have several advantages including

low costs, absence of mammalian pathogens and no endotoxic side effect.

Recently, it has been reported how recombinant HSP70 (r-AtHSP70) of plant origin, purified from

bacterial cultures, was able to protect rat cardiac and hepatic function under ischemic and sepsis

conditions [20].

Starting from such evidence, in the present paper we used the recombinant HSP70, obtained from

heat stressed Arabidopsis thaliana cDNA, to evaluate, for the first time, its potential effect on the

survival response in the normal MCF-10A human mammary epithelia cells and in both MCF-7 and

MDA-MB-231 human breast-derived cells with low and high tumorigenic potential, respectively.




5

MATERIALS AND METHODS

Materials

DMEM/F12, DMEM, L-glutamine, penicillin, streptomycin, fetal bovine serum, horse serum, and

PBS were purchased from GIBCO. Insulin, hydrocortisone, cholera toxin, epidermal growth factor

Acetone, Tris-HCl, SDS, Bromophenol, Glycerol, Gelatin, Commassie Brilliant Blue R250 were

from SIGMA. Acrylamide/bis acrylamide, 40% solution were from Applichem. Anti-antibodies

HSP70, Cyclin D1, TP53, Survivin, phospho ERK1/2, ERK1/2, β actin and endostatin were

purchased from Santa Cruz Biotechnology, while anti-antibodies phospho-Akt, Akt, phospho-

p38MAPK, p38MAPK from Epitomics. Anti-antibody phospho-TP53 (Ser-15) was from Cell

Signaling. The His Tag antibody was purchased from GenScript.

Cell cultures

MCF-7, MDA-MB-231, MCF-10A and BJhTERT cells were obtained from American Type Culture

Collection (Manassas, VA, USA) where they were authenticated, stored according to the supplier’s

instructions, and used within 4 months after frozen aliquot resuscitations. Every 4 months, cells

were authenticated by single tandem repeat analysis at our Sequencing Core; morphology, doubling

times, estrogen sensitivity, and mycoplasma negativity were tested (Myco Alert; Lonza, Basel,

Switzerland). ER-positive MCF-7 human breast cancer cells were cultured in Dulbecco’s Modified

Eagle’s Medium/Nutrient Mixture F-12Ham (DMEM/F-12) supplemented with 5% fetal bovine

serum (FBS); L-glutamine (1%) and Penicillin/Streptomycin (1%).

MDA-MB-231 is a metastatic human breast cancer cell line triple-negative for ERα, PR and HER-

2. MDA-MB-231 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)

supplemented with 5% fetal bovine serum (FBS); L- glutamine (1%) and Penicillin/Streptomycin

(1%).

The MCF-10A cell line is a non-tumorigenic human epithelial breast cell line responsive to insulin,

glucocorticoids, cholera toxin, and epidermal growth factor (EGF). The MCF-10A cells were




6

cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F-12)

supplemented with 5% Horse serum (HS); L-glutamine (1%), Penicillin/Streptomycin (1%), 100

ng/ml cholera toxin; Hydrocortisone (0,5μg/ml); Insulin (10μg/ml) and EGF (20ng/ml).

The BJhTERT human fibroblast cell line was cultured in Minimum Essential Medium (MEM) with

10% FBS, L-glutamine (1%) and Penicillin/Streptomycin (1%). All cells were grown at 37°C under

5% CO2 humidified incubator.

Normal Lymphocytes.

Peripheral blood was obtained from healthy volunteers. Peripheral blood mononuclear cells

(PBMC) were isolated by Ficoll-Paque Plus (GE Healthcare) gradient centrifugation from 10ml of

blood and then washed once with cold phosphate buffered saline (PBS). Total protein extracts from

PBMC were lysed in Ripa buffer and used for Western blot.

HSP-70 His-tag Arabidopsis

The recombinant HSP70 protein, tested in our experiments, derives from Arabidopsis thaliana

plants. The coding region of the gene HSP70-4 was amplified by PCR from cDNA coming from

leaf tissues exposed to heat stress. The amplified sequence was confirmed by sequencing and

cloned in the E. coli expression vector pET27+ resulting in pETAtHSP70. Competent BL21 cells

were transformed with the pETAtHSP70 plasmid. The recombinant AtHSP70 protein was extracted

and purified as previously reported [20]. Briefly, the protein was purified by affinity

chromatography and then dialyzed. The purity of r-AtHSP70 was checked by SDS-PAGE and

Western blot as previously described [20].

These experiments were performed at the Research Center “Metapontum Agrobios” (Metaponto,

Matera) which supports research and development of the agri-food system through plant

biotechnology. In all experiments, purified r-AtHSP70 was solubilized in Tris-HCl pH7.5

phosphate buffer. The same buffer alone was added to the control samples.




