bioRxiv preprint doi: https://doi.org/10.1101/2020.05.10.087304; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Extracellular Thimet Oligopeptidase is Released with Extracellular Vesicles from Human
Prostate Cancer Cells
Yu Liu1,2; Lisa Bruce1,3; Adele J. Wolfson1
Author Affiliations: 1. Department of Chemistry, Wellesley College, Wellesley, MA (Liu, Bruce, Wolfson)
2. Saint Louis University School of Medicine, Saint Louis, MO (Liu)
3. Stealth Biotherapeutics, Newton, MA (Bruce)
Correspondence to: Adele J Wolfson Wellesley College, Department of Chemistry, Wellesley, MA 02481 ([email protected])
1 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.10.087304; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
ABSTRACT
Androgen signaling plays a central role in the development of prostate cancer. Androgen
hormone synthesis is tightly governed by the hypothalamic–pituitary–gonadal (HPG) axis,
including gonadotropin-releasing hormone (GnRH). Thimet oligopeptidase (TOP) is a
biologically significant peptidase known to cleave GnRH and potentially regulate its activity.
Thus, TOP can play an important role in the HPG axis through regulating the downstream
production and release of gonadal steroid hormones, including androgens, which may further
affect prostate cancer development. TOP is known to be secreted out to the extracellular space.
Here, we report that extracellular TOP can be associated with extracellular vesicles (EVs).
Western blot analysis of EVs isolated from PC3 or DU145 prostate cancer cells revealed that
TOP protein is, indeed, carried by the EVs. Budding of EVs from stimulated PC3 prostate cancer
cells can also be visualized by confocal microscopy. Significantly, the TOP enzyme carried by
EVs is enzymatically active. The present study shows that EV-associated TOP is a novel form of
this extracellular peptidase that may play a role in the disease progression of prostate cancer
cells.
2 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.10.087304; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
1. Introduction
Prostate cancer is the second leading cause of cancer-related death in men in the US. It is
estimated that 1 in 6 men will be diagnosed with prostate cancer and approximately 1 in 33 (3%)
will die of it 1. It has long been known that androgen signaling plays a central role in the
development of prostate cancer 2, 3 and androgen deprivation therapy (ADT) remains a major
therapeutic strategy for advanced prostate cancer 2-4. Androgen hormone synthesis is tightly
governed by the hypothalamic–pituitary–gonadal (HPG) axis. The pulsatile release of
gonadotropin-releasing hormone (GnRH) stimulates the secretion of luteinizing hormone (LH)
from the anterior pituitary gland, which then signals the testes to produce testosterone, the major
androgen hormone in men. Therefore, GnRH is the major regulator in androgen production 3.
GnRH antagonists act as competitive inhibitors of GnRH and decrease the secretion of LH from
the pituitary, thereby leading to a decrease in testosterone secretion from Leydig cells in the testes
5. Furthermore, GnRH antagonists are currently FDA-approved frontline treatments of prostate
cancer and are widely used in clinical applications 39.
Thimet oligopeptidase (TOP, E.C.3.4.24.15) is a well-characterized zinc metallo-
endopeptidase that hydrolyzes only short peptides with less than 20 residues, and recognizes
greatly variable cleavage sequences depending on the specific substrate 6-9. Studies have indicated
that GnRH is a substrate of TOP 6, 10, which can cleave and regulate activity of GnRH 9-11. Via
regulation of GnRH, TOP modulates the hypothalamus–pituitary–gonadal axis as well as the
downstream production and release of gonadal steroid hormones, like testosterone 11, 12, 41.
Furthermore, TOP is highly expressed and regulated in prostate tissue, and its actions are increased
in androgen-dependent prostate cancer compared to androgen-independent cancer11. Thus, TOP is
involved in both etiology and pathophysiology of prostate cancer 11.
3 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.10.087304; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
TOP has been found to be distributed in different subcellular locations depending on cell
types, i.e. nuclear localization 13, 14, membrane association 15-17, cytosolic and secreted forms 13-16,
18-21. Most of peptide processing is initiated in the intracellular space of parental cells, 6 while many
peptides need to be further processed and activated in the extracellular milieu by enzymes, such as
TOP 6. Although cytosolic TOP account for 75% of enzyme activity, a significant portion of TOP
released to the extracellular milieu is responsible for the extracellular processing of neuropeptides
such as GnRH 22. In fact, studies show that TOP can be secreted to the extracellular surroundings
6, 18, 19 and TOP activity has been detected in the media of DHT-treated LNCaP androgen
responsive cells 11.
