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Secretion of extracellular hsp90α via exosomes increases cancer cell motility: a role for plasminogen activation
© McCready et al; licensee BioMed Central Ltd. 2010
Received: 8 October 2009
Accepted: 16 June 2010
Published: 16 June 2010
Metastasis is a multi-step process that is responsible for the majority of deaths in cancer patients. Current treatments are not effective in targeting metastasis. The molecular chaperone hsp90α is secreted from invasive cancer cells and activates MMP-2 to enhance invasiveness, required for the first step in metastasis.
We analyzed the morphology and motility of invasive cancer cells that were treated with exogenous exosomes in the presence or absence of hsp90α. We performed mass spectrometry and immunoprecipitation to identify plasminogen as a potential client protein of extracellular hsp90α. Plasmin activation assays and migration assays were performed to test if plasminogen is activated by extracellular hsp90α and has a role in migration.
We found that hsp90α is secreted in exosomes in invasive cancer cells and it contributes to their invasive nature. We identified a novel interaction between hsp90α and tissue plasminogen activator that together with annexin II, also found in exosomes, activates plasmin. Extracellular hsp90α promotes plasmin activation as well as increases plasmin dependent cell motility.
Our data indicate that hsp90α is released by invasive cancer cells via exosomes and implicates hsp90α in activating plasmin, a second protease that acts in cancer cell invasion.
Approximately 90% of cancer deaths are not from the primary tumor but due to metastasis to distant sites . Current treatments do not target metastatic disease. Towards developing anti-metastasis drugs, a functional proteomic screen was performed to identify surface proteins required for tumor cell invasion, the first step in metastasis . One of the proteins identified was the molecular chaperone heat shock protein 90α (hsp90α) . Intracellular hsp90α aids in the folding, assembly-disassembly and activation of a variety of client proteins including kinases, steroid hormone receptors and transcription factors . We discovered that extracellular hsp90α acts in tumor cell invasion through its activation of the pro-invasive protein matrix metalloproteinase-2 (MMP-2). Since the publication of this study, additional reports in the literature have demonstrated the importance of extracellular hsp90α in both physiological and pathological states. Extracellular hsp90α is required for both dermal fibroblast  and neuronal motility  as well as for melanoma migration , invasion and metastasis .
The secretion method of extracellular hsp90α from invasive cancer cells has not been fully elucidated. Hsp90α has been found in exosomes in immune and other physiologically normal cell types [8–11] and suggested to be in exosomes in diabetic cells . Exosomes are small vesicles, approximately 30-100 nM in diameter, that are part of the endocytic pathway. They are secreted as intact vesicles that form within multivesicular bodies (MVB) and are released from cells when the membrane of the MVB fuses with the plasma membrane. Exosomes function in the immune system and in acellular communication . Recent reports indicate that exosomes contribute to the aggressive nature of gliomas by transferring the mutated EGFRvIII receptor between cells . The presence of hsp90α in exosomes of other cells types and the observation that exosomes contribute to glioma aggressiveness suggested to us that hsp90α in exosomes might contribute to cancer invasiveness.
In this study, we demonstrate that hsp90α is secreted from invasive cancer cells via exosomes and increases cancer cell migration. We show that extracellular hsp90α is necessary for the activation of a second extracellular protease, plasmin, and that fibrosarcoma cell movement is dependent on this activation.
A172, HT-1080, and MDA-MB231 cells were obtained from ATCC and maintained in DMEM supplemented with 10% FBS, 1% NEAA, and 1% P/S. SUM159 cells were a kind gift from Charlotte Kuperwasser and were maintained in Hams F12 media supplemented with 5% FBS, 5 μg/mL insulin, 10 ng/mL EGF and 1% P/S. All cells were grown in a 37°C incubator with 7.5% CO2.
Quantitative Real time PCR
Total RNA was extracted from MDA-MB231 breast cancer cell lines with TRIzol (Invitrogen, California) and 2 μg of RNA was reverse transcribed into cDNA with Superscript III (Invitrogen) following the instructions supplied by the supplier. Real time PCR was performed at the Tufts Univesity Center for Neuroscience Research using the Stratagene real time cycler. Primer sequences were as follows: HSP90AA1-1 forward 5'-GGCAGAGGCTGATAAG-AACG-3' and reverse 5'CCCAGACCAAGTTTGATCATCC-3'; HSP90AA1-2 forward 5'-CATCTGATGGTGTCTGGATCC-3' and reverse 5'-AATGGCTGCAGATCCTTGTAG-3'. Samples were analyzed using the 2-ΔΔCT method (29) with GAPDH as the reference.