7

Western Blot

MCF-7, MDA-MB-231, MCF-10A and BJhTERT cells were grown in 10 cm dishes to 70-80%

confluence, treated or not with r-AtHSP70 for 72h and lysed. Total cellular extracts were subject to

SDS-PAGE and transferred to a nitrocellulose membrane by electroblotting as previously described

[21, 22]. Finally, the membranes were incubated with horseradish peroxidase-coupled secondary

antibodies for 1 h at room temperature. The membranes were washed with TBST 1X (Tris-Buffered

Saline and Tween 20) after each antibody binding reaction. Detection of each protein was

performed using an ECL kit (Santa Cruz Biotecnhology).

Immunofluorescence

As to immunofluorescence analysis, the cells were fixed with 4% paraformaldehyde, permeabilized

with 0.2% Triton X-100, followed by blocking with BSA (3%, 30 min), and then incubated with

anti-r-AtHSP70 His-tag antibody (4°C, overnight) and with fluorescein-conjugated secondary

antibody (30 min, room temperature). IgG fluorescein-conjugated secondary antibody was used as

negative control (NCtrl). DAPI staining was used for nuclei detection (Sigma). Protein cellular

localization was observed under a fluorescence microscope (Olympus BX51 fluorescence

microscope; x 100 objective).

MTT assay

For viability assay MCF-7, MDA-MB-231 and MCF-10A cells were seeded (1x104) in 96-well

plates and serum-starved for 24h before the addition of treatment. On the second day, the cells were

treated with r-AtHSP70 at 5, 10 and 20μg/ml and incubates at 37° C for 72 hours. At the end of

treatment the medium was removed and replaced with MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-

diphenyltetrazolium bromide (2mg/ml) and incubated at 37°C for 3-4 hours [22]. During this

incubation, viable cells with active metabolism convert MTT into a purple colored formazan




8

product with an absorbance maximum near 570 nm. Finally, the absorbance was measured on each

well in a multi-plate reader (Multiscan ex Thermo Scientific).

Clonogenic assay

Clonogenic assay was performed by plating MCF-7, MDA-MB-231 and MCF-10A cells in 6 well

plates at a density of 3000 cells/well. The day after, the cells were starved and then r-AtHSP70

(10µg/ml and 20µg/ml) was added to cultures. The medium was replaced with fresh medium

containing r-AtHSP70 every 3 days. On the 10th day, the medium was removed and cell colonies

were stained with crystal violet (0.1% in 20% methanol). Colonies containing >50 normal-

appearing cells were counted and photographed through a digital camera. The experiment was

repeated three times in triplicates.

Detection of MMPs by zymography

The MCF-7, MDA-MB-231 and MCF-10A cells were treated or untreated with r-AtHSP70 at final

concentration of 10 and 20μg/ml for 24 and 48h at 37°C and, at the end of incubation, the

conditioned medium was collected and subjected to zymographic analysis [23]. Briefly, 40µl of

each supernatant were precipitated with cold acetone at 20°C for 1h, centrifuged and the pellets

were solubilized with a loading buffer (Tris-HCl pH6,8 125M, SDS 4%, Bromophenol 0,10%,

Glycerol 0,25%). The obtained samples were separated in 7.5% polyacrylamide gels copolymerized

with 0.1% (w/v) gelatin. Stacking gels contained 5.4% polyacrylamide. After electrophoresis, the

gels were washed in 2.5% (w/v) Triton X-100, 10mM CaCl2, 50mM Tris-HCl, pH 7.4 and then

incubated overnight at 37°C in 1% (w/v) Triton X-100, 50mM Tris-HCl, 10mM CaCl2, pH 7.4

(developing buffer). After staining and de-staining of the gels, gelatinase activity was detected as a

lysis area on the blue background of the gel fractionated by SDS-PAGE on a 7,5% gel




9

polyacrylamide 40% (Acrylamide/bis acrylamide, 40% solution, Applichem) containing 1.0mg/ml

gelatin (Sigma) under non reducing conditions.

After washing in Tris buffer, the gel was stained with Coomassie Brilliant Blue R-250, and

subsequently destained with 10% (v/v) acetic acid. The non-staining bands resulting from digestion

of the substrate by gelatinase enzymes were then visualized.

Cell migration assay

Cell migration was assessed in scratch assays. Briefly, confluent MCF-7, MDA-MB-231 and MCF-

10A cells plated on tissue culture dishes were manually scratched with a 10µl pipette tip, washed

with PBS, to remove cells in suspension, and then incubated at 37°C in the absence or presence of

r-AtHSP70 (10μg/ml). Phase contrast images at specific scratch sites were captured at 0, 12, 24 and

48h.

Transwell migration assay

Cell migration was performed using transwell chambers (Corning, NY, USA) with a pore size of

8μm. For the migration assay, a total of 2×104 cells/ml were placed into the upper chamber in

serum-free medium, and in the bottom chamber r-AtHSP70 (10μg/ml) was added. After 12h of

incubation at 37°C with 5% CO2, the cells on the upper surface of the chamber were removed using

cotton swabs, and then, the migrated cells on the bottom surface were fixed in 4%

paraformaldehyde (Sigma-Aldrich), stained with 0.1% crystal violet (Sigma-Aldrich) and counted

under a light microscope in five random fields. Data are expressed as number of cell migrating

standardized to the respective untreated control set to 100%. The experiment was repeated three

times in triplicates.