Extracellular vesicles (EVs) are small cellular membrane vesicles that are either released from
intracellular space (exosomes) 27, or budded from the plasma membrane surface of almost all cell
types upon cell activation or programmed cell death (microvesicles) 23-27. In the past several years,
the biomedical research field has shown growing interest in the biological roles of EVs due to their
emerging roles in pathophysiological conditions and various human diseases 26, 27. Studies show
that EVs carry a heterogeneous array of intracellular and membrane-associated biologically active
molecules, such as proteins, lipids, and DNA or RNA 23-27. Previous studies demonstrate that EVs
carry transmembrane protease of the matrix metalloproteinase superfamily (MMP14) 23. Given
that TOP has been visualized on the cell plasma membrane surface by confocal microscopy 17, we
sought to investigate if TOP can be released with EVs, in addition to its free form 11, to the
extracellular space. Soluble molecules can be diluted by the large pool of circulating blood, while
EV-associated molecules can be kept in a relatively concentrated microenvironment around the
parental cells that release these EVs. Additionally, EVs can interact with cells/tissues across vast
4 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.10.087304; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
distances 28, 29. Therefore, we propose that EV-associated TOP may be important in GnRH
processing and activity regulation in a systemic manner.
2. Material and methods
2.1. Cell culture and treatment
The PC3 or DU145 cell lines were cultured at 37 °C and at 5% CO2 and grown in RPMI-1640
medium with 10% Fetal Bovine Serum at pH 7.8. PC3 cells were treated without (control) or
with 5, 10, or 20 µM calcium ionophore (A23187) for 24 hours. DU145 cells were treated with
0.3 µM DHT or its vehicle (ethyl alcohol) for 24 hours. Cell lysates were collected to extract
TOP, while the supernatant was harvested for EV purification.
2.2 Purification of MVs from cell culture supernatants
The supernatants from above control and treated cells were initially centrifuged twice at 3000
rpm for 15 min to remove residual cells, following by ultracentrifugation at 100,000 g for 30
minutes (Gyorgy et al., 2011; Li et al., 2011, 2012). Then the EV pellets were re-suspended in
PBS with 2% SDS.
2.3 Western blotting analysis of TOP protein
Western blot was conducted, as described before 23, for EVs (at the maximum lane volume) to
detect whether the EVs carry TOP protein, cell lysates (10 µg/lane) were used as control.
Transferred membrane was blocked in 5% milk, followed by overnight immuno-probing at 4°C
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with 1:300,000 anti-TOP (rabbit IgG) and 1:250,000 anti-actin (mouse IgG) primary antibodies.
Secondary anti-TOP and anti-actin antibodies (GE Healthcare) were used at 1:10,000. The
probed membrane was visualized using the Storm Scanner apparatus. Band intensity was
quantified using ImageQuant software.
2.4 Immunocytochemistry analysis
Cells without or with A23187 treatment were blocked in 20% normal goat serum (NGS) and 1%
BSA in 0.05M TBS, then incubated in 1:5000 monoclonal Rabbit-anti-TOP antibody with 1%
NGS in 0.05M TBS with 0.02% sodium azide, 0.1% gelatin (pH 7.6) for 24 hrs. The cells were
washed with 0.05M TBS with 0.02% sodium azide, 0.1% gelatin, and 10% Triton-X , then
incubated in secondary Donkey-anti-Rabbit Red Alexa 594 (Invitrogen) at 1:100 and FITC-
labeled Annexin V with 1.5% NGS in 0.05M TBS with 0.02% sodium azide and 0.1% gelatin for
90 minutes, then washed with the same buffer, and staining with DAPI for 30 minutes. The
slides were cover slipped with Gel/Mount (biomeda, cat# M01). Imaging: The cells were imaged
using a Leica TCS SP5 II confocal microscope.
2.5 Assessment of TOP Activity with Quenched Fluorescence Assay
Cell lysates and purified EV suspensions were used for TOP activity assessment. The assay
buffer was 25mM TRIS HCl (0.125mM KCl, 1µM ZnCl2, 1mM TCEP, 10% glycerol). Wild
type TOP (0.1µM) was used as assessment functioning control. TOP substrate Methyl
Coumarinyl 18 Acetate (MCA) was used at a concentration of 1.4 mM. The competitive inhibitor
of TOP, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP) was used at a
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concentration of 16.5 µM to assess breakdown in prostate cancer cells not due to TOP cleavage.