Brefeldin A Treatment
MDA-MB231 cells were treated with 10 μg/mL Brefeldin A (BFA), (Sigma, Missouri) or vehicle control for 16 hours. Conditioned media was collected, concentrated and subjected to SDS-PAGE followed by a Western blot probed with MMP-2 antibody (EMD Biosciences, New Jersey), anti-hsp90α or β-actin antibody (Sigma, Missouri). β-actin protein should be absent in conditioned media samples isolated from intact, alive cells.
MDA-MB231 cells were transfected with either control siRNA (non-targeting) or 100 nM siRNA directed against the HSP90AA1-2 (sense 5'-GTTAACTGGTACCAAGAAA-dTdT-3') isoform using Oligofectamine (Invitrogen). RNA was extracted as indicated above and the results are graphed as percentage knockdown setting the control at 100%.
Exosomes were isolated from A172, HT-1080, MDA-MB231, and SUM159 cells as previously described . Briefly, 5 × 106 cells were plated in 10% DMEM and allowed to settle overnight. Cells were then washed with HBSS and re-fed with serum free media or serum free media containing 15 nM dimethyl amiloride (Sigma). Media was collected 48 hours after the addition of serum free DMEM and spun at 300 × g to collect any cellular debris. This media was then filtered with a 0.2 μM filter and spun for 1 hour at 110,000 × g. The pellet was washed with PBS and spun for 1 hour at 110,000 × g. One μg of protein was subjected to Western Blot probed for hsp90α (Assay Designs, Michigan). Samples were also probed with an anti-Annexin II antibody (BD Biosciences, California) and Flotillin (Cell Signaling Technology, Massachusetts) as positive controls and vATPase subunit B (Molecular Probes, California) as a negative control.
1 × 104 MDA-MB231 cells were plated into an 8-well chamber slide and treated with exosomes isolated from MDA-MB231 cells or 0.5 μg recombinant hsp90α (Assay Designs) for 16 hours. Cells were fixed in PBS/4% paraformaldehyde/4% sucrose, permeabilized in 0.1% TritonX-100/PBS, blocked in 1%BSA/PBS and stained with Alexa546-labeled phalloidin (Invitrogen, CA) for 30 minutes to visualize F-actin.
Cell shape analysis
The cell shape and area of MDA-MB231 cells were measured and calculated with OpenLab software (Improvision). Cell shape was defined using the equation (4 × cell area)/cell perimeter2, where greater than 1 indicates a perfect circle and values less than 1 indicate a more irregular shape.
Wound healing assay
1 × 105 SUM159 breast cancer cells or A172 glioma cells were plated in an 8 well chamber slide. Cells were wounded by scratching a sterile yellow pipette tip lengthwise along the chamber. The cells were washed twice with 1× PBS and serum free media was placed in each well with either the vehicle control PBS, 0.5 μg recombinant hsp90α protein, 1 μg exosomes isolated from SUM159 cells, or 1 μg exosomes isolated from SUM159 cells plus 40 μg/mL anti-hsp90 antibody (SPS-771, Assay Designs). Pictures were taken immediately after cell wounding (0 hours) and 16 hours after cell wounding. Wound width was calculated using OpenLab software and is represented as μm between the cells at 16 hours for each treatment.
4 × 106 MDA-MB231 breast cancer cells were plated in a 150 mm tissue culture dish and allowed to settle for 24 hours. Cells were then refed with serum free media and incubated for 48 hours at 37°C. Conditioned media was concentrated by centrifugation (Millipore, MA) and a protein assay was performed (BioRad, CA). 1 mg of protein was pre-cleared with protein A beads after which 1 ug of hsp90α antibody (Assay Designs) was added to the samples. Samples were washed with RIPA B buffer (50 mM Tris, 150 mM NaCl, 0.5% NP40, 0.25% DOC) boiled, subjected to SDS PAGE, stained with Coomassie Blue and removed from the gel for mass spectrometry analysis. The excised gel bands were analyzed by mass spectrometry as previously described . MS results were verified using antibodies for hsp90α and tPA (Abcam, MA).
Plasminogen activation assay
Plasminogen activation assays were performed as previously described . Briefly, HT-1080 fibrosarcoma cells were plated in 10% DMEM and refed with serum free media 24 hours after plating. DMSO, 0.5 μM [Glu] plasminogen (American Diagnostica Inc, CT), or 40 μg/mL DMAG-N-oxide (a gift from Len Neckers) were added for five hours at 37°C. DMAG-N-oxide was used in this experiment because the large amount of antibody required for this experiment precluded its use. It has been previously characterized as an inhibitor of extracellular hsp90α . Conditioned media was concentrated (Millipore) and a protein assay was performed (Bio-Rad). 25 μg of each sample was loaded into a 0.1% gelatin zymogram. The zymogram was washed twice for two hours each in wash buffer (50 mM Tris-HCl, 150 mM NaCl, 2.5% (v/v) Triton X-100, pH 7.4), three times for 5 minutes each in water and then incubated in wash buffer for 12 hours at 37°C. The zymogram was stained with 0.5% coomassie, destained, and densitometry was performed to determine the plasminogen activation levels of each condition.