10

UV treatment

MCF-7 and MCF-10A cells were subjected to UVB (λ=365nm) for a cycle of 30 minutes and then

treated with vehicle or with of r-AtHSP70 (10μg/ml). After that, cell cultures were stowed in the

incubator at 37°C for the following 72 h. At the end of this period, the cells were lysed and used for

the Western blot analysis.

Statistical analysis

Each datum point represents the mean ± S.D. of at least 3 independent experiments. Data were

analyzed by Student’s t test using the Graph Pad Prism 4 software program (Graph Pad Software,

La Jolla, CA, USA). P<0.05wasconsidered as statistically significant.




11

RESULTS

Heterologous HSP70 crosses the cells and positively regulates the viability

The basal levels of endogenous HSP70 protein were analyzed in MCF-7 and MDA-MB-231 breast

tumoral cells as well as in normal MCF-10A mammary cells, BJhTERT human fibroblast cell line

and human lymphocytes. The neoplastic cells, especially the more aggressive tumoral MDA-MB-

231 cell line, showed a slightly increased expression of the endogenous HSP70 compared to normal

cells (Fig. 1A) in line with previous data showing HSPs increase in mammary tumors [24]. To

evaluate whether r-AtHSP70 used in the treatment, could enter the cells or, otherwise, could affect

the expression level of endogenous protein, we used the specific anti-polyHistidine (His-tag)

antibody that recognizes the polyHistidine tail linked to the protein. As shown in Fig. 1B, the MCF-

7 cells incubated for 72h with r-AtHSP70 result positive to the anti His-tag antibody, confirming

that the heterologous HSP70 protein does cross the membrane. To test the specificity of this

reaction, we reprobed the filter with the anti-human HSP70. In untreated MCF-7 cells, the

endogenous HSP70 is well expressed. A slight increase of this protein level is obtained by the

treatment with r-AtHSP70, indicating that the treatment might also affect the endogenous protein

expression. Immunofluorescence analysis shows a high positivity to anti-polyHistidine r-HSP70-

FITC in treated MCF-7 and MCF-10A cells, with respect to the empty vehicle. The image reveals a

broad immunoreactivity of the r-AtHSP70 at nuclear level as well around the cytoplasm, confirming

the entrance into the cells. No reaction was noticed in the nuclei and in the cells processed without

primary antibody (NCtrl) (Fig. 1C).

Since exogenous r-AtHSP70 was able to migrate within the cell, we investigated the effects of this

protein on cell growth. With this aim, we tested different concentrations of the molecule after 72h

of incubation in MTT assay. As shown in Fig. 2A, treatment with r-AtHSP70 increased cell

proliferation of both breast cancer cell lines, while no effect was revealed in normal MCF-10A

cells. Analogously, the clonogenic assay, performed after 10 days, has confirmed a better survival

of breast tumor cells under r-AtHSP70 treatment (Fig. 2B, C).




12

Effects of r-AtHSP70 on breast cancer progression and invasion

Next, we evaluated the expression levels of Cyclin D1, Survivin and TP53 as they are important

regulators of cell growth and apoptosis.

Western Blot analysis revealed that r-AtHSP70, in the examined cell lines, induces an up-regulation

of both Cyclin D1 and Survivin proteins, with prevalent effects in breast cancer cell phenotypes

(Fig. 3). On the other hand, a lowering of TP53 expression is evidenced in MDA-MB-231 cells and

under the highest doses of r-AtHSP70 also in MCF-7 cell line. Then, we evaluated the functional

status of the MAPKs (ERK1/2 and p38), since they play a central role in proliferative responses,

differentiation and stress. The phosphorylation of ERK1/2 tends to increase by the exogenous r-

AtHSP70, particularly in MDA-MB-231 and MCF-10A cells, while the same treatment decreases

the basal level of phospho-p38 (Fig. 4). Overall, these findings, together with MTT and clonogenic

assays, support the assumption that extracellular r-AtHSP70 signal gives an important contribution

to breast cancer proliferation and survival.

MMPs play a critical role in the multiple steps of tumor progression, including angiogenesis,

invasion and metastases. Based on these concepts, we wanted to investigate whether extracellular

AtHSP70 can trigger the activity of MMP-2 and MMP-9. The zymography results, as reported in

Fig. 5A, show a different basal level of metalloproteases among the three cell lines, with barely

detectable levels of MMP-2 and MMP-9 in MCF-7 cells. On the contrary, the MDA-MB-231 cell

line, expresses constitutively high levels of MMP-9, which result to be clearly activated by

extracellular r-AtHSP70 already after the first 24h. In the same cells, the MMP-2, which is less

expressed than MMP-9, is weakly induced by 24h of treatment (Fig. 5A). Surprisingly, even in the

normal MCF-10A cell line, the exogenous r-AtHSP70 induces the up-regulation of MMP-9 and

MMP-2. With the prolongation of the incubation up to 48h, in MCF-7 cells, the MMP-9 is slightly

induced by the highest concentration of exogenous r-AtHSP70, while the MMP-2 tends to decrease.

