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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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])

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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 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.

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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 metallo-

endopeptidase that hydrolyzes only short 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.

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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 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 , 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 (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 of the matrix 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

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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

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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 -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

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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

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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, and , 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

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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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