HT-1080 fibrosarcoma cells were plated in 10% DMEM. 48 hours after plating the cells were labeled with CMTMR (Invitrogen) and 1 × 105 labeled cells were plated into a 24-well Fluoroblok plate (BD Biosciences, CA). Cells were treated with either 40 μg/mL rabbit IgG, 40 μg/mL anti-hsp90α (SPS-771, Assay Designs), or 0.5 μg plasmin (Molecular Innovations, MI). Cells were allowed to migrate for 24 hours after which the number of cells that migrated to the bottom chamber were photographed and counted.
Hsp90α is secreted via exosomes in invasive cancer cells
Exosomes induce a change in morphology of breast cancer cells
Exosomes increase cancer cell motility
Extracellular hsp90α immunoprecipitates with tissue plasminogen activator
Hsp90α aids in the conversion of plasminogen to plasmin
Inhibition of extracellular hsp90α decreases tumor cell migration
In this study we present evidence that extracellular hsp90α, secreted via exosomes, activates a novel client protein and increases tumor cell motility. Previously published work from our lab and others indicate that extracellular hsp90α contributes to the activation of both MMP-2  and HER-2 , two proteins involved in cancer metastasis. We now present data indicating that extracellular hsp90α is necessary for the activation of a second protease, plasmin, also involved in tumor metastasis . Inhibiting extracellular hsp90α in vivo inhibits both wound healing  and tumor invasion . Our current findings suggest however that other exosomal proteins may also contribute to these processes. Hsp90α protein alone does not elicit as complete an effect on cell morphology or movement as the addition of exosomes (Figures 3 and 4). Hsp90α binds to tPA and inhibiting hsp90α decreased plasmin activation and cell migration. Therefore, we speculate that hsp90α is part of an extracellular complex including annexin II, tPA and plasminogen that functions to increase cell movement. Annexin II is found in exosomes and has an established role in aggressive tumors and binds both tPA and plasminogen thereby enhancing the conversion of plasminogen to active plasmin . Extracellular hsp90α increases annexin II at the cell surface in rat aortic cells, leading to an increase in plasmin production in these cells . Also, cell surface annexin II expression levels are increased in metastatic tumors and it interacts with multiple extracellular proteases that have been implicated in tumor progression . Although plasmin is known for its role in cellular invasion it has not been well studied in migration, one component of the multi-step process of tumor invasion. Plasmin may be contributing to cell migration by contributing to the local remodelling of the extracellular matrix exposing cryptic cell attachment sites necessary for cellular migration, similar to that seen in smooth muscle cells during wound healing . It is also possible that plasmin contributes to cell migration by interacting with currently unknown targets.
We suggest that exosome contents are released outside the tumor cell in close proximity to each other and other inactive extracellular pro-invasive proteins such as plasminogen. Once released from the exosomes, extracellular hsp90α assists in the activation of pro-MMP2 as well as plasminogen. Beyond MMP-2 and plasmin it is possible that hsp90α could activate other extracellular proteins as most of the proteins identified by mass spectrometry in this study were found in their inactive pro-forms. It is therefore interesting to speculate that extracellular hsp90α could activate a cassette of proteins that function collectively in cancer cell migration. These proteins would act in concert to enhance breakdown and remodeling of the extracellular matrix and permit the tumor cell to invade its microenvironment. Thus, inhibition of extracellular hsp90α could inhibit a growing number of proteins that are responsible for increased tumor cell movement making extracellular hsp90α an attractive target for drug therapy to limit tumor invasion.
In summary, we have identified that exosomes increase cell motility. One mechanism for this increased motility is the activation of plasmin by extracellular hsp90α. The discovery of a second protease activated by extracellular hsp90α suggests the possibility that the one role of extracellular hsp90α in cancer cells is the activation of precursor proteins that contribute to cellular migration and invasion.
The authors would like to thank J Dice, M Forgac, L Liscum, and T Bagci for their advice during the preparation of this manuscript. This work is supported by a grant from the NIH: R01 CA 116642 (DG Jay) and an NIH training grant T32 DK007542-21 (J McCready).
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