In the most aggressive MDA-MB-231 cell phenotype, the MMP-9 at 48h results as the highest

activated, in order to reach the saturation level, regardless of the experimental conditions.




13

In untransformed MCF-10A cells, at the same time, the MMP-9 is no longer up regulated by

exogenous r-AtHSP70, while the weak activation of the MMP-2 persists (Fig. 5A).

However, the unexpected activation of MMPs also in normal breast cells, has led us to examine the

metalloprotease-related endostatin, an anti-angiogenetic protein. As presumed, in MCF-10A cells,

and, in a lower extent, in MCF-7 cells, the extracellular r-AtHSP70 induces an up-regulation of

endostatin, while in MDA-MB-231 cells the protein expression is reduced and maintained at a

lower level under treatment (Fig. 5B).

Next, we performed scratch assay and here we reported the images captured at the most significant

time points of r-AtHSP treatment (T0, 12, 24 and 48h). The data, analyzed with the progress of

time, validate that r-AtHSP70 promoted net movement of MCF-7 and especially of MDA-MB-231

cells, to almost close the gap created (Fig. 6A). The migratory potential of the cells was also

determined through the Boyden chamber assay (Fig. 6B). The r-AtHSP70 placed below the cell

permeable membrane is able to attract MCF-7 and, in a greater extent, MDA-MB-231 cells.

However, it is worth highlighting how MCF-10A cells, under stimulation, show no migratory

potential to both tests used (Fig. 6A, B).

In UV-treated cells HSP70 preserves cell survival

Since elevated HSP70 expression has been shown to provide a selection advantage to cancer cells,

also preventing cell death under stressful conditions, we wanted to study whether in UV-treated

cells the extracellular r-AtHSP70 might better preserve cell survival. To this aim, MCF-7 and MCF-

10A cells were subject to UV ray (λ=365nm) for 30 minutes and then treated or untreated with

HSP70 for the following 72h. As presumed, the survival factor phospho-Akt results to be down

regulated under UV ray, while on the same irradiated cells, the co-incubation with HSP70 retrieves

the phosphorylation levels of the protein than the previous condition (Fig. 7). Moreover, Cyclin D1

follows the same pattern of expression of phospho-Akt. Such stressful condition increases TP53 and

its phosphorylation (Ser 15) in MCF-7 cells; however, the addition of HSP70 to cells previously




14

undergone to UV, reduces the oncosuppressor protein at levels similar to the controls. All these

events are emphasized in cancer cells compared to normal MCF-10A cell line (Fig. 7).

DISCUSSION

Elevated expression levels of HSPs in tumoral cells play a cytoprotective role by preventing

apoptosis and by restoring protein homeostasis to promote survival of several client proteins [25,

26]. Indeed, high levels of HSPs are closely associated with invasive potential of breast cancer and

with a poor prognosis [27]. Among different HSPs members, in our study we focused on HSP70,

which results to be more expressed in breast cancer cells, and particular in the more aggressive

MDA-MB-231 phenotype, compared to non-cancer cell types.

Therefore, inhibition of these classes of proteins is an important strategy of anticancer therapy. In

addition to the role of intracellular HSPs, even those present in the extracellular compartment

profoundly affect the tumor aggressiveness. The results obtained in this investigation are heading in

this direction, since the exogenous r-AtHSP70 protein, in a singular manner in transformed breast

cells, is able to keep the proliferative and survival signals, both in basal and stress-induced

conditions. It is worth to keep also in mind as the neoplastic cells, compared to normal cells, show

an overexpression of proteins belonging to the family of HSPs that boosts cell survival [11]. The

recombinant Arabidopsis thaliana protein, used in this study, is able to cross the membrane of both

normal and tumor cell types and to localize in the cytosol and around the nucleus affecting cell

behavior, particularly in neoplastic cells. In this respect, it was proposed as the HSP70 crosses the

membrane, through its ability to bind to phosphatidylserine and formation of pores, as shown in

artificial lipid membranes [28, 29]. The endolysosomal and exosomal transport has been implicated

in HSP70 trafficking in various cell types, thereby influencing many cellular activities and signaling

cascades [30-38]. Here, we have shown that recombinant plant HSP70 induces an increase of cell

viability in breast tumoral MCF-7 and MDA-MB-231 cells, together with up-regulation of Cyclin

D1 and Survivin. In contrast, the expression of TP53 is down regulated.




15

It is well known in literature that Survivin and Cyclin D1, both transducers of proliferative and

growth signals, are more expressed in neoplastic cells and closely related with the increase of

chemoresistance [39, 40]. Our results confirm an over-expression of these proteins in the neoplastic

clones compared to untransformed MCF-10A cells. Moreover, increased levels of Survivin have

been found in the more aggressive MDA-MB-231 cell phenotype. In addition to upregulating these

proteins, r-AtHSP70 also leads to activation of ERK1/2. This kinase plays a central role in cell

proliferation control, in fact it is necessary for G1 to S phase progression and for the induction of

positive regulators of the cell cycle [41]. One key target of the pathway is D-type Cyclins and our

results are in line with this, since both functional signals are up regulated under HSP stimulus. On

the contrary, p38 MAPK, being most frequently associated with a tumor-suppressor function,

results decreased following treatment with the highest concentrations of r-AtHSP70, coincidently

when cell survival is more preserved.

Owing to the ability of HSP70 to protect cells from a wide range of apoptotic signals and to drive

tumorigenesis, we wanted to investigate if the protein was able to influence the metastatic and pro-

invasive properties of the neoplastic breast cells. To this, we have determined the activity of

metalloproteinases, MMP-2 and MMP-9, which, as widely known, are the main markers of cell

invasion and migration [11, 42]. The zymographic analysis performed on the conditioned medium

of the cells, collected after treatment with AtHSP70 showed a strong induction of metalloproteases,

most evident at 24h of treatment. At this time, only in the high tumorigenic and metastatic MDA-

MB-231 cells, there is a consistent up-regulation of MM-9. The unexpected activation of MMP-2

and MMP-9 in the wild type MCF-10A cells, correlates well with the sustained expression of the

antiangiogenic protein, endostatin. In fact, data from the literature have documented as

metalloproteinases can act as negative regulators of angiogenesis by releasing anti-angiogenic

proteins endostatin, angiostatin and tumostatin [43, 44]. As we demonstrated, endostatin is already

more expressed in MCF-10A cells both in basal condition as well as under HSP70 stimulation.

Therefore, the activation of MMP-2 and MMP-9 found in these cells, is not correlated to metastatic




16

reaction, but rather to anti-angiogenic response. In contrast, in the two tumoral phenotypes, as

expected, the endostatin is barely detectable, especially in the more aggressive MDA-MB-231 cells,

in which, however, the MMPs are significantly active.

Further confirmation of the pro-metastatic effect of HSP70 comes from scratch and transwell

migration assays. In fact, while normal cells show no migratory response, tumor cells, and, in

particular, the MDA-MB-231 cells are highly mobile enough to fill the space created. Besides,

many studies on HSPs and the family of HSP70 reported that these proteins are involved in the

formation of metastases and dissemination of tumor cells.

After assessing the role of extracellular HSP70 on the growth and on the pro-metastatic properties

of the cells, we studied the effects of the exogenous r-AtHSP70 in MCF-7 and MCF-10A cells

under UV-stress condition. As expected, the decreased levels of Akt phosphorylation in irradiated

MCF-7 cells were well restored by r-AtHSP70 and these data correlate with the pattern of Cyclin

D1 expression.

According to the above data, the increase of TP53 and its phosphorylation at Ser 15, that has been

extensively used as a marker for TP53 stability and function, in UV-induced MCF-7 cells, is lost

under r-AtHSP70 protein exposure [45, 46]. This reinforces the concept of the cytoprotective and

anti-apoptotic role of 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 Arabidopsis thaliana dramatically improves cell survival, particularly in the

transformed clones. Within these clones, the most significant effects have been documented in the

more tumorigenic MDA-MB-231 cells. In doing so, the heterologous r-AtHSP70 potentiates the

directional movement as well as the metastatic potential of the tumoral cells. Therefore, the study of

these proteins and the characterization of their expression in the transformed phenotypes could be

useful to identify molecules capable of interfering on their biological action in order to increase the

probability of success and effectiveness of some antitumor drugs.




17

Since the protein mainly influences the behavior of cancer cells, as here reported, the use of specific

inhibitors may be of clinical importance because of their selectivity of action towards the

transformed cells, thus reducing the side effects in normal tissues.

Whereas r-AtHSP70 seems to enter cells, it could be used as a tool for studying mechanisms of

action of this protein on tumor progression. Indeed, mutants of r-AtHSP70 produced in

heterologous systems might help in identifying the critical sequences of the protein responsible for

structure/function relationships with tumor induction and maintenance.

Acknowledgements

We thank Dr. Sturino D for the English revision, Professor of English, University of Calabria,

Cosenza.

Conflicts of interest

The authors have nothing to disclose.




18

REFERENCES

[1] De Maio A, Vazquez D. Extracellular heat shock proteins: a new location, a new function.

Shock 2013; 40:239-46.

[2] Ortega E, Giraldo E, Hinchado MD, Martínez M, Ibáñez S, Cidoncha A, et al. Role of Hsp72

and norepinephrine in the moderate exercise-induced stimulation of neutrophils’ microbicide

capacity. Eur J Appl Physiol 2006; 98:250-5.

[3] Ortega E, Hinchado MD, Martín-Cordero L, Asea A. The effect of stress-inducible extracellular

Hsp72 on human neutrophil chemotaxis: a role during acute intense exercise. Stress 2009; 12:240-9.

[4] Wang R, Kovalchin JT, Muhlenkamp P, Chandawarkar RY. Exogenous heat shock protein 70

binds macrophage lipid raft micro domain and stimulates phagocytosis, processing, and MHC-II

presentation of antigens. Blood 2006; 107:1636-42.

[5] Abboud PA, Lahni PM, Page K, Giuliano JS Jr, Harmon K, Dunsmore KE, et al. The role of

endogenously produced extracellular hsp72 in mononuclear cell reprogramming. Shock 2008;

30:285-92.

[6] Pockley AG, Muthana M, Calderwood SK. The dual immunoregulatory roles of stress proteins.

Trends Biochem Sci 2008; 33:71-9.

[7] Srivastava P. Interaction of heat shock proteins with peptides and antigen presenting cells:

chaperoning of the innate and adaptive immune responses. Annu Rev Immunol Review 2002;

4:395-425.




19

[8] Beere HM, Wolf BB, Cain K, Mosser DD, Mahboubi A, Kuwana T, et al. Heat-shock protein 70

inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell

Biol 2000; 2:469-75.

[9] Wu FH, Yuan Y, Li D, Liao SJ, Yan B, Wei JJ, et al. Extracellular HSPA1A promotes the

growth of hepatocarcinoma by augmenting tumor cell proliferation and apoptosis-resistance. Cancer

Lett 2012; 317:157-64.

[10] Eustace BK, Sakurai T, Stewart JK, Yimlamai D, Unger C, Zehetmeier C, et al. Functional

proteomic screens reveal an essential extracellular role for hsp90 alpha in cancer cell invasiveness.

Nat Cell Biol 2004; 6: 507-14.

[11] Sims JD, McCready J, Jay DG. Extracellular heat shock protein (Hsp) 70 and Hsp90α assist in

matrix metalloproteinase-2 activation and breast cancer cell migration and invasion. PLoS One

2011; 6:e18848.

[12] Gabai VL, Budagova KR, Sherman MY. Increased expression of the major heat shock protein

Hsp72 in human prostate carcinoma cells is dispensable for their viability but confers resistance to a

variety of anticancer agents. Oncogene 2005; 24:3328-38.

[13] Juhasz K, Lipp AM, Nimmervoll B, Sonnleitner A, Hesse J, Haselgruebler T, et al. The

Complex Function of Hsp70 in Metastatic Cancer. Cancers 2013; 6:42-66.




20

[14] Kluger HM, Lev DC, Kluger Y, McCarthy MM, Kiriakova G, Camp RL, et al. Using a

Xenograft Model of Human Breast Cancer Metastasis to Find Genes Associated with Clinically

Aggressive Disease. Cancer Res 2005; 65:5578-87.

[15] Evdonin AL, Kropacheva IV, Medvedeva ND. Extracellular Hsp70 stimulates multiple

signaling pathways in A431 carcinoma cells. Biochemistry (Moscow): Membrane and Cell Biology

2009; 3:291-7, Suppl Ser A

[16] Jijon HB, Buret A, Hirota CL, Hollenberg MD, Beck PL. The EGF receptor and HER2

participate in TNF-α-dependent MAPK activation and IL-8 secretion in intestinal epithelial cells.

Mediators Inflamm 2012; 2012:207398.

[17] Lee K, Kim YM, Kim DY, Jeoung D, Han K, Lee ST, et al. Release of heat shock protein 70

(Hsp70) and the effects of extracellular Hsp70 on matrix metalloproteinase-9 expression in human

monocytic U937 cells. Exp Mol Med 2006; 38:364-74.

[18] Wells A. Tumor invasion: Role of growth factor-induced cell motility. Adv Cancer Res 2000;

78:31-101.

[19] Lindquist S. The heat-shock response. Annu Rev Biochem 1986; 55:1151-91.

[20] Pasqua T, Filice E, Mazza R, Quintieri AM, Cerra MC, Iannacone R, et al. Cardiac and hepatic

role of r-AtHSP70: basal effects and protection against ischemic and sepsis conditions. J Cell Mol

Med 2015; 9:1492-503.




21

[21] Panno ML, Giordano F, Rizza P, Pellegrino M, Zito D, Giordano C, et al. Bergapten induces

ER depletion in breast cancer cells through SMAD4-mediated ubiquitination. Breast Cancer Res

Treat 2012; 136:443-55.

[22] Mauro L, Pellegrino M, De Amicis F, Ricchio E, Giordano F, Rizza P, et al. Evidences that

estrogen receptor α interferes with adiponectin effects on breast cancer cell growth.

Cell Cycle 2014; 13:553-64.

[23] Latronico T, Brana MT, Merra E, Fasano A, Di Bari G, Casalino E, et al. Impact of manganese

neurotoxicity on MMP-9 production and superoxide dismutase activity in rat primary astrocytes.

Effect of resveratrol and therapeutical implications for the treatment of CNS diseases. Toxicol Sci

2013; 135:218-28.

[24] Calderwood SK, Gong J. Molecular chaperones in mammary cancer growth and breast tumor

therapy. J Cell Biochem 2012; 113:1096-103.

[25] Brooks SA, Lomax-Browne HJ, Carter TM, Kinch CE, Hall DM. Molecular interactions in

cancer cell metastasis. Acta Histochem 2010; 112:3-25.

[26] Nguyen DX, Bos PD, Massagué J. Metastasis: from dissemination to organ-specific

colonization. Nat Rev Cancer 2009; 9:274-84.