All fluorescence assays were conducted using a Molecular Devices SpectraMax M3 microplate
reader with emission wavelength at 400nm, and excitation wavelength at 325nm. Activity was
monitored every minute for up to 30 minutes. The slope of the graph (fluorescence
intensity/minute) was used to determine the activity of TOP. The activity from samples was
subtracted from the blank, which consisted of 1ul of MCA and 199ul of assay buffer.
3. Results
To address whether extracellular TOP is associated with EVs, cell models were established by
treatment of cells using calcium ionophore A23187 which is known to induce EV release from
cells by increasing intracellular calcium and membrane phospholipid redistribution30,31. Therefore,
PC3 cells were stimulated with 5 or 20 µM A23187 to induce cell activation and EV release (Fig.
1A). The culture supernatant samples from untreated control and A23187-treated cells were
subjected to centrifugation twice with low-speed to remove any floating cells and cell debris,
followed by ultracentrifugation to isolate EVs from supernatant of two treatment groups and the
control group. Western blot analysis showed TOP being detected within the isolated EVs and their
parental cell lysates of both A23187-treated and control cells (Fig. 1A). Results indicate that the
amount of TOP were undetectable on EVs from cells that were treated with 5 µM A23187 and
those of the control group. In contrast, the EVs from cells that were treated with 20 µM A23187
showed a clear TOP protein band (Fig. 1A) as compared to corresponding TOP bands from their
parental cells and the positive control recombinant TOP protein (Fig. 1A). In support of the above
findings, we found that stimulation of PC3 prostate cancer cells with 20 µM A23187 can cause up
to 90% of cells to undergo apoptosis (not shown). This is consistent with the observed phenomenon
7 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.10.087304; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
that EVs may be released by cells undergoing apoptosis. Furthermore, this is in line with previous
studies showing that tobacco smoke extract treatment can cause human macrophages to undergo
apoptosis and release EVs that carry matrix metalloproteinase-14 protein 23.
Having detected TOP expression in DU-145 cells, another prostate cancer cell line 32, 33, we
next sought to determine whether the EVs released from these cells also carry TOP protein.
Immunoblot results indicated that EVs from the culture supernatants of both control and the DU-
145 cells treated by 0.3 µM DHT do indeed carry TOP protein (Fig. 1B). Therefore, the fact that
TOP enzyme is carried by EVs in the extracellular space helped us to confirm our initial hypothesis
that extracellular TOP can be associated with EVs, and not only in the soluble form. However, this
novel finding is not surprising since EVs bleb off of the plasma membrane at random locations,
where TOP molecules may be associated with the membrane surface.
After detecting TOP molecules from the isolated EVs, TOP in both EVs and cell membrane
surface was further visualized by confocal microscopy of PC3 cells that were treated without
(control) or with 10 µM A23187 for 24 hours. Interestingly, the confocal cross-sectional images
highlighting the cellular surface of PC3 cells not only showed membrane-associated TOP, but
revealed co-staining of phosphotidylserine-rich domains in small, well-circumscribed blebs (Fig.
2A). Phosphotidylserine is a marker of EV generation 34. The sizes of these membrane surface
domains are consistent with the recognized size range for membrane microvesicles
(approximately 0.1 to 1 µm) 26,27,35. Thus, A23187 treatment appears to induce the budding and
release of EV-associated TOP protein. Furthermore, the images show a concentration of cell-
surface TOP induced on the cell surface of A23187-treated cells compared to the untreated
control cells (Fig. 2A). These results confirm and extend our findings with western analysis of
the isolated EVs (Fig. 1A-B). EVs are known for their heterogeneity and carriage of various
8 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.10.087304; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
molecular cargos. This heterogeneous nature is clearly demonstrated in Figure 2A, which shows
a large concentration of membrane-surface TOP as a result of induction apoptosis by A23187.
The visualized, circumscribed cell-surface domains that stained intensely for both TOP and
externalized phosphotidylserine also suggest that the TOP-associated EVs are budding from
apoptotic cell surfaces (Fig. 2A). Furthermore, as seen in the merged image, co-localization of
TOP and phosphotidylserine is variable, thus illustrating the heterogeneity of EVs and the array
of molecular cargo they carry.
Finally, we sought to evaluate whether TOP carried by EVs is enzymatically active. The
purified EV samples isolated from A23187-treated or control cells were then analyzed via TOP
enzymatic assay. Enzyme rates were calculated in terms of change in fluorescence per unit time.
Robust TOP activity was detected in the EVs isolated from A23187-treated prostate cancer cells
as compared to the EVs from the untreated control cells (Fig. 2B). These interesting findings not
only confirm the presence of TOP protein on membrane EVs, but also clearly demonstrate that
the EV-associated form of extracellular TOP is enzymatically active.