[27] Grenert JP, Johnson BD, Toft DO. The importance of ATP binding and hydrolysis by hsp90 in

formation and function of protein heterocomplexes. J Biol Chem 1999; 274:17525-33.




22

[28] Arispe N, Doh M, De Maio A. Lipid interaction differentiates the constitutive and stress-

induced heat shock proteins Hsc70 and Hsp70. Cell Stress Chaperones 2002; 7:330-8.

[29] Arispe N, Doh M, Simakova O, Kurganov B, De Maio A. Hsc70 and Hsp70 interact with

phosphatidylserine on the surface of PC12 cells resulting in a decrease of viability. FASEB J 2004;

18:1636-45.

[30] Gastpar R, Gehrmann M, Bausero MA, Asea A, Gross C, Schroeder JA, et al. Heat shock

protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural

killer cells. Cancer Res 2005, 65:5238-47.

[31] Nylandsted J, Gyrd-Hansen M, Danielewicz A, Fehrenbacher N, Lademann U, Høyer-Hansen

M, et al. Heat shock protein 70 promotes cell survival by inhibiting lysosomal membrane

permeabilization. J Exp Med 2004; 200:425-35.

[32] Bausero MA, Gastpar R, Multhoff G, Asea A. Alternative mechanism by which IFN-gamma

enhances tumor recognition: Active release of heat shock protein 72. J Immunol 2005; 175:2900-

12.

[33] Hunter- Lavin C, Davies EL, Bacelar MM, Marshall MJ, Andrew SM, Williams JH. Hsp70

release from peripheral blood mononuclear cells. Biochem Biophys Res Commun 2004; 324:511-7.

[34] Lancaster GI, Febbraio MA. Exosome-dependent trafficking of HSP70: a novel secretory

pathway for cellular stress proteins. J Biol Chem 2005; 280:23349-55.




23

[35] Mambula SS, Calderwood SK. Heat shock protein 70 is secreted from tumor cells by a non

classical pathway involving lysosomal endosomes. J Immunol 2006; 177:7849-57.

[36] Lv LH, Wan YL, Lin Y, Zhang W, Yang M, Li GL, et al. Anticancer drugs cause release of

exosomes with heat shock proteins from human hepatocellular carcinoma cells that elicit effective

natural killer cell antitumor responses in vitro. J Biol Chem 2012; 287:15874-85.

[37] Radons J, Multhoff G. Immunostimulatory functions of membrane-bound and exported heat

shock protein 70. Exerc Immunol Rev 2005; 11:17-33.

[38] Evdonin AL, Guzhova IV, Margulis BA, Medvedeva ND. Extracellular heat shock protein 70

mediates heat stress-induced epidermal growth factor receptor transactivation in A431 carcinoma

cells. FEBS Lett 2006; 580:6674-8.

[39] Mobahat M, Narendran A, Riabowol K. Survivin as a preferential target for cancer therapy. Int

J Mol Sci 2014; 15:2494-516.

[40] Casimiro MC, Velasco-Velázquez M, Aguirre-Alvarado C, Pestell RG. Overview of cyclins

D1 function in cancer and the CDK inhibitor landscape: past and present. Expert Opin Investig

Drugs 2014; 23:295-304.

[41] Meloche S, Pouysségur J. The ERK1/2 mitogen-activated protein kinase pathway as a master

regulator of the G1- to S-phase transition. Oncogene 2007; 26:3227-39.




24

[42] Köhrmann A, KammererU, KappM, Dietl J, Anacker J. Expression of matrix

metalloproteinases (MMPs) in primary human breast cancer and breast cancer cell lines: New

findings and review of the literature. BMC Cancer 2009; 9:188.

[43] Nilsson UW, Dabrosin C. Estradiol and tamoxifen regulate endostatin generation via matrix

metalloproteinase activity in breast cancer in vivo. Cancer Res 2006; 66:4789-94.

[44] Nilsson UW, Garvin S, Dabrosin C. MMP-2 and MMP-9 activity is regulated by estradiol and

tamoxifen in cultured human breast cancer cells. Breast Cancer Res Treat 2007; 102:253-61.

[45] Shieh SY, Ikeda M, Taya Y, Prives C. DNA damage-induced phosphorylation of p53 alleviates

inhibition by MDM2. Cell 1997; 91:325-34.

[46] Ashcroft M, Kubbutat MH, Vousden KH. Regulation of p53 function and stability by

phosphorylation. Mol Cell Biol. 1999; 19:1751-8.

[47] Gupta S, Deepti A, Deegan S, Lisbona F, Hetz C, Samali A. HSP72 protects cells from ER

stress-induced apoptosis via enhancement of IRE1alpha-XBP1 signaling through a physical

interaction. PLoS Biol 2010; 8:e1000410.

[48] Wang X, Yuan B, Dong W, Yang B, Yang Y, Lin X, et al. Induction of heat-shock protein 70

expression by geranyl geranyl acetone shows cytoprotective effects in cardiomyocytes of mice

under humid heat stress. PLoS One 2014; 9:e93536.

[49] Guo S, Wharton W, Moseley P, Shi H. Heat shock protein 70 regulates cellular redox status by

modulating glutathione-related enzyme activities. Cell Stress Chaperones 2007; 12:245-54.




25

Figure Legends

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 and subjected to

immunoblot analysis with rabbit antiserum against human HSP70. The histograms represent the

mean ± S.D. of three separate experiments in which band intensities were evaluated in terms of

optical density arbitrary units. (B) Upper panel: 40μg extracts of MCF-7 cells untreated (Ctrl) or

treated (r-AtHSP70) for 72h with His-tailed r-AtHSP70 and Immunoblotted with α-His-tag r-

AtHSP70 antibody. Middle panel: the filter was reprobed with anti-human HSP70 (α-hHSP70).

Bottom panel: β actin was used as control for protein loading. (Ctrl+) correspond to 40μg of pure r-

AtHSP70 loaded onto the gel.

(C) Immunofluorescence of recombinant-AtHSP70 through α-His-tag r-AtHSP70 (FITC-

conjugated) in MCF-7 and MCF-10A cells treated for 48h with vehicle (Ctrl) or 10μg of r-

AtHSP70. DAPI staining was used to visualize the cell nucleus. NCtrl, negative control, coincides

to the sample processed without primary antibody.

Representative results are shown.

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

with vehicle (Ctrl) or increasing doses of r-AtHSP70 as indicated for 72 h. Cell proliferation is

expressed as fold change ± S.D. relative to vehicle-treated cells of three different experiments each

performed in triplicate. *p < 0.05 compared to vehicle.

(B) Clonogenic assay in MCF-7, MDA-MB-231 and MCF-10A cells. The cells were grown in the

absence (Ctrl) or presence of r-AtHSP70 (10μg/ml and 20μg/ml) for 10 days. The results shown are

representative of three independent experiments performed in triplicates. (C) The histogram is the




26

number of colonies expressed as percentage relative to control. *P<0.01, compared with untreated

cells (Ctrl).

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 and

MCF-10A cells treated for 72h with vehicle (Ctrl) or increasing doses of r-AtHSP70 (5, 10 and

20µg/ml). β actin was used as control of equal loading and transfer. (B) Histograms represent the

average fold change in Cyclin D1, TP53 and Survivin relative to Ctrl. *p < 0.05 compared to Ctrl.

Figure 4. Role of r-AtHSP70 on MAPK signal.

(A) Western blot analysis on protein lysates from MCF-7, MDA-MB-231 and MCF-10A cells

treated for 72h with vehicle (Ctrl) or increasing doses of r-AtHSP70 (5, 10 and 20µg/ml) showing

ERK and p38 activation. The immunoblots were stripped and reprobed with total ERK and total

p38. β actin was used as loading control. The results are the mean ± S.D. of three separate

experiments in which the band intensities were evaluated in terms of optical density arbitrary units

and expressed as the percentage of the control assumed to be 100%. (B) Histograms represent the

average fold change in pERK/total ERK and pp38/total p38 ratio relative to Ctrl. *p < 0.05

compared to Ctrl.

Figure 5. Effect of r-AtHSP70 on the activity of matrix metalloproteinase and endostatin

expression in transformed and untransformed breast cells.

(A) The cells were treated with r-AtHSP70 (10 and 20µg/ml) for the indicated time. Conditioned

media was collected and analyzed by gelatin zymography. Representative zymogram of three

separate experiments is shown. (B) Western blotting determination of endostatin levels at 48h. β

actin was used as loading control. (C) Histograms represent the mean ± S.D. of three separate




27

experiments in which band intensities were evaluated in terms of optical density arbitrary units and

expressed as the percentage of the control assumed as 100%. *p < 0.05 compared to Ctrl.

Figure 6. Cell migration induced by r-AtHSP70

(A) Cell migration was assessed by Scratch assay for control and 10μg/ml r-AtHSP70-treated cells.

The filling of scratch by migrated cells at time 0, 12, 24 and 48h was imaged. MDA-MB-231 cells

have a more motile appearance to migrate than MCF-7 and MCF-10A. These images are

representative of three independent experiments.

(B) Transmigration assay through Boyden chambers in all three cell lines treated with 10μg/ml r-

AtHSP70 for 12h. White histogram: untreated cells; gray histogram: treated cells.

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

(ƛ=365nm) for a cycle of 30 minutes and then treated for 72h with vehicle (Ctrl) or r-AtHSP70

(10µg/ml) showing the phosphorylated form of Akt-Ser473 (pAkt), TP53-Ser15 (pTP53). The

immunoblots were stripped and reprobed with Cyclin D1, total Akt and TP53. β 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 to Control.




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

Updated version

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

Access the most recent version of this article at:

Manuscript

Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/early/2016/03/02/1535-7163.MCT-15-0830To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-15-0830

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/early/2016/03/02/1535-7163.MCT-15-0830


Recombinant Arabidopsis HSP70 sustains cell survival and … · 2016. 3. 2. · 1 Recombinant...

Documents

Transcript of Recombinant Arabidopsis HSP70 sustains cell survival and … · 2016. 3. 2. · 1 Recombinant...