4. Discussion
TOP is generally considered to be a soluble molecule in the extracellular milieu 11.
However, the current study highlights for the first time a previously unrecognized novel form of
extracellular TOP that is carried by the membrane EVs. Most importantly, we showed that
extracellular EV-associated TOP exhibits considerable enzymatic activity (Fig. 2B). We have
shown that EV-carried extracellular TOP can be released from two different human prostate
cancer cell lines, both PC3 and DU145 prostate cancer cells (Fig. 1A-B). The DU145 results
9 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.10.087304; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
suggest that EV-associated TOP may also be released under natural conditions without additional
stimulation with DHT hormone (Fig. 1B). As noted, many physiological substrates of TOP, such
as GnRH, bradykinin and neurotensin, are found either in the extracellular space or in the
systemic circulation. In order to fully modulate its physiological substrates, TOP must be
accessible to the extracellular space, either by exposure on the cell membrane surface or via
release from the cell as a soluble protein. Previous studies have localized TOP to the cell surface
of the plasma membrane 17 and have shown that TOP is secreted to the extracellular
surroundings as a soluble free protein 6,18,19. The current study highlights a previously
unrecognized, novel form of extracellular TOP that is carried by membrane EVs.
Studies have shown that EVs released by cancer cells appear to play an important role in
tumor progression, diagnosis, and therapeutic potential 36-38. The role played by a number of
TOP-specific substrates in the proliferation of prostate cancer cells is a topic of interest in cancer
research. Early stage prostate cancer proliferation is dependent on male sex hormones
(androgens) and can be effectively treated with androgen-ablation therapy. Analogs of TOP
substrate GnRH-I have been utilized in the treatment of prostate cancer since the early 1980s 39,
40, for which they are used to suppress the HPG axis during the early androgen dependent phase
of prostate cancer. GnRH antagonists are currently FDA-approved frontline treatments of
prostate cancer and are widely used in clinical applications 39.
As a key modulator of the activities of GnRH and its other physiological substrates,
extracellular TOP stands as an important subject of study for further understanding of the
pathophysiological role of these hormones and monitoring the progression of prostate cancer. In
contrast to the soluble form of TOP, which easily diffuses into the circulation and can be diluted
by large volumes of circulating blood, the EV-carried TOP may stay in the tumor
10 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.10.087304; this version posted May 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
microenvironment in a relatively high concentration. Thus, this EV-associated format of TOP
may enable the enzyme to work more potently on its substrates, therefore contributing to the
progression of pathological conditions. Our study paved the way for further understanding of the
activities and functions of EV-associated TOP in the extracellular space and the specific
mechanisms by which TOP becomes bound to the plasma membrane and is subsequently
released with EVs. Our novel finding may provide insight into the development of more
effective therapeutic strategies and monitoring methods in the treatment of prostate cancer.
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Figures and legends
Figure 1. Detection of TOP molecules in isolated EVs from PC3 and DU-145 cells. (A) The PC3 cells were treated without (untreated control) or with 5 or 20 µM A23187 for 24h, then the EVs isolated from supernatants (sup.) or from the cells were lysed and analyzed via Western blotting. Recombinant wild type (WT) TOP served as positive control for detection of TOP. (B) The DU145 cells were either untreated (control) and treated with 0.3 µM DHT for 24h, then the EVs isolated from supernatants (sup.) or the cells were lysed and analyzed via Western blotting. TOP was probed by rabbit anti-human TOP primary antibody.
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Figure 2. TOP-positive EVs were budding from plasma membrane surface of A23187- activated PC3 prostate cancer cells. (A) Cells were either untreated (control) or treated with 10 µM A23187 for 24 hrs. Cells were then chemically fixed and detected with anti-TOP antibody (Red, Alexa 594 labeled) and FITC- labeled Annexin V (Green, which preferentially binds externalized phosphatidylserine). The yellow color in the merged images (Merge) demonstrates co-localization of the two labels indicating the TOP-positive EVs. Scale bar 1 µm. (B) To assess TOP activity in cells and the isolated EVs, cells were treated without (control) or with 10 µM A23187 for 24 hrs. Then the cell lysate and EV suspension were used for detection of TOP activity. Enzymatic activity of TOP was determined using quenched fluorescence kinetic assay, by the cleaving of fluorescent-tagged artificial substrate (7-methoxycoumarin-4-yl) acetate (MCA). Enzyme rates, as depicted above, were obtained by calculating the slopes (fluorescence intensity, arbitrary units). Error bars were calculated using standard deviation